Preparation and physicochemical characterization of omeprazole:methyl-beta-cyclodextrin inclusion...

HealthMEDVolume 4 / Number 3 / 2010 ISSN 1840-2291

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Published by DRUNPP, Sarajevo Volume 4 Number 3, 2010 ISSN 1840-2291

HealthMEDVolume 4 / Number 3 / 2010


Sadržaj / Table of ContentsAttitudes of medical freshmen’s about some principles of professionalism .............................................................. 485-492Karaoglu Nazan, Okka Berrin, Şeker Muzaffer

* * *Comparing the detection of HIV antigen P24 by human and mouse monoclonal antibodies ..................... 493-498Masoomeh Hashemi, Seyed Mehdi Boutorabi, Reza Hajihoseini, Ali Mirjalili, Maryam Razaghi, Shirin Norolahi, Mehrdad Jalalian

* * *The uninsured ill in a developing nation ................ 499-514 Paul Andrew Bourne

* * *Preparation and physicochemical characterization of ar-temisinin-gelucire 44/14 solid dispersions ......... 515-521Gamal Osman Elhassan, Kah Hay Yuen, Jia Woei Wong,Jiyauddin Khan, Samer Al-Dhalli

* * *Evaluation of serum levels of interleukin-6 andinterleukin-8 in patients with recurrent aphthousulcerations ............................................................ 522-525Behnoush Bakhtiari, Pejman Bakianian Vaziri, MehrdadHajilooi, Hamed Mortazavi

* * *Objective and self-perceived weight status in Province of Vojvodina ........................................................... 526-532Grujic Vera, Dragnic Natasa, Harhaji Sanja, Cankovic Sonja, Radic Ivana, Cankovic Dusan

* * *Hemodynamic Instability due to IntratumoralHemorrhage in Retroperitoneal AlveolarRhabdomyosarcoma in Adult ............................ 533-535Atakan Sezer, Mehmet Ali Yagcı, Ahmet Rahmi Hatipoglu,Irfan Coskun, Fedai Calta, Irfan Cicin, Ufuk Usta, OsmanTemizoz, Teyfik Aktoz

* * *Anti-Tubercular Chemotherapy and DrugInduced Hepatitis ................................................ 536-544Amer Hayat Khan, Syed Azhar Syed Sulaiman, Mohamed Azmi Hassali, A. Razzak Muttalif

* * *Diagnosis of helicobacter pylori infection by ELISA stool antigen and comparison with the other diagnostic met-hods ....................................................................... 545-551Maryam Razaghi, Seyyed Mehdi Boutorabi, Ali Mirjalili, Shirin Norolahi, Masoumeh Hashemi, Mehrdad Jalalian

HealthMED journal with impact factor indexed in: - Thomson Reuters ISI web of Science - Science Citation Index-Expanded - EBSCO Academic Research Premier - Index Copernicus - getCITED, and etc.

Sadržaj / Table of Contents* * *

Characteristics of AIDS Patients with Mycobacterium Co-Infection: A Pilot Clinical Trial ...................... 552-558Ruichao Lu, Yong Zhang, Weiping Cai, Hongzhou Lu

* * *Treatment benefits on metabolic syndrome with diet and physical activity ...................................................... 559-566Gani Dragusha, Abdulla Elezi, Shpend Dragusha, Daut Gorani, Luljeta Begolli, Valton Sahiti

* * *Analysis of the Relationships between the Determinants In-fluential in performance of Pre-hospital Emergency System of Iran using the DEMATEL Approach .............. 567-572Amir Ashkan Nasiripour, Mohammadkarim Bahadori,Shahram Tofighi, Mahmoudreza Gohari

* * *Evaluation of clinical features and complications of dengue infected patients in Penang general hospital,Malaysia ................................................................... 573-579Khurshid Alam, Syed Azhar Syed Sulaiman, Asrul AkmalShafie, Rozina Ghazali

* * *Endocrine Dysfunction in Beta-ThalassaemicMajor Patients at Rawalpindi, Pakistan ........... 580-585Dilshad Ahmed Khan, Asma Naseer Cheema, Masood Anwar, Farooq Ahmad Khan

* * *Serum Interleukin-6 as a Serologic Marker of Chronic Periapical Lesions; A Case-control Study ......... 586-590Behnoush Bakhtiari, Hamed Mortazavi, Mehrdad Hajilooi, Shahrzad Nazari

* * *Use Of Constructivist Approach for MedicalEducation: Review of Literature ........................... 591-594Kurtulus Ongel, Sevinc O.Erdal, Selnur Erdal, Haluk Mergen, Nazan Karaoglu

* * *Therapeutic Adherence with Methadone Treatment:Evaluation of therapeutic adherence and withdrawalmanagement in methadone maintenance treatmentprogram in Penang, Malaysia ......................... 595-604Wasif. S. Gillani, Azhar. S. Sulaiman, Forouzan Bayat Nejad, Tahir Mehmood Khan, Amir Hayat Khan

* * *Basal Cell Carcinoma: Cultivation Potential and Resultsof Chromosome Aberrations Analysis ................ 605-609Ibrulj S., Haveric A., Haveric S., Rahmanovic A., Alendar F.

* * *Therapy effect on ejection fraction in patientswith hypo- and hyperthyroidism ....................... 610-614Ismana Surkovic, Ismet Suljevic, Azra Kudumovic

* * *Comparisson of the results from continuous glucosemonitoring and results from Capillary Glycaemiain children with type 1 diabetes ........................ 615-622Snjezana Hasanbegovic

* * *Glucose intolerance is the major risk factorsfor coronary artery disease ................................. 623-630Hajder M., Samardzic R., Kudumovic A.

* * *MRI of rectum – a new diagnostic modality indifferentiating malignant from benign lesions .... 631-637Amela Sofic, Nedzad Sehovic, Serif Beslic, Besim Prnjavorac, Damir Sofic

* * *Comparison of arterial blood pressure values indysfunction of thyroid gland before and afterthe therapy ............................................................ 638-642Ismana Surkovic, Ismet Suljevic, Azra Kudumovic

* * *Negative effects of laboratory chemicals onthe reproductive health ........................................... 643-651Alicelebic Selma

* * *Simple algorithmic approach to the treatmentof chylothorax ...................................................... 652-657Safet Guska, Ilijaz Pilav, Safet Musanovic

* * *Diagnostic characteristics of neuroradiologicaltests in lumbar disc herniation ........................... 658-663Mirza Moranjkic, Zlatko Ercegovic, Mirsad Hodzic,Harun Brkic, Farid Ljuca

* * *Basal Hyperprolactinemia, Occult Hyperprolactinaemia and Gonadotropins secretion in infertile women ......... 664-671Hajder E., Hajder M., Zukic E., Samardzic R.

* * *Biomarkers of plaque instability in acutecoronary syndrome patients ............................... 672-679Jasmina Nurkic, Farid Ljuca, Midhat Nurkic, Farid Barakovic, Denijel Tulumovic, Elmir Jahic, Amra Dzankovic

* * *Disorder at depth perception of the traumatichead injury ................................................................ 680-683Raif Serdarevic

* * *The effects of Antenatal corticosteroids onneonatal morbidity and mortality ....................... 684-688Maksic H, Heljic S, Dizdarevic J, Misanović V, Catibusic F, Dzinovic A.

* * *Effect of Weak Static Magnetic Fields onthe Number Red Blood Cells into FatteningTurkey Peripheral Blood Samples Under In Vitro and In Vivo Conditions ........................... 689-697Zijad Muharemovic, Muhamed Katica, Jasmin Musanovic, Nejra Hadzimusic, Sabina Catic, Kenan Caklovica, Dzelil Korkut

* * *Cholesterol and Lifestyle: The Stanford HealthImprovement Program (HIP) Experience ......... 698-700Yann A. Meunier

* * *

Instructions for the autors ..................................... 701-702

HealthMED - Volume 4 / Number 3 / 2010

Journal of Society for development in new net environment in B&H 485

Abstract

Objectives: The baseline of teaching professi-onalism is teaching cognitive bases of professi-onalism and internalization of its values by stu-dents. It is necessary to know the current status of professionalism and then educating about lapses of it and assessing the results. This study aims to determine the proper attitudes and the effect of six month faculty experience to these attitudes.

Study Design: An anonymous and voluntary questionnaire consisting of brief socio-demograp-hic variables and 25 sentences about some princi-ples of professionalisms was applied to the new entrants of Selcuk University Meram Medical Fa-culty at the first and terms of 2007-2008 academic years. Chi-square test was used in statistical anal-yses and significance accepted as p=0.05.

Results: The first questionnaire represented 76.9% (107/139), and second 179.8% (111/139) of new entrants. There were 54 male (50.5%), 53 female (49.5%) in the first group and 62 male (55.9%) and 49 female (44.1%) in the second gro-up. In answers of sentences there was a significant negative change about professionalism in seven items and a non-significant negative change in 11 items.

Conclusion: This study shows negative chan-ges in the attitudes of new entrants towards some principles of professionalism. It must be the first aim to integrate these lapsing points to the curri-culum and to evaluate the results.

Key words: Professionalism, medical educa-tion, freshmen, medical students, faculty climate

Introduction

Although the importance of teaching professio-nalism in medicine is known and considered essen-tial there are two main factors hindering attempts about integrating professionalism to the curricula: absence of a common definition of medical pro-fessionalism and a commonly accepted theore-tical model for integrating it into the curriculum [1,2]. While professionalism is defined as generic behavioral characteristics by some authors, some definitions enlarge it to patient-doctor relationship and to an ethical responsibility of the medical pro-fession to the community [3]. Some also notes that professionalism is not just a list of definitions but it is learning based on cognitive bases that mean knowing and realizing the nature, historical roots, and the reasons society uses the professionalism and the obligations necessary to sustain professio-nal status[4]. Some authors claims that professiona-lism is based on attitudes and professional attitu-des goes to professional behaviors [1,5]. In the root of professional attitudes humanistic principles like honesty, respect, compassion, empathy etc. takes place [1]. It is also suggested that professionalism is a whole of a group of ethic values and behaviors that constructing the social contract between doc-tor and the community [6]. But this social contract seems to be more challenging by the increasing complexity of the medical practice, by the entran-ce of government, law and the many other sectors to the health care field [7].

The most common acceptance about professi-onalism is the necessity of being professional in medical practice [2]. Secondly; teaching professi-onalism in early years of medical education re-

Attitudes of medical freshmen’s about some principles of professionalismKaraoglu Nazan1, Okka Berrin2, Şeker Muzaffer1

1 Department of Medical Education and Informatics, Meram Faculty of Medicine, Selcuk University, Konya, Turkey,2 Department of Medical History and Ethics, Meram Faculty of Medicine, Selcuk University, Konya, Turkey,3 Department of Medical Education and Informatics, Meram Faculty of Medicine, Selcuk University, Konya, Turkey

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sults in better performance in clinical years and better medical practice after graduating [5]. Besi-des efforts to teach professional behaviors, asses-sment of this process is also on debate [8]. The need for institutional initiatives to design and support a professional climate is noted [1,7,9,10]. In the re-port of General Medical Council (GMC) named ‘Tomorrow’s Doctor’ in 2003 it was suggested that the suitable attitudes and behaviors of a doc-tor should be gained during medical education and all the graduated doctors should know and have the main principles of professionalism [11]. The si-milarity of unprofessional behaviors observed in medical students and in physicians in practice also shows the importance of determining and correc-ting the lapses in professionalism during medical education [1]. Not to begin teaching before clinical years of education assumed to loose an opportu-nity about gaining generic values of professiona-lism [12]. To pass over an unprofessional behavior will open a road to more unprofessional behaviors [13,14,15].

Besides the ‘formal curriculum’ there is a powerful hidden curriculum consisting of unscrip-ted, unplanned and highly interpersonal forms of teaching and learning [4,16]. The hidden curriculum describes a set of influences that function at the level of organizational structure and culture and teaching professionalism on the bases of positi-ve attitudes without altering the negative social norms often expressed through the hidden curri-culum, professionalism education would be unad-dressed [1,4].

In the recent years competency in professio-nalism is suggested to be an imperative skill for graduating from medical schools [17]. Because of these reasons as some authors noted studies evalu-ating the ideas, behaviors and attitudes of medical students about professionalism can give informa-tion about the climate of medical education and is important in designing medical curriculum [10]. Although there is an increasing interest and in-creasing amount of studies about professionalism education in preclinical years, nothing much is known about “how to teach professionalism” and “the effect of hidden curriculum on professiona-lism” which is suggested to be tightly related to unprofessional behaviors [18,19].

Goals

The first aim of this study is to determine the attitudes and beliefs of new entrant medical stu-dents in the first semester when they got the theo-retical bases of professionalism through some lec-tures which also mean to evaluate our roots about professionalism education. The second aim is to determine if there is a change during the medical education by evaluating them in the second seme-ster when they are mostly on the effects of hidden curriculum which also means to evaluate and gain data for the climate of our institution.

Methods

The new entrants of Selcuk University Meram Medical Faculty in 2007-2008 academic years con-stituted the study population. Although there were 164 new entrant medical students in that academic year the 25 of the students were excluded. Twenty of them were the failed students of one year before and they were excluded with the idea of the more effect of hidden curriculum on them. Five of them were the foreign students and their native language was not Turkish so language problems with a Tur-kish questionnaire could result in misunderstanding.

In the curriculum of the first year in first three months “medical history and ethics”, “medical education and informatics” and “behavioral scien-ces” have some lectures and professionalism prin-ciples is mentioned as a part of these lectures not in the name of “professionalism” but in the name of “doctor and community”, “patient-doctor relati-onship”, “doctor-doctor relationship” etc.

We waited for first three months to provide them a basic theoretical knowledge and we also know that first three months is generally an adap-tation period for the new entrants. They have new friendships but not knowing the upper classes and the hospital climate. They are not a part of medi-cal environment yet and hidden curriculum besi-des role models has none or has the least effect on them. Approximately six months later, in the se-cond semester we applied the questionnaire again for the purpose of evaluating the change on their attitudes and beliefs about some principles of pro-fessionalism they had been educated about.



The first part of the questionnaire was consi-sting of two demographic variables (gender and age) and two questions about past and the future ideal (ideal of being a doctor and ideal after gradu-ation). Second part of the questionnaire was con-stituted as 25 descriptive sentences about some main principles of professionalism composed by the authors depending on the literature and the concepts of the lectures they got in the first three months because of the absence of a validated que-stionnaire [3,7,11,19]. These sentences were about pa-tient-physician relationship, physician- physician relationship, physician -health care personnel re-lationship and unsuitable behaviors for a medical student and a physician. The questionnaire was tested with twenty second and third year medical students and revised. The internal consistency of the questionnaire as measured by Cronbach’s Alp-ha was 0.873. A five point Likert scale was used to ensure that students could express themselves completely (completely disagree to completely agree). Informed consent was obtained orally sin-ce the questionnaire was self-administered and anonymous and participation was voluntary. The students were asked to complete the questionnaire in a class at the end of a lecture and returned them to the author in the same session.

Although five-point Likert scale was used at the time of statistical analyses the answers “comple-tely disagree and disagree” and “agree and com-

pletely agree” were combined at the same cate-gory. To avoid all the responses to be in a positive end we put nine negative sentences and reversed the scores of these sentences while calculation. The demographic variables and the answers of 25 sentences represented as number and percentages. For the comparison of independent groups chi-square analyses were used. All tests were two tai-led and p<0.05 accepted as statistically significant.

Results

In the first application 107 and in the second application 111 fulfilled questionnaires analyzed. When the foreign students (n=5) and the failed ones (n=20) were excluded the analyzed questi-onnaires were representing 76.9% (107/139) and 79.8% (111/139) of the new entrants respectively. The mean age of the first group was 19.19±1.29 years and 19.91±1.04 years in the second group. In respect to gender there were 54 male (50.5%), 53 female (49.5%) in the first and 62 male (55.9%), 49 female (44.1%) in the second group. According to first part of the questionnaire the groups were similar (p>0.05). Table 1 shows the answers of the students to the first part of the questionnaire in the first and second application.

When the answers were analyzed totally the-re was statistical significance in eight sentences.

Table 1. The comparison of the first part of the questionnaire in the first and second application.

Age (year)First application

n (%)*

19.19±1.29

Second applicationn (%)*

19.91±1.04p**

Gender♂♀

54(50.5)53(49.5)

62(55.9)49(44.1)

0.425

My ideal was being a doctor;YesNo

80(74.8)27(25.2)

70(63.1)41(36.9)

0.062

Ideal after graduation;Specialization in a clinical branchSpecialization in basic sciencesNot decided yet

85(79.4) 8(7.5)

14(13.1)

93(83.8) 5(4.5) 13(11.7)

0.602

TOTAL 107(100.0) 111(100.0)* The percentage of rows. ** Chi-square test

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Table 2. The comparison of the students’ answers to the second part of the questionnaire about some principles of professionalism in the first and second application

SENTENCESFIRST APPLICATION

Disagree Unsure Agree n(%) n(%) n(%)

SECOND APPLICATIONDisagree Unsure Agree

n(%) n(%) n(%)P

1- All living ones desires and should see respect from a doctor. 2(1.9) 5(4.7) 100(93.5) 3(2.7) 4(3.6) 104(93.7) 0.901

2-Talking about a colleague in front of others in a public place is not an unsuitable behavior. 98(91.6) 4(3.7) 5(4.7) 98(88.3) 10(9.0) 3(2.7) 0.824

3-The rules and routine procedures should be told to the patient during his/her stay in the hospital. 5(4.7) 3(2.8) 99(92.5) 6(5.4) 8(7.2) 97(87.4) 0.368

4-It is not possible for a doctor to make the medical information understandable for the patient. 91(85.0) 12(11.2) 4(3.7) 83(74.8) 15(13.5) 13(11.7) 0.0245-The medical records and personal information should be kept hidden even after the death of the patient by the doctor.

6(5.6) 14(13.1) 87(81.3) 6(5.4) 15(13.5) 90(81.1) 0.997

6- Sometimes it is necessary to give information to a patient about a medical issue we are not competent. 98(91.6) 4(3.7) 5(4.7) 85(76.6) 19(17.1) 7(6.3) 0.020

7-Patients have the right to learn from where and how they could get the best health care opportunities. 0(0.0) 3(2.8) 104(97.2) 6(5.4) 5(4.5) 100(90.1) 0.013

8- It is patient’s right to take an advice from another doctor about the disease he or she has. 1(0.9) 4(3.7) 102(95.3) 4(3.6) 25(22.5) 82(73.9) 0.000

9-Making jokes about patients and colleagues are need of a doctor. 105(98.1) 2(1.9) 0(0.0) 101(91.0) 7(6.3) 3(2.7) 0.016

10-A doctor should renew his/her vocational knowledge and share them with the colleagues during professional life.

1(0.9) 10(9.3) 96(89.7) 6(5.4) 2(1.8) 103(92.8) 0.803

11-It is an unsuitable behavior to eat or drink something in the corridors of hospital. 12(11.2) 16(15.0) 79(73.8) 24(21.6) 15(13.5) 72(64.9) 0.061

12- A doctor should sometimes apply a procedure to a patient although not competent. 94(87.9) 11(10.3) 2(1.9) 95(85.6) 8(7.2) 8(7.2) 0.253

13-Patient-doctor relationship is a social contract and doctor should be honest. 2(1.9) 4(3.7) 101(94.4) 3(2.7) 3(2.7) 105(94.6) 0.893

14-Making private talks in the corridors of hospital is not suitable. 7(6.5) 23(21.5) 77(72.0) 15(13.5) 22(19.8) 74(66.7) 0.175

15-A patient may ask questions to a doctor in every period of the health care. 6(5.6) 20(18.7) 81(75.7) 15(13.5) 16(14.4) 80(72.1) 0.192

16-Sometimes it is necessary to argue with health care personnel in front of patients and patients’ intimates. 95(88.8) 10(9.3) 2(1.9) 98(88.3) 11(9.9) 2(1.8) 0.936

17-A doctor must see and act equally to all human beings. 4(3.7) 6(5.6) 97(90.7) 1(0.9) 1(0.9) 109(98.2) 0.026

18-Discussing the practice of a colleague with patient and patient’ intimates is not suitable. 6(5.6) 2(1.9) 99(92.5) 2(1.8) 8(7.2) 101(91.0) 0.693

19-A doctor must comply with the current norms. 6(5.6) 21(19.6) 80(74.8) 9(8.1) 10(9.0) 92(82.9) 0.47920-The economic aspect of the procedures are not responsibility of a doctor to talk with the patient. 97(90.7) 7(6.5) 3(2.8) 93(83.8) 11(9.9) 7(6.3) 0.116

21-Not talking to the colleague before reporting him/her to administrative units is not a suitable behavior. 2(1.9) 6(5.6) 99(92.5) 10(9.0) 16(14.4) 85(76.6) 0.001

22-Alternatives of the proposed procedures and treatment are medical issues and should not be a matter of a patient to discuss.

101(94.4) 5(4.7) 1(0.9) 93(83.8) 13(11.7) 5(4.5) 0.012

23-It is an unsuitable behavior for a doctor to talk about patients in public areas. 2(1.9) 5(4.7) 100(93.5) 2(1.8) 8(7.2) 101(91.0) 0.617

24- Informed consent is not necessary for every procedure. 99(92.5) 5(4.7) 3(2.8) 100(90.1) 6(5.4) 5(4.5) 0.477

25-Although we are a student not correcting the misconception of a patient who supposed we are a doctor is not a suitable behavior.

11(10.3) 28(26.2) 68(63.6) 17(15.3) 31(27.9) 63(56.8) 0.222



While seven of them were pointing a significant negative change (4,6,7,8,9,21,22), one of them was showing a significant positive change (17). There were no change in four of the sentences (1,5,13,16). In 11 sentences the responses of students showed a negative change although not significant (2,3,11,12,14,15,18,20,23,24,25). In two sentences answers showed a positive change (10,19). In table 2 the comparisons of the answers to the second part of the questionnaire are shown.

Discussion

Although there is an increasing interest to the professionalism education in preclinical years, there are a limited number of studies about the effect of the preclinical climate on the students. Main reason suggested to be the idea that lapses in professionalism generally occurs in clinical years [10,19]. But as noted before professionalism educati-on should begin from preclinical years in cogniti-ve bases and during the medical education should be provoked to be internalized [4]. This study we are presenting here is the first experience from Turkey and one of the limited numbered studies determining the effect of preclinical climate and indirectly the hidden curriculum. These kind of studies are important because its known that suita-ble attitudes are getting worse during the medical education by the lose of idealism or by the effect of hidden curriculum and efforts needed to change this challenge [1,16].

In a previous study after a six month effect of hidden curriculum medical students began to think some unsuitable behaviors as acceptable which they thought as unprofessional before [10]. Similarly, we found that approximately six month later answers of students significantly getting wor-se about the behaviors in the questionnaire. When we evaluated every sentence only the answer of the sentence “a doctor must see and act equally to all human beings” changed positively, but seven answers significantly changed to a negative direct. Besides, although the change was not statistically significant answers to 11 sentences changed to a negative manner (Table 2). Why because the for-mal education about professionalism was the same and although much more students said that being a

doctor was not their ideal in the second group the-re were not a significant difference from group one we may suggest that this worsening is the indirect effect of the hidden curriculum of our faculty. We couldn’t claim it as a decline of idealism because the students in the groups were not all the same. It was seen that from the beginning of the medical school most of the students had some expectations and most of them wanted to be specialist in a clini-cal branch (Table 1). We think that these expectati-ons also show the importance of early steps about professionalism education and the importance of its probable reflection in the future.

In a previous study; authors pointed to gene-ral concept medical faculties gave theirs students from the beginning of medical education [12]. “You will be a doctor” concept reminds students that they are in a special, elite, separate position and they are privileged ones. Herein we see this con-cept in especially sentence 25 which was saying “although we are a student, not correcting the mis-conception of a patient who supposed we are a doctor is not a suitable behavior”. While 63.6% of the students in the first group said that they agree, in the second group 56.8% of the students agreed this sentence. Besides, the proportion of “disagree and unsure” answers were high in the first group and became higher in the second group. It may show that belonging to a special group concept began to internalize by them.

In sentence six; “Sometimes it is necessary to give information to a patient about a medical issue we are not competent” and in sentence eight; “its patients right to take an advice from another doc-tor about the disease he or she has”, the answers of students significantly changed to a negative ma-nner which may reflect the erosion in the professi-onalism principles. Those may also be the result of the conception which medical faculties give their students as white coat owners. Some authors describe this position as the white coat’s symbo-lic representation of a shield or boundary between medical students and everyone else [12].

GMC describes medical professionalism as; good clinical care, maintaining good medical practice, successful relationships with patients, working with colleagues, developing the skills, attitudes and practices of a competent teacher, probity and not allowing own health to put pati-

490



ents and others at risk [11]. Our questionnaire gene-rally depended on these principles and especially detailed in relationships with patients and physici-ans. Sentences one, 13 and 17 which were about respect and honesty to the patient were answered as “agree” with a high proportion reflecting that the bases of professionalism about respect and honesty was well thought and well understood (Table 2). But giving information to patient is an important part of honesty, respect, good commu-nication and good medical practice and negative change in the answers of students in sentences 3,4,7,8,15,20,22,24 which were about this impor-tant part of professionalism was interesting. Espe-cially sentence 24 “Informed consent is not neces-sary for every procedure” disappointed us because it is one of the popular and important issues of me-dical profession in the recent years and it was one of the most discussed lectures of first year curri-culum in the first three months in our curriculum. Quick erosion in these sentences may also be due to the misconception of belonging to an elite and unquestionable profession.

Working with colleagues is another impor-tant aspect of medical profession. The sentences two, 8, 9, 18, 21 were related to this theme and all of them changed negatively and three of them (8,9,21) showed a significant negative change. This change may show the necessity for interven-tions in our curriculum about working with the colleagues with respect beyond the jealousy. The sentence two; “Talking about a colleague in front of others in a public place is not an unsuitable be-havior”, sentence 18 “discussing the practice of a colleague with patient and patient intimates is not suitable” and sentence 21 “not talking to the colle-ague before reporting to administrative units is not suitable” also may point to an important matter of medical profession we must try to work on more in the future. Can we teach professionalism? All the medical faculties try to select the most successful ones of the exams for their hard and long educa-tion process but performance on the exam is not correlated with being professional [14]. It is also a misconception to think that if the scores of exams are okay everything is okay. Systematic education and assessment of professionalism is fundamen-tal of medical education [7]. In a study in Medical Faculty of Hacettepe University authors showed

the positive attitude change after a course program about professionalism and noted that professiona-lism can be thought in a formal curriculum [5].

It is known that attitudes affect the behaviors and provocation and maintaining proper attitu-des of medical students affect the quality of he-alth care [16]. In a case control study relationship between problematic behaviors during medical education and interdisciplinary crimes of medical practice after graduation was shown [17]. Similarly in another study authors took notice to the relation of interdisciplinary crimes in medical practice and unprofessional attitudes as dishonesty, no-liabili-ty, not developing skills etc. [20]. But an author no-ted the conflict of a physician between necessities of medical profession and necessities of own as a human being [21]. According to this author it is im-possible to find a medical student who passed five semesters in education and made no jokes about a friend or a teacher. Joking is a simple way of co-ping with stress and it prevents burn out or depre-ssion. While trying to be a professional, a doctor should neglect own self and family [21]. From this point of view maybe students in our study were reflecting the need to cope with their stress with their answer for the sentence nine (p=0.016).

Traditionally professionalism transferred with role-models [4]. The importance of role-models on professionalism education either negatively or positively especially in hidden curriculum is noted in the literature [18,22-24]. In general, students noted such qualities in their chosen role-models as taking time with patients and students, listening carefully and patiently, extending themselves in small ways to ensure the patient’s comfort and understanding, being reinforcing and encouraging toward patients and students, and seeming to love their work [23]. But some authors claims that succe-ss chance of education with role-models would be very limited in today’s world [4]. We know that we couldn’t be able to show the effect of role-models in the hidden curriculum by this study and with new entrants but all those made us to be aware of institutional climate, with the words of an another article “ecology of professionalism” [25].

In a previous study the student’s perceptions about professionalism in the preclinical and clini-cal period were evaluated and acceptance of some unprofessional behaviors in the second evaluation



was noted [10]. The negative change in student’s attitudes was attributed to easy participation of the student to an unprofessional behavior which is thought acceptable or because of participation to an unprofessional behavior student converts the thought from unacceptable to acceptable in anot-her study [19]. Similarly, we are reporting a degra-dation of student’s attitudes about professionalism although these students were in very early stage of their education pointing out the importance of ear-ly interventions we need for our faculty climate. These interventions need an institutional culture with students, residents, faculty, staff, and the in-stitution itself [25].

In conclusion; the study we reported herein showed a negative change in attitudes and beliefs of new entrants about some principles of professi-onalism in approximately six months period. Of course further studies needed to see the process of these students and more detailed assessment of the climate about professionalism. The curricu-lum, culture of the institute or the sources of the faculties could be different but the main principles of the professionalism are the same and it could be adapted to the all medical faculties [4,7,26,27].

The results of this study only reflects the stu-dents’ views of the institute the study was made in so it can not be generalized to the other institu-tions but it could be a guide for the other institu-tions. The voluntary participation may be call to bias but the number of students volunteered and their portion among new entrants were conside-rably high. The questionnaire constituted by the authors was designed in the constructions of the theoretical lectures and because of it some may claim that it couldn’t assess all the aspects of pro-fessionalism. It is the first questionnaire in Turkey about professionalism evaluation and its reliabili-ty was accepted well. Despite hearing the words “professionalism” many times, students founded to have no real idea what this word meant [25]. The necessity of behavioral approach instead of a holi-stic approach was suggested [14]. Because of these reasons we didn’t use the word “professionalism” in the questionnaire and we used some sentences about attitudes and beliefs.

Acknowledgement

We want to thank to the administrative units and new entrants of Selcuk University.

The baseline of teaching professionalism is teaching cognitive bases of professionalism and internalization of its values by students. It is nece-ssary to know the current status of professionalism and then educating about lapses of it and assessing the results. This study aims to determine the pro-per attitudes and the effect of six month faculty experience to these attitudes.

References

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2. Ainsworth MA, Szauter KM. Medical students’ professionalism: are we measuring the right be-haviors? A comparison of Professional lapses by students and physicians. Acad Med 2006; 81 (10 Suppl): S83-6.

3. MacKenzie CR. Professionalism and medicine. HSSJ 2007; 3: 222–7.

4. Cruess RL, Cruess SR. Teaching professionalism: general principles. Med Teach 2006; 28: 205 -8.

5. Elcin M, Odabasi O, Gokler B, Sayek I, Akova M, Kiper N. Developing and evaluating professionali-sm. Med Teach 2006; 28: 36-9.

6. O’Sullivan AJ, Toohey SM. Assessment of professi-onalism in undergraduate medical students. Med Teach 2008; 30: 280 -6.

7. Steinert Y, Cruess SR, Cruess RL, Snell L. Faculty development for teaching and evaluating professi-onalism: from program design to curriculum chan-ge. Med Educ 2005; 39: 127–36.

8. Arnold L. Assessing professional behavior: yester-day, today, and tomorrow. Acad Med 2002; 77:502-15.

9. Wasserstein AG, Brennan PJ, Rubenstein AH. Insti-tutional leadership and faculty response: fostering professionalism at the University of Pennsylvania School of Medicine. Acad Med 2007; 82: 1049–56.

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10. Humphrey HJ, Smith K, Reddy S, Scott D, Madara JL, Arora VM. Promoting an environment of pro-fessionalism: The University of Chicago “Road-map”. Acad Med 2007; 82: 1098–107.

11. General Medical Council. Tomorrow’s Doctors: The principles of professional practice. London: GMC 2003.

12. Gindsburg S, Kachan N, Lingard L. Before the white coat: perceptions of professional lapses in the pre-clerkship. Med Educ 2005; 39: 12-9.

13. Hickson GB, Pichert JW, Webb LE, Gabbe SG. A complementary approach to promoting professi-onalism: identifying, measuring, and addressing unprofessional behaviors. Acad Med 2007; 82: 1040-8.

14. Stern DT, Frohna AZ, Gruppen LD. The predic-tion of professional behaviour. Med Educ 2005; 39: 75-82.

15. Hodgson CS, Teherani A, Gough HG, Bradley P, Papadakis MA. The relationship between measu-res of unprofessional behavior during medical sc-hool and indices on the California Psychological Inventory. Acad Med 2007; 82(10 Suppl): 4-7.

16. Woloschuk W, Harasym PH, Temple W. Attitude change during medical school: a cohort study. Med Educ 2004; 38: 522–34.

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19. Reddy ST, Farnan JM, Yoon JD et al. Third-year medical students’ participation in and percepti-ons of unprofessional behaviors. Acad Med 2007; 82(10 Suppl): 35–9.

20. Teherani A, Hodgson CS, Banach M, Papadakis MA. Domains of unprofessional behavior during medical school associated with future disciplinary action by a State Medical Board. Acad Med 2005; 80(10 suppl): 17-20.

21. Reis DC. Who am I and why am I here? Profe-ssionalism research through the eyes of a medical student. Acad Med 2008; 83: S111-12.

22. Du Preez RR, Pickworth GE, Van Rooyen M. Tea-ching professionalism: a South African perspecti-ve. Med Teach 2007; 29: 284-91.

23. Shapiro J, Rucker L, Robitshek D. Teaching the art of doctoring: an innovative medical student elective. Med Teach 2006; 28: 30-5.

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25. Goldstein EA, Maestas RR, Fryer-Edwards K et al. Professionalism in medical education: an in-stitutional challenge. Acad Med 2006; 81: 871–6.

26. Arnold EL, Blank LL, Race KEH, Cipparrone N. Can professionalism be measured? The deve-lopment of a scale for use in the medical envi-ronment. Acad Med 1998; 73: 1119-21.

27. Quaintance JL, Arnold L, Thompson GS. Deve-lopment of an instrument to measure the climate of professionalism in a clinical teaching envi-ronment. Acad Med 2008; 83(10 Suppl): S5-S8.

28. Dudic M. Promoting the significance of knowled-ge through education in the new societ, TTEM 2009; 2: 104-108.

29. 29. Kudumovic M, Kudumovic Dz, Mesanovic N, Huremovic E. Modern Information Comunication Technologies and educational technologies appli-ed to education of medicine, HealthMED 2010;1: 158-162

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Corresponding author Nazan Karaoglu, Selcuk Universitesi Meram Tip Fakültesi, Akyokus, Meram, 42080, Konya, Turkey E-mails: [email protected] [email protected]



Abstract

Previous studies have shown that antigen p24 appears in the primary stages of the infection befo-re there is any evidence of antibodies. Therefore, the evaluation of antigen P24 can be an appropri-ate index for diagnosing the infection in its first stages.

Three hundred negative blood samples were te-sted with the third-generation kit for determining the antibody against HIV. Thirty positive samples were collected from the AIDS Research Center, and it was confirmed that the patients were HIV positive using Immunoblot and nucleic acid am-plification test (NAT) methods. All the samples were investigated with the kit designed for deter-mining antigen p24 in the ELISA method.

The average of optical density of the positive samples in the antigen test was 1.6, whereas the average optical density for the negative samples was 0.08. The difference between these two ave-rage values of optical density was statistically si-gnificant (p<0.005). By using human monoclonal antibodies, one unit per milliliter of WHO (World Health Organization) antigen, and two picograms per milliliter of recombinant antigen, the analytic sensitivity of the measurements was obtained. According to results obtained for the negative samples, the measurement specificity is 100%. In

pretreatment, the samples of serum positive and the samples of BBI (Boston Biomedica Inc. ) pa-nels for which the antigen, antibody, and polyme-rase chain reaction (PCR) tests were positive; the 1.5 M glycine buffer with a pH of 2 increased the diagnostic sensitivity from 70% to 93%.

This test has high sensitivity and specificity in diagnosing HIV and is simpler, faster, more accu-rate, and more economical than other diagnostic methods.

Key words: ELISA test; Antigen P24; Immu-nity measurement; HIV

Introduction

HIV is a virus with a single-string RNA geno-me that has about 10000 nucleotides. HIV is the cause of AIDS, and its initial origin was injecting contaminated blood and blood products (1). The method currently proposed for diagnosing indivi-duals with HIV is to determine the antibody that works against the virus. However, this test has false positive results. The Western Blot test (1) was used for examining the false positive cases (2, 3). The diagnosis of the infection in the ear-ly stages is very important for preventing secon-dary infections in blood transfusions. Therefore, a method is required for diagnosing HIV in blood

Comparing the detection of HIV antigen P24 by human and mouse monoclonal antibodiesMasoomeh Hashemi1, Seyed Mehdi Boutorabi2, Reza Hajihoseini3, Ali Mirjalili4, Maryam Razaghi5, Shirin Norolahi5, Mehrdad Jalalian6,7

1 Department of Biochemistry, Payamenour University of Tehran, Iran2 Research Lab., Pishtaz Teb Inc., Iran3 Payamenour University of Tehran, Iran4 Department of Biotechnology, Razi Vaccine and Serum Research Institute, Iran5 Department of Microbiology, Faculty of basic Sciences, Islamic Azad University of Zanjan, Iran6 Research Center of Iranian Blood Transfusion Organization, Khorasan-e Razavi Blood Center, Iran7 Department of Community Health, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor D.E., Malaysia

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samples, especially during the early stages of in-fection (4). Monoclonal and polyclonal antibodi-es are being used increasingly for detecting virus antigens, especially antigen P24 of the virus. Two of the objectives of the present research were to use the ELISA test for detecting this antigen with high sensitivity and specificity and to compare its effectiveness to other methods for diagnosing the infection in its early stages. Based on the results of our study of the ELISA test, the diagnostic pro-blems may have been solved.

Materials and methods

Sample collection

Three hundred samples of serum that were ne-gative for HIV were selected randomly from in-dividuals who had no symptoms. Thirty positive samples were collected from the AIDS Research Center, and it was confirmed that the patients were HIV positive by the use of Immunoblot and NAT methods.

Testing antibodies against HIV

By using the kit produced by Pishtaz Teb Za-man Co., all 300 collected samples were tested in compliance with the method specified in the brochure of the kit. The optical densities of these samples were less than the cutoff determined for the kit (i.e., an average optical density of negative control + 0.2). In this kit, gp120, gp41, and P24 were used for detecting antibodies.

Designing the diagnostic kit for antigen P24 in the ELISA Method

Plate Preparation: We acquired human mo-noclonal antibodies produced by Biomaric Co. (catalog Nos. 200.004 and 200.008) and mouse monoclonal antibodies (catalog Nos. 200.005 and 201.004) with concentrations of 1 and 2.5 mg/ml. Each of these products was dissolved in coating buffer. One hundred microliters of the solution were placed in a suitable vessel and incubated at a

temperature of 4 oC for one night. The wells were maintain at room temperature for one hour after washing the ELISA plate with a phosphate buffer containing Tween 20 and a blocker solution that contained 1% bovine serum albumin and carbo-hydrate. Then, the contents of the wells were disc-harged and kept at room temperature for six hours in order to dry them. The plates were kept in foil containing damp absorbent in the range of 2-8 oC until the tests were performed.

Conjugated Solution Preparation

Conjugated solution 1. Biotinated monoclonal antibodies produced by Biomaric Co. were used. Considering the initial and final volumes of the antibodies, a concentration of 0.1 microgram in per milliliter was calculated for the biotinated an-tibodies. Conjugated solution 2. Different concen-trations of the conjugated Strepto Avidine HRP (Hydrogen Proxidas) produced by Sigma Co. were used for identifying the antigen-antibody complex. This conjugated solution was diluted in a stabilizing solution produced by Pishtaz Teb, which is used especially for HRP preservation.

The Testing Procedure

One hundred microliters of the serum sample and 50 microliters of conjugated solution 1 were added to each well. The antigen P24 of HIV pro-duced by the Russian RPC Co. (catalog no.90/636) with a concentration of 100 picograms in per milli-liter was used for positive control. The wells were incubated at a temperature of 37 oC for one hour.

After the incubation, the wells were washed five times with a solution containing the phosp-hate buffer and Tween; then, the washing solution was completely discharged and 100 microliters of conjugated solution 2 were added to each well and incubated at a temperature of 37 oC for 30 minu-tes. After the incubation was completed, the wells were washed five times with washing buffer and 100 microliters. Then, substrate chromogen so-lution was added to each well after the washing solution (the substrate chromogen solution conta-ining tetramethylbenzidine and hydrogen peroxi-



de produced by Pishtaz Teb) had been completely discharged. After 15 minutes, the solution became blue in color, indicating that the reactions had ta-ken place. One hundred microliters of blocker (1N hydrochloric acid) were added to stop the reacti-on, and color changed to yellow. At a wavelength of 450 nm, the optical densities of the contents of the wells were read against reference filter 630.

Cutoff

The average and standard deviation of the opti-cal densities of the negative samples were calcula-ted using the +3SD formula in order to determine the cutoff and the mean cutoff for the kit.

Dissociation of the antigen-antibody complex

For preventing the probable interaction of the antibodies with antigen P24 in the samples, serum samples, the antibodies, antigen, and PCR tests of the positive samples were adjoined with various solutions including glycin buffer with a pH of 2, 0.5 N hydrochloric acid, Triton X100 with a con-centration of 0.1%, alkaline buffer no.1 contai-ning 2 M amine ethanol, 2.5% Triton X100, 0.15 M NaCl with a pH of 10, and alkaline buffer no.2 containing 4 M urea, 1% Supanin, 0.02% ethanol, and 0.15 M NaCl, and 0.01 M Na3PO4 with a pH of 10. One hundred microliters of the sample with 100 microliters of the abovementioned solutions were mixed and were kept in the incubator in a temperature of 37 oC for periods of 30, 60, 90, and 120 minutes. Concerning the last two solutions, 20 microliters of the solution were mixed with 100 microliters of the serum sample. The pH of the sample with the Tris buffer with a pH pf 10.6 was adjusted to approximately 7. Several negative samples were examined along with the positive samples in order to investigate the probable effect of these solutions on background optical density.

In addition, the antibody test was repeated, and the results were compared with a titer of the anti-bodies in the serum sample without pretreatment with the solution in order to be sure of the reduc-tion of the effect of the antibodies in the serum on samples adjoined with acidic solutions and Triton.

Diagnostic Sensitivity

Seroconversion panels no. PRB926 (Mixed ti-ter) and PRB108 (M) were chosen and were in-vestigated with the designed kit for studying the diagnostic sensitivity by using BBI international panels.

Analytic Sensitivity

In this stage, the analytic sensitivity was deter-mined by two methods:

1. The measurement of analytic sensitivity designed with human monoclonal antibodies and mouse monoclonal antibodies was investigated by diluting the series of P24 antigens recombinant.

2. The measurement of analytic sensitivity was investigated by using the dilutions of P24 antigen series proposed by the World Health Organization (WHO).

Imprecision Test

Three samples with different concentrations of antigen were used for investigating the impreci-sion of measurement in which ten working series (duplicate for any series) were studied during five different days.

Results

The average optical density of the samples that tested negative for antibodies was 0.07, and the standard deviation was 0.023.

From the average optical density of the negati-ve samples, the cut-off optical density for distin-guishing negative from positive samples by using the +3SD approach was determined to be 0.15. Samples that had optical density values greater than 0.15 were considered to be positive. The ran-ge of the optical densities of the positive samples was 0.35-2.91, and the average was 1.6.

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The effect of the pretreatment of serum samples with acidic, alkaline solutions and X-100 Triton

The titer of the antibodies available in serums adjoined with acid was reduced significantly com-pared to the serum sample without adjoining. But, in samples that were adjoined with X-100, no titer reduction was found. In samples that had negative antigen test results and in which the existence of antibodies was confirmed, the titer of the antibodi-es was reduced significantly after being adjoined with acidic solutions.

Considering the comparison of the optical density of the samples with positive antibody test and negative antigen test, pretreatment with glyci-ne buffer (pH = 2) at a temperature of 37 oC for one hour caused an increase in the optical density compared to the hydrochloric acid at 37 oC. Pre-treatment with Triton X100 and solutions 1 and 2 had no effect in enhancing optical density. No in-crease in optical density was found in the samples with negative antibody tests, and they were used as control samples. This indicates that the enhan-cement of optical density in samples with positive antibodies after being adjoined with acidic solu-tion for the antigen test is a specific phenomenon that results from the separation of antibodies from the antigen and the detection of antigen P24 by the measurement methods.

Considering the results obtained, the diagnostic sensitivity and specificity were increased by 93% and 100%, respectively.

The values of different markers of HIV in sam-ples of commercially-available seroconversion panels were studied. These panels contain a group of serial samples that has been sampled at various times, and they indicate the time required to detect antibodies in the samples.

The international panels specified in the fo-llowing tables were used for investigating diagno-stic sensitivity.

Analytic sensitivity of the measurement

In this stage, the analytic sensitivity was deter-mined by two methods:

1. The measurement of analytic sensitivity designed with human monoclonal antibodies and mouse monoclonal antibodies was investigated by diluting the series of P24 antigens recombinant.

2. The measurement of analytic sensitivity was investigated by using the dilutions of P24 antigen series proposed by the World Health Organization (WHO).

Considering the dilution of the serial of re-combinant antigen P24 in the sample of negative serum for evaluation of the analytic sensitivity of measurement and with regard to the cutoff defined for negative samples, the minimum detectable va-lue by this measurement technique was two pico-grams in per milliliter.

Specificity test

For assessing of specificity, we observed the cross reaction between antibodies and other viral structural antigens. In this method, we prepared the antigens gp41 and gp120 at concentrations of 200 pg/ml. We also examined 300 negative cases in the same way. Before treatment by glycin buffer, we found antigens in 21 of the 30 positive cases when we designed the kit using human monoclonal anti-bodies, and we found the antigen in 18 cases when using the mouse monoclonal antibodies. After trea-tment with glycin buffer, the sensitivities were 93% (28 positive cases) and 90% (27 positive cases), res-pectively. We performed the same test on random negative cases, and the specifity of the kit was 100% for both methods, and no false positive cases were observed. Our findings showed that the diagnostic sensitivity and specificity of the human monoclonal antibody tests before treatment by glycin were 70% and 100%, respectively. However, after treatment with 1.5 M glycin buffer, the sensitivity and specifi-city were calculated as 93% and 100%, respectively.

Imprecision Test

The results of the imprecision of the intra-assay measurement obtained from three samples with dif-ferent concentrations of antigen showed that the de-



signed kit had appropriate imprecision for various limits of antigen. The range of the imprecision of the measurements was calculated on the basis of the coefficient of variables (CV%) as 4.2 to 12.8.

Discussion and conclusions

The ELISA method is applicable for detecting antigen p24 of HIV through using the specific an-tibodies that trap antigen. The studies showed that antigen p24 of the virus are detectable within one day after the appearance of RNA (5, 6).

Mouse monoclonal or polyclonal antibodies have been used for the detection of antigen p24 (7, 8. In our research, the comparison of two mou-se and human antibodies showed that the analytic sensitivity of measurement, which is an important parameter in determining diagnostic sensitivity, is improved when human antibodies are used.

Separation of the complex of antigens and an-tibodies has been done using different methods in various research studies (9). In a study done by M. A. Rodrigues et al. in 2003, antigen p24 was detected in 554 serum samples (509 samples from individuals who were HIV positive and 45 control samples from people who were HIV negative). They used the acid treatment method for separa-ting the complex of antigen p24 and its antibody. In individuals with no symptoms, a significant in-crease in antigen P24 detection was found (48.2% versus 8.4%), and the same results were obtained for the patients (85.7% versus 37.1%). This indi-cates that acid treatment increases the sensitivity of the technique for detecting antigen p24 (10).

In this study, it was found that samples that were treated with glycine buffer before measu-rements were taken had an increase of diagnostic sensitivity in both measurements (70% before pre-treatment versus 93% after adjoining).

The increase in the percentage of measurement sensitivity after treating the samples with 1.5 M glycine buffer indicated the removal of the effect of antibody intervention of antigen P24 existing in the serum.

These antibodies prevent the connection of an-tigen to trapping antibodies in measurement thro-ugh the formation of a complex with antigen and covering antigenic epitope (11, 12).

Pretreatment with a special buffer has also been used in Perkin Elmer, Abbott, and Coulter kits. In a study done by Fedyuk et al. in1993, The ELISA Test was used for detecting antigen p24. They inve-stigated 97 serum samples from HIV positive pati-ents. They studied the effect of the formation of the complex of antigen p24 and its antibody in detecting antigen P24. Their results showed that this complex causes confusion in antigen detection. After acid treatment, 50% of the serum samples were positive, while, before treatment, only 5% was positive (13).

The sensitivity and specificity of the measure-ment techniques we studied were 93% and 100%, respectively, after pretreatment with 1.5 M glycine solution, which shows optimal sensitivity and spe-cificity that compare favorably with results obtai-ned from other studies. Analytic sensitivity of me-asurement in using human monoclonal antibody was one unit per milliliter with consideration to the standard proposed by the World Health Orga-nization; while using mouse monoclonal antibody, this value was four units per milliliter. This dif-ference indicates that the analytical sensitivity of human monoclonal antibodies is improved, which is due to the high affinity and avidity of the human antibodies for the antigen. The analytic sensitivi-ty of this measurement and the comparison of the results with the results of other detection kits of antigen P24 are illustrated in Table 4.

Considering the results obtained in this study using the seroconversion panels produced by BBI Co., employing P24 antigen detection is accompa-nied with significant reduction of serologic win-dow period (14).

The ELISA method for determining antigen p24 is a direct method for diagnosing HIV infecti-on. This method is economical and easily feasible in most laboratories, and no specialized personnel are required to conduct the tests. There is no cross-sectional contamination in PCR, and the preserva-tion of the sample is easier than RNA because of the greater durability of the antigen.

Acknowledgment

Pishtaz Teb Production & Research Co. provi-ded financial support for this research. We thank the honorable managing director of the Company,

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Mr. Behrouz Hajian Tehrani, and all of the per-sonnel who work in the Research & Development Department of the Company.

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1. Clavel J, Guetard D, Burn-Vezinet F, et al. Isolation of a new human retrovirus from west African pati-ents with AIDS. Science,1986;233:343-346.

2. Storch G.A. Essentials of diagnostic virology. 1th edition; USA, Churchill Churchill Livingstone, 2000.

3. Knipe D.M, Howley P.M, Fields virology, 4 th edi-tion, USA, Lippincott Williams&Wilkins, 2001; Vol 2.

4. Scarlatti G, Lombardi V, Plebani A, et al. Polyme-rase Chain Reaction, virus isolation and antigen assay in HIV-1 antibody positive mothers and their children. AIDS, 1991;5:1173-1178.

5. Sutthent R, Gaudart N, Chokpaibulkit K, et al. P24 antigen detection assay modified with a booster step for diagnosis and monitoring of human immu-nodeficiency virus type-1 infection. J Clin Microbi-ol, 2003;41:1016-1022.

6. Schupbach J, Tomasik Z, Nadal D, et al. Use of HIV-1 p24 as a sensitive, precise and in expensive mar-ker for infection, disease progression and treatment failure.Int J Antimicrob Agents,2000;16:441-445.

7. Henrard D.R, Philips J, Windor J, et al. Detection of human immunodeficiency virus type-1 p24 an-tigen and plasma RNA relevance to indeterminant serologic tests. Transfusion,1994;43:376-380.

8. Ly T.D, Edlinger C, Vabert A, et al. Contribution of ombineddetection assay of p24 antigen and anti-human immunodeficiency virus antibody in diagno-sis of primary HIV infection by routine testing. J Clin Microbiol, 2002 ;40 :1938-1946.

9. Goudsmit J, Lange D, Paul D, et al. Antigenemia and antibody titers to core and envelope antigen in AIDS, AIDS-related complex and subclinical hu-man immunodeficiency virus infection. J Infect Dis, 1987; 155 :558-560.

10. Rodriguez-Iglesias, Alvarez J.ZR, Vergara A, et al.Improved Detection of HIV p24 Antigen in se-rum after Acid pretreatment. 2003; 11:849-851.

11. Gallarda JL, Henrard DR, Liu D, Harrington S, Stramer SL. Early detection of antibody to human immunodeficiency virus type-1 by using an anti-gen conjugate immunoassay correlates with the presence of immunoglobulin M antibody.J Clin Microbiol.1992;30:2379-2384.

12. Schupbach J. Measurment of HIV-1 p24 antigen by signal-amplification boosted ELISA of heat de-natured plasma is a simple and inexpensive alter-native to test for viral RNA. AIDS Rev, 2002;4;83-92.

13. Fedyuk N, Polrovsky A, Garaev M, International Conference on AIDS.Detection of HIV-1 p24 anti-gen in sera of HIV-infected patients from Russian federation.1993 jun 6-11;9:260-263.

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Corresponding author Masoomeh Hashemi, Department of Biochemistry, Payamenour University of Tehran, Iran, E-mail: [email protected]



Abstract

Background: Empirical studies have used a piecemeal approach to the examination of health, health care-seeking, uninsured people and the he-alth status of those who are chronically ill, but no study emerged in an extensive literature search, on the developing nations, and in particular Latin America and the Caribbean, that has investigated health and health care-seeking behaviour among uninsured ill people in a single research.

Aims: The current study aims to narrow this divide by investigating health, self-reported dia-gnosed health conditions, and health care-seeking behaviour among uninsured ill Jamaicans, and to model factors which account for their moderate-to-very good health status as well as health care-seeking behaviour.

Methods and materials: The current study utilises cross-sectional survey data on Jamaicans which was collected in 2007. The survey is a mo-dification of the World Bank’s Living Standard Household Survey. This work extracted a sample of 736 respondents who indicated that they were ill and uninsured from a sample of 6,783 respon-dents. Logistic regression analyses examined 1) the relationship between moderate-to-very good health status and some socio-demographic, eco-nomic and biological variables; as well as 2) a correlation between medical care-seeking behavi-our and some socio-demographic, economic and biological variables.

Results: Sixty out of every 100 uninsured ill Jamaicans were females; 43 out of every 100 were poor; 59 out of every 100 uninsured ill persons dwelled in rural areas; 1 of every 2 utilised public health care facilities, two-thirds had chronic he-alth conditions, and 22 out of every 100 reported at least poor health. Moderate-to-very good health status was correlated with age (OR = 0.97, 95%

CI = 0.95-0.98); male (OR = 0.60, 95% CI = 0.37-0.97); middle class (OR = 0.45, 95% CI = 0.21-0.95); logged income (OR = 2.87, 95% CI = 1.50-5.49); area of residence (Other Town – OR = 2.33, 95^% CI = 1.19-4.54; Urban – OR = 2.01, 95% CI = 1.11-3.62), and health care-seeking behavi-our (OR = 0.45, 95% CI = 0.27-0.74). Sixty-one of every 100 uninsured respondents with ill health sought medical care. Medical care-seeking beha-viour was significantly related to chronic illness (OR = 2.25, 95%CI = 1.31-3.88); age (OR = 1.03, 95%CI = 1.01-1.04); crowding (OR = 1.12, 1.01-1.24); income (OR = 1.00, 95% CI = 1.00-1.00); and married people (OR = 0.48, 95% CI = 0.28-0.82). Uninsured ill Jamaicans who resided in ru-ral areas had the lowest moderate-to-very good health status, but there was no difference in health care-seeking behaviour based on the geographical location of residence.

Conclusion: Despite the fact that there is he-alth insurance coverage available for those who are chronically ill and elderly in Jamaica, there are still many such people who are without health insurance coverage. The task of public health spe-cialists and policy makers is to fashion public edu-cation and interventions that will address many of the realities which emerged in this research.

Keywords: Uninsured, uninsured ill, chronic illness, health status, health care-seeking behavi-our, health disparity, inequality in health, develo-ping nation.

Introduction

In all cultures, people desire good health and long life. Ill-health, therefore, is a challenge to the aim of healthy life expectancy, and is the rationale for investments in health options such as exerci-se, diet, nutrition, science and technology, medical

The uninsured ill in a developing nationPaul Andrew Bourne

Department of Community Health and Psychiatry, Faculty of Medical Sciences, The University of the West Indies, Mona, Kingston, Jamaica

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consultation and/or health care utilisation. All li-ving organisms will experience ill-health as well as good health over their life courses; and when ill-health threatens the quality and length of life, it becomes the justification for humans’ willingness to rectify, address and possibly postpone illnesses. Ill-health (i.e. illness, sickness or ailment) thre-atens existence, productivity, development, the individual and the wider society, and because of that humans demand the best health care options. Demand for health care must be paid for by (1) a combination of health insurance coverage and out-of-pocket payment, (2) the state, (3) out-of-pocket payments or (4) relatives, associates and/or family members. Ill-health can be a burden to the individual, family, community and the nation, and it is a probability against which people and the society seek to protect themselves. All illnesses require some typology of treatment, and while this does not necessarily have to be a traditional medi-cal practitioner, curing illness means that the indi-vidual must forego consuming something in order to restore his/her good health.

Some illnesses such as the common cold may not require a trained medical practitioner to cure, but often the individual will be required to spend money on over-the-counter medications, use a home remedy or utilise non-traditional healers in the quest to restore his/her former healthy state. There are other illnesses such as diabetes melli-tus, heart disease, kidney problems, hypertension, HIV/AIDS, sexually transmitted infections, and other chronic and non-communicable diseases, which require the attention of traditional medical experts to address their cure.

The traditional medical practitioners require payment in the form of cash and/or health insuran-ce coverage. Because individuals desire to restore their health, they are expected to provide payment for health care, which for particular health conditi-ons can be exorbitantly high. It is this reality which may result in premature mortality if the state does not provide health care coverage for those who are economically challenged and/or vulnerable. The World Health Organization (WHO) [1] opined that 80% of chronic illnesses were in low and middle income countries, suggesting that illness interfa-ces with poverty. The WHO continued that 60% of global mortality was caused by chronic illness,

and this should be understood within the context that four-fifths of chronic dysfunctions are in low-to-middle income countries [1]. It also postulated that “In reality, low and middle income countries are at the centre of both old and new public health challenges” [1]. Embedded in the realities outli-ned by the WHO are the incapacity of the poor, the association between poverty and illness, between poverty and premature mortality, poverty and hu-man suffering, and poverty and future retardation of economic growth, and the fact that health in-surance provides some cushion against this, for the individual and for society. Other studies have equally found that there is a significant statistical relationship between poverty and illness [2-4] and poverty and chronic illness, [5] which means that illness can make the vulnerable less likely to sur-vive and the wealthy become poor.

The high risk of mortality in developing coun-tries is owing to food insecurity, low water qua-lity and low sanitation coupled with inadequate access to material resources. Poverty makes it an insurmountable hurdle for poor people to effecti-vely address illness unless health care services are free. Hence, those in the lower socioeconomic cla-ss will be expected to have poorer health, as they are crippled by their material deprivation and low health options. The WHO captures this aptly “... People who are already poor are the most likely to suffer financially from chronic diseases, which often deepen poverty and damage long term eco-nomic prospects”. [1] Among the challenges for people living in poverty is access to health insu-rance coverage. Such a possibility means that the burden of health care is an out-of-pocket payment that cannot be provided by the poor, and this will eliminate life in the process. Cass et al. [6] found that infant mortality in Peru for those in the poo-rest quintile (i.e. poorest 20%) was almost 5 times more than for those in the wealthiest quintile (i.e. wealthiest 20%). This indicates the extent of the health challenge of the poor, and the role that the lack of health insurance and income play in the demise of individuals and even their children.

Another research paper revealed that life expec-tancy between the poorest 20% and the wealthiest 20% was 6.3 years, and this figure rose to 14.3 years for disability-free life expectancy, [7] sug-gesting that access and lack of access to resources



explain health and healthy life expectancy in and among the social classes in a society. Grossman [8] found a positive correlation between income and health status, indicating that money makes a difference in health, health care-seeking behavio-ur, physical milieu and health care coverage. Smi-th and Kington, [9] on the other hand, went further than Grossman when they postulated that money buys health. This viewpoint is somewhat decep-tive, as money provides access to good physical milieu, the best health care options, nutrition, die-tary choices and health information which are not readily available to the poor, but it does not buy health. Health is not a commodity for sale, and so it cannot be purchased, but money allows for access to better health choices and by extension can change health outcomes. Those issues could be the intent of Smith and Kington, when they say that money buys health, and they further exem-plify the challenges if an individual does not have access to it.

Material deprivation is such that the poor will be far from concerned with health insurance co-verage, proper diet and nutrition, health care choi-ces, but more with survivability. This denotes that they will be living on the margins of survivability and the decision to purchase health insurance will be the opportunity cost of food, clothing, shelter, minimal education and health options. Within the context of material and widespread health depri-vation for those in the lower socioeconomic strata, the state must play a role in aiding improvements in the healthy life expectancy of those therein. It is through this avenue that public health must act in order to fulfill the aim of the state in improving the quality of life of all residents in the nation.

Public health uses information from within and outside the society to improve the health and quality of people’s lives, and this requires conti-nuous research findings. According to the WHO, “In Jamaica 59% of people with chronic diseases experience financial difficulties because of the-ir illness...” Hence, poverty and illness, poverty and chronic illness, and poverty and low access to material resources are well established in rese-arch literature, but a dearth of information existed in Latin America and the Caribbean, and in parti-cular Jamaica, on the sick and uninsured. Can we assume that they are all poor people, and use this

to plan for them in a developing nation? An exten-sive review of the literature in developing nations, and in particular Latin America and the Caribbean, did not produce a single study that has examined health, and health care-seeking behaviour among uninsured ill people. The current study aims to narrow this divide by investigating health, self-re-ported diagnosed health conditions and health ca-re-seeking behaviour, at the same time examining who are the unhealthy and uninsured, and mode-lling factors which account for the moderate-to-very good health status of uninsured ill Jamaicans, in order to provide public health specialists with pertinent information that can be used to address some of the challenges within the society.

Methods and material

Data

The current study utilised the latest cross-sec-tional survey data in Jamaica to examine health, self-reported diagnosed health conditions and he-alth care-seeking behaviour, and to model factors which account for the moderate-to-very good he-alth status of unhealthy and uninsured Jamaicans. The Jamaica Survey of Living Conditions (JSLC) began collecting data from Jamaicans in 1988 and the latest dataset available is for 2007. The JSLC is a modification of the World Bank’s Living Standard Household Survey [10, 11]. This work extracted a sample of 736 respondents who indica-ted that they were ill and not insured, from a sam-ple of 6,783 respondents [12]. The cross-sectional survey was conducted between May and August 2002 in the 14 parishes across Jamaica, and inclu-ded 6,783 respondents of all ages. The JSLC used a stratified random probability sampling technique to draw the original sample of respondents, with a non-response rate of 26.2%. The sample was weighted to reflect the population.

The design was a two-stage stratified random sampling design where there was a Primary Sam-pling Unit (PSU) and a selection of dwellings from the primary units. The PSU is an Enumerati-on District (ED), which constitutes of a minimum of 100 dwellings in rural areas and 150 in urban areas. An ED is an independent geographical unit

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that shares a common boundary. This means that the country was grouped into strata of equal size based on dwellings (EDs). Pursuant to the PSUs, a listing of all the dwellings was made, and this became the sampling frame from which a Master Sample of dwellings was compiled, which in turn provided the sampling frame for the labour force. One third of the 2007 Labour Force Survey (i.e. LFS) was selected for the survey.

Study instrument

The JSLC used an administered questionnaire where respondents were asked to recall detailed information on particular activities. The questio-nnaire was modelled on the World Bank’s Living Standards Measurement Study (LSMS) household survey. The questionnaire covered demographic variables, health, education, daily expenses, non-food consumption expenditure, and other varia-bles. Interviewers were trained to collect the data from household members.

Statistical methods

Descriptive statistics were used to provide so-cio-demographic characteristics of the sample. Chi-square analyses were used to examine the association between non-metric variables. Anal-ysis of variance was used to test the statistical si-gnificance of a metric and non-dichotomous va-riable. Logistic regression analyses examined 1) the relationship between good health status and some socio-demographic, economic and biologi-cal variables; as well as 2) a correlation between medical care-seeking behaviour and some socio-demographic, economic and biological variables. The statistical package SPSS 16.0 was used for the analysis. A p-value less than 5% (2-tailed) was used to indicate statistical significance.

The correlation matrix was examined in order to ascertain if autocorrelation and/or multicolline-arity existed between variables. Based on Cohen and Holliday [13] correlation can be low (weak) - from 0 to 0.39; moderate – 0.4-0.69, and strong – 0.7-1.0. Any variable that had at least moderate (r > 0.6) was re-examined in order to address mul-

ticollinearity and/or autocorrelation between or among the independent variables [14-16]. Anot-her approach in addressing collinearity (r > 0.6) was to independently enter variables in the model to determine which one should be retained during the final model construction. The method for reta-ining or excluding a variable from the model was based on its contribution to the predictive power of the model and its goodness of fit [17]. Wald sta-tistics were used to determine the magnitude (or contribution) of each statistically significant vari-able in comparison with the others, and the Odds Ratio (OR) for the interpreting of each significant variable.

Measurement

Health status is a binary measure where 1= mo-derate-to-very good health; 0= otherwise which is determined from “Generally, how do you feel about your health”? Answers to this question were analyzed on a Likert scale ranging from excellent to poor. Medical care-seeking behaviour was ta-ken from the question ‘Has a health care practi-tioner, healer, or pharmacist been visited in the last 4 weeks?’ with there being two options: Yes or No. Medical care-seeking behaviour therefore was coded as a binary measure where 1=Yes and 0= otherwise. Crowding is the total number of in-dividuals in the household divided by the number of rooms (excluding kitchen, verandah and bat-hroom). Sex: This is a binary variable where 1= male and 0= otherwise. Age is a continuous varia-ble which is the number of years alive since birth (using last birthday). Age group is a non-binary measure: children (aged less than 15 years); yo-ung adults (ages 15 to 30 years); other-aged adults (ages 31 to 59 years); young elderly (ages 60 to 74 years); old elderly (ages 75 to 84 years) and oldest elderly (ages 85 years and older). Social hierarchy: This variable was measured based on income quintile: The upper classes were those in the wealthy quintiles (quintiles 4 and 5); middle class was quintile 3 and the poor were those in the lower quintiles (quintiles 1 and 2).

Chronic illnesses: These are ailments or dise-ases that are prolonged, not likely to be resolved spontaneously, and are infrequently cured.



Inequity denotes differences that are unneces-sary and avoidable, but are also thought to be un-fair and unjust, and these are adjudged based on the context of the customs operating in the society in general.

Equity in health means (1) equal access to care for equal needs, (2) equal access to utilisation for equal needs, and (3) equal quality of care for all in the society.

Inequalities in health mean patterns of socioeco-nomic disparities in health outcome which are sy-stematic, avoidable and important within a country.

Model

The multivariate model used in this study is in keeping with wanting to capture the multi-dimen-sional concept of health and the health care-see-king behaviour of uninsured ill people. Utilising logistic regression on secondary cross-sectional data, the present study modelled moderate-to-very good health status and the health care-seeking be-haviour of uninsured ill Jamaicans. Using a p-va-lue of less than 0.05 to indicate statistical signi-ficance, each model reflects only those variables that are statistically significant.

Health Model

Hit = f(Ait, Xi, SSit, lnYit, ARit, HSBit, εit) ..................................[1]

Health Care-seeking Behaviour Model

Hit = f(Ait, CIit, Hit, lnYit, CRit, MSit, εit) ......... [2]

Where Hti is current moderate-to-very good he-alth status of uninsured ill person i in time period t; Ai is age (in years) of person i in time period t; Xi is gender of person i; SSit is social class of person i in time period t; lnYit is logged income of person i in time period t; ARit is area of residence in time period time t; HSBit is health care-seeking behavi-our in time period t; CRi is crowding in the hou-sehold of person i in time period t; CIit is chronic illness of person i in time period t; MSit is marital status of person i in time period t; εit is residual error of person i - in time period t.

Results

Table 1 presents information on the demograp-hic characteristics of the sample. The sample was 736 respondents (i.e. 10.85% of the initial survey) who indicated that they were both sick and uninsu-red, and of which 40.5% were males. Concurrin-gly, of the sample 95.4% had at most primary level education and 0.8% had tertiary level education. Children constituted 28.7% of the sample; young adults, 10.2%; other adults, 31.3%; young-old, 16.4%; old-old, 10.5%; and oldest-old, 3.0%. The median age was 42.0 years (range = 0 – 99 years). The median total annual expenditure was USD 5,689.89 (range = USD 261.56 – 32,780.78; US$ 1.00 = J$ 80.47 - at the time of the survey). The number of visits made to medical practitioner(s) was 1.4 ± 1.0), while the amount of time spent in private care facilities was 3.0 ± 2.8 compared to 5.2 ± 5.0 for public care facilities). The mean cost of public medical care was USD 4.44 ± USD 16.14 compared to USD 13.64 ± USD 28.22 for private medical expenditure.

Of those who utilised public health care facili-ties, 22.9% of them purchased the prescribed me-dication compared to 78.8% who visited private health care facilities.

Table 2 highlights information on health care-seeking behaviour, health care utilisation, self-re-ported illness and area of residence by social hi-erarchy. Based on Table 2, there were significant statistical associations between (1) health care-seeking behaviour and social hierarchy; (2) public health care centre utilisation and social hierarchy, and (3) private health care centre utilisation and social hierarchy.

Table 3 highlights information on monthly food expenditure, per capita consumption, length of illness, number of visits made to health practi-tioners, medical expenditure and self-reported di-agnosed illness by area of residence. Based on Ta-ble 3, there were significant statistical associations between (1) monthly food expenditure and area of residence and (2) per capita consumption and area of residence – P < 0.05. However, there were no significant statistical relationships between the other variables and area of residence – P > 0.05.

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Table 1. Demographic characteristic of sample, n=736

Characteristic n %Sex Male 298 40.5 Female 438 59.5Marital status Married 161 30.8 Never married 276 52.8 Divorced 14 2.7 Separated 10 1.9 Widowed 62 11.9Social hierarchy Poorest 20% 170 23.1 Second poor 146 19.8 Middle 165 22.4 Second wealthy 142 19.3 Wealthiest 20% 111 15.4Area of residence Urban 176 23.9 Semi-urban 128 17.4 Rural 432 58.7Injury in last 4-weeks Yes 23 3.1 No 712 96.9Self-reported diagnosed illness Acute conditions Influenza 124 18.6 Diarrhoea 26 3.9 Asthma 73 10.9 Chronic conditions Diabetes mellitus 69 10.3 Hypertension 147 22.0 Arthritis 40 6.8 Other 189 28.3Health care-seeking behaviour Yes 446 61.4 No 280 38.6Health care utilization Public hospital (yes) 146 29.9 Private hospital (yes) 27 5.5 Public health care centres (yes) 96 19.6 Private health care centres (yes) 212 43.4 Other (yes) 8 1.6Purchased medication Yes 411 58.6

There was a statistical association between he-alth care-seeking behaviour and age group of res-

pondents – χ2 = 11.1, P = 0.048. As uninsured ill people become older, they are more likely to seek medical care: Children, 54.8%; old-adults, 54.8%; other-age adults, 64.0; young-old, 63.3%; old-old, 73.3%; and oldest old, 66.7%.

There was a statistical relationship between ha-ving chronic illness and being the household head – χ2 = 63.3, P < 0.0001. Almost 55% of those with chronic illnesses were household heads, compa-red to 22.4% who did not have chronic illness but were household heads.

A significant statistical association existed between sex and having chronic illness - χ2 = 4.7, P < 0.031. More females had chronic illness (69.8%) than males (61.7%).

There was a significant statistical association between health status and typology of illnesses (i.e. acute and chronic conditions) - χ2 = 62.3, P < 0.0001. Thirty-seven percent of those with chronic illnesses reported at least poor health status com-pared to 12.2% of those with acute conditions. On the other hand, 61.1% of those with acute conditi-ons reported at least good health status compared to 31.3% of those with chronic conditions.

A statistical difference was found between the mean income of those in the different social hi-erarchies – F statistic = 277.50, P < 0.0001. The mean income for those in the poorest 20% was USD 666.07 ± 175.40 followed by the second poor, USD 1,090.68 ± 132.14; middle class, USD 1,489.69 ± 169.07; second wealthy, USD 2,131.55 ± 254.49 and the wealthiest 20%, USD 4,201.39 ± 235.26.

Multivariate analysis

Table 5 shows variables which are correlated (or not) with the moderate-to-very good health sta-tus of uninsured ill respondents. Seven variables emerged as significantly associated with modera-te-to-very good health status – Model χ2 = 83.70, P < 0.001, -2 Log likelihood = 482.9 – and they accounted for 23% of the variability in health sta-tus. The model is a good fit for the data - Hosmer and Lemeshow goodness of fit χ2= 3.72, P = 0.88.



Table 2. Health care-seeking behaviour, health care utilization, self-reported illness and area of residen-ce by social hierarchy

Characteristic

Social hierarchy

PPoorest 20%

Second poor Middle Second

wealthyWealthiest

20%

n (%) n (%) n (%) n (%) n (%)

Health care-seeking behaviour 0.046 Yes 90(54.2) 86(59.7) 98(60.1) 91(65.0) 81(71.7) No 76(45.8) 58(40.3) 65(39.9) 49(35.0) 32(28.3)Health care utilization Public hospitals 0.337 Yes 32(37.2) 30(35.3) 35(35.7) 30(33.7) 19(23.5) No 54(62.8) 55(64.7) 63(64.3) 59(66.3) 62(76.5) Private hospitals 0.451 Yes 5(5.7) 5(5.9) 3(3.1) 6(6.7) 8(9.9) No 83(94.3) 80(94.1) 95(96.9) 83(93.3) 73(90.1) Public health care centres 0.016 Yes 29(33.3) 21(24.7) 21(21.4) 15(17.0) 10(12.3) No 59(67.0) 64(75.3) 77(78.6) 73(83.0) 71(87.7) Private health care centres 0.001 Yes 28(31.5) 35(41.2) 49(50.0) 52(58.4) 48(59.3) No 61(68.5) 50(58.8) 49(50.0) 37(41.6) 33(40.7) Self-reported diagnosed illness 0.200 Acute conditions Influenza 24(15.0) 25(19.1) 34(22.7) 26(20.3) 15(15.2) Diarrhoea 3(1.9) 9(6.9) 7(4.7) 3(2.3) 4(4.0) Asthma 21(13.1) 17(13.0) 17(11.3) 6(4.7) 12(12.1) Chronic conditions Diabetes mellitus 15(9.4) 15(11.5) 9(6.0) 15(11.7) 15(15.2) Hypertension 38(23.8) 23(17.6) 37(24.7) 27(21.1) 22(22.2) Arthritis 15(9.4) 8(6.1) 7(4.7) 6(4.7) 4(4.0) Other 44(27.5) 34(26.0) 39(26.0) 45(35.2) 27(27.3)Area of residence <0.0001 Urban 19(11.2) 21(14.4) 35(21.2) 42(29.6) 59(52.2) Semi-urban 16(9.4) 25(17.1) 30(18.2) 36(25.4) 21(18.6) Rural 135(79.4) 100(68.5) 100(60.6) 64(45.1) 33(29.2)

Length of illness (i.e. in days) mean± SD 10.6±11.6 12.9±22.7 11.1±15.9 31.5±116.3 14.9±21.8 0.006

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Table 3. Monthly food expenditure, per capita consumption, length of illness, number of visits made to health practitioner, medical expenditure and self-reported diagnosed illness by area of residence

CharacteristicArea of residence

PUrban Semi-urban Ruraln (%) n (%) n (%)

†Monthly food expendituremean ± standard deviation 280.71±192.00 277.45±162.97 237.07±145.59 0.002

Per capita consumptionmean ± standard deviation 2425.23±1992.18 1923.62±1241.60 1441.30±1179.85 < 0.0001

Length of illness in daymean ± standard deviation 9.5±19.1 13.5±23.0 17.7±65.4 0.256

Number of visits made to health care practitioner in last 4-weeksmean ± standard deviation

1.4±0.7 1.4±1.3 1.4±1.0 0.927

†Medical expenditure Public mean ± standard deviation 3.47±7.07 4.72±16.51 4.78±18.65 0.787

Private mean ± standard deviation 13.58±13.21 15.38±15.60 13.14±35.37 0.851

Self-reported diagnosed illness 0.162 Acute conditions Influenza 19(12.3) 34(28.8) 17(17.9) Diarrhoea 3(1.9) 4(3.4) 19(4.8) Asthma 21(13.6) 9(7.6) 43(10.9) Chronic conditions Diabetes mellitus 16(10.4) 13(11.0) 40(10.1) Hypertension 37(24.0) 24(20.3) 86(21.7) Arthritis 10(6.5) 6(5.1) 24(6.1) Other 48(31.2) 28(23.7) 113(28.5)

†Quoted in USD (USD 1.00 = Ja. $ 80.47 at the time of the survey)

Table 4. Self-reported diagnosed health conditions of uninsured ill respondents by age cohort

Characteristic

Age cohort

PChildren Young adults

Other-aged adults

Young old Old-old Oldest-old

n (%) n (%) n (%) n (%) n (%) n (%)Self-reported diagnosedillness < 0.0001

Acute conditions Influenza 83(45.6) 10(15.6) 19(9.1) 6(5.1) 6(8.1) 0(0.0) Diarrhoea 13(7.1) 2(3.1) 6(2.9) 2(1.7) 2(2.7) 1(4.5) Asthma 42(23.1) 11(17.2) 13(6.2) 4(3.4) 2(2.7) 1(4.5) Chronic conditions Diabetes mellitus 1(0.5) 2(3.1) 32(15.3) 21(17.9) 10(13.5) 3(13.6) Hypertension 0(0.0) 4(6.3) 55(26.3) 41(35.0) 36(48.6) 11(50.0) Arthritis 0(0.0) 0(0.0) 12(5.7) 18(15.4) 9(12.2) 1(4.5) Other 43(23.6) 35(54.7) 72(34.4) 25(21.4) 9(12.2) 5(22.7)



Table 5. Logistic regression: Variables of moderate-to-very good health status of uninsured ill respondentsVariable Coefficient Std. Error Wald statistic Odds ratio 95.0% C.I.

Age -0.033 0.008 18.605 0.967*** 0.95 - 0.98 Average Medical Expenditure 0.000 0.000 1.668 1.00 1.00 - 1.00 Male -0.511 0.244 4.374 0.60* 0.37 - 0.97 Middle Class -0.807 0.387 4.345 0.45* 0.21 - 0.95 Upper class -1.029 0.553 3.465 0.36 0.12 - 1.06†Lower class 1.00 Married 0.140 0.278 0.253 1.15 0.67 - 1.98 Divorced, separated or widowed -0.421 0.349 1.455 0.66 0.33 - 1.30†Never married 1.00 Logged Income 1.053 0.332 10.063 2.87** 1.50 - 5.49 Urban area 0.696 0.300 5.365 2.01* 1.11 - 3.62 Other town 0.844 0.342 6.092 2.33* 1.19 - 4.54†Rural area 1.00 Head household 0.218 0.250 0.761 1.24 0.76 - 2.03 Dummy health care-seekers -0.803 0.255 9.882 0.45** 0.27 - 0.74 Chronic illness -0.456 0.351 1.696 0.63* 0.32 - 0.86

Model χ2 (12) = 83.70, P < 0.001 -2 Log likelihood = 482.96, Nagelkerke R2 = 0.23Hosmer and Lemeshow goodness of fit χ2= 3.72, P = 0.88Overall correct classification = 75.1%Correct classification of cases of self-rated moderate-to-very good health status = 93.4%Correct classification of cases of not self-rated not moderate-to-very good health status = 26.5%†Reference group, *** P < 0.0001, **P < 01, *P < 0.05

Table 6. Logistic regression: Variables of self-reported health care-seekers of uninsured ill respondents

Variable Coefficient Std. Error Wald statistic Odds ratio 95% CIChronic illness 0.812 0.277 8.609 2.25** 1.31 - 3.88 Age 0.024 0.008 9.593 1.03** 1.01 - 1.04 Moderate-to-very good health -0.857 0.281 9.274 0.42** 0.24 - 0.74 Secondary education 1.117 0.762 2.148 3.06 0.69 - 13.60 Tertiary education 1.278 1.222 1.094 3.59 0.33 - 39.42†Primary and below education 1.00 Male -0.358 0.244 2.154 0.70 0.43 - 1.13 Crowding 0.114 0.053 4.694 1.12* 1.01 -1.24 Logged income 0.000 0.000 4.138 1.00* 1.00 - 1.00 Length of illness 0.000 0.002 0.013 1.00 1.00 - 1.00 Married -0.733 0.274 7.181 0.48** 0.28 - 0.82 Divorced, separated, or widowed -0.692 0.384 3.248 0.50 0.24 - 1.06†Never married 1.00 Urban area 0.171 0.286 0.359 1.19 0.68 - 2.08 Other town -0.336 0.302 1.238 0.72 0.41 - 1.29†Rural area 1.00

Model χ2 (13) = 47.85, P < 0.001 -2 Log likelihood = 486.1, Nagelkerke R2 = 0.15Hosmer and Lemeshow goodness of fit χ2= 8.11, P = 0.62Overall correct classification = 69.0%Correct classification of cases of self-reported health care-seekers = 89.4%Correct classification of cases of self-reported health care-nonseekers = 32.2%†Reference group, *** P < 0.0001, **P < 01, *P < 0.05

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Table 6 presents information on variables and self-reported health care seeking behaviour of uninsured respondents. Six variables emerged as significant statistical correlates of self-reported health care-seeking behaviour - Model χ2 = 47.9, P < 0.001, -2 Log likelihood = 486.1. The model is a good fit for the data - Hosmer and Lemeshow goodness of fit χ2= 8.11, P = 0.62.

Discussion

The current research used a sample of respon-dents who indicated both experiencing ill-health and having no health insurance coverage. Of the sample of respondents (i.e. n = 736), 60 out of every 100 were females, 43 out of every 100 were poor, 35 out of every 100 were in the upper social class, 59 out of every 100 dwelled in rural areas, 3 out of every 100 had been injured during the last 4 weeks, 61 out of every 100 sought medical care, 50 out of every 100 utilised public health care, two-thirds re-ported being diagnosed with a chronic illness, 31 out of every 100 were elderly, and 29 out of every 100 were children. Those in the lower socioecono-mic class were more likely to dwell in rural areas. Those in the poorest 20% were more likely to use public health centres, and the wealthiest 20% were more likely to utilise private health care centres. Fif-ty-four percent of those in the poorest 20% sought medical care in the last 4 weeks compared to 72% of those in the wealthiest 20%. Concurringly, of the sample, 78.4% indicated at least fair health status. Moderate-to-very good health status was explained by age, sex, social class, income, area of residence and health care-seeking behaviour. Rural residents had the least moderate-to-very good health status among uninsured ill Jamaicans. People who dwe-lled in Other Towns were 2.3 times more likely to indicate moderate-to-very good health compared to those in rural areas, and those in urban areas were 2.0 times more likely to claim moderate-to-very good health status. Those who indicated having a chronic illness were 37% less likely to report mo-derate-to-very good health. In addition, the present sample represents 70% of those who indicated ha-ving an illness in Jamaica for 2007.

Statistics from the Planning Institute of Jama-ica and the Statistical Institute of Jamaica [10]

showed that 15.5% of Jamaicans reported ill-he-alth in 2007. Within the context of the current fin-dings and that of PIOJ and STATIN, it computes that 71% of those who were experiencing illness were without health insurance coverage. Given that 50% of those who claimed to be experiencing ill-health utilised the public health care system and the fact that two-thirds of the illnesses were chro-nic conditions (3 females for every 2 males were uninsured and ill, and 6 out of every 10 uninsured ill people were of the dependent age cohort - less than 15 years or 60+ years), the public health care sector in Jamaica needs to recognize the impen-ding challenges of uninsured unhealthy people.

Van Agt et al. [5] found that the chronically ill were more likely to be poor, a statement with whi-ch this study concurs. In this paper, 43.2% of the chronically ill were poor (25.2% of poorest 20%) compared to 35.2% of the upper class (15.3% of the wealthiest 20%). This study went further than Van Agt et al.’s work, as the chronically ill were more likely to be elderly (42.5% of the chronically ill were 60+ years), to seek more medical care, were more likely to utilise public health facilities, more likely to live in rural areas (59.1%), more likely to be household heads (54.8%) and more likely to be females (63%). Clearly the poor are highly vulnerable to chronic illness [1, 5] and ma-terial deprivation [4], which accounts for more of them not having health insurance coverage whi-le suffering from ill-health. Hence, those who are uninsured and ill must interface with chronic heal-th conditions as well as income deprivation.

Income is well established in the health litera-ture as being associated with health [4, 8, 9], and this explains the fact that those in the lower socioe-conomic class have poorer health than those in the upper class [18, 19]. This paper found that uninsu-red ill people with more income are 2.9 times more likely to report moderate-to-very good health sta-tus, and they are also more likely to seek medical care. The challenge for those in the lower class is more than lower health status; it is also being de-prived of the health care that they need. Statistics revealed that poverty in Jamaica is substantially a rural phenomenon (prevalence of poverty in rural areas, 15.3%; semi-urban poverty, 4.0%; urban po-verty 6.2%) [10]. This study highlights that tho-se who are ill and uninsured are likely to dwell in



rural zones, explaining how financial deprivation accounts for lower ownership of health insurance coverage, the worst health being found among tho-se in rural areas compared to city dwellers. Using per capita consumption to measure income in this study, it was revealed that urban residents had 1.7 times more income than rural residents, and that semi-urban residents had 1.3 times more inco-me than rural dwellers, suggesting that the health disparities between the geographical dwellers is explained by this income inequity. It is therefore this access to more income that accommodates the greater health status of the urban and semi-urban respondents, compared to the rural dwellers, and it highlights a real need to correct income inequali-ty among the socioeconomic groups in the nation. A study by Stronks et al. [20] found an interrela-tionship between income, health and employment status, which further argues for greater health for urban and semi-urban dwellers, as rural residents are more likely to be seasonally employed, self-em-ployed or have low-income employment.

While income is related to better health status, which is also the case among uninsured ill people, concurring with the literature on a population [8, 9, 20], the great health disparity between the diffe-rent social classes is more related to income than place of residence. Such a finding provides clari-fication for a study done by Vila et al. [21] which stated that great health disparities in the city of Milwaukee were associated with area of residence by different social hierarchy. Income has a greater influence on better health than area of residence, and it even correlates with health care-seeking behaviour among the uninsured ill, unlike area of residence. Money matters in the health of uninsu-red Jamaicans as well as the general populace, as it offers a better explanation for peoples’ choices, accounting for the greater health of those who are able to choose, than their place of residence. Lack of access to money, therefore, in any geographical locality, explains health and material deprivation. Hence, it is not the fact of being in a rural area that accounts for poor health, but material and ot-her deprivations are greater in rural areas, a factor which provides an understanding for the massive health disparity between them and city residents.

Poverty is associated with premature mortality, and the current research provides some explanation

for this established fact. This paper is on uninsu-red ill Jamaicans, and the findings highlighted that 54% of those in the poorest 20% visited a health care practitioner, 58% of the poor compared to 65% of the second wealthy and 72% of the wealthiest 20%. While the affluent class has access to mate-rial and other resources to address health concerns, the poor are not as privileged as the upper class. This research found that 70.1% of those in the poo-rest 20% had at least one chronic health condition, the second poor, 61.2%; the second wealthy, 72.7% and the wealthiest 20%, 68.7%, which means that non-utilisation of medical care is likely to lead to complications and possible premature mortality. The WHO had stated that 60% of global mortali-ty is caused by chronic illness, but clearly pover-ty, non-treatment of chronic illnesses and cultural practices are all a part of the rationale for mortality, and not merely the condition.

Although those who suffer from chronic condi-tions in Jamaica are able to access public health in-surance which can reduce out-of-pocket payments for treatment and medication, clearly the culture prevents some people from accessing this facili-ty. This work showed that a large percentage of uninsured ill people dwelled in rural areas, where poverty was 2.5 times more than urban poverty and 3.8% more than semi-urban poverty, arguing for the role of the culture in preventing them from accessing assistance from the state. With this pre-ponderance of unwillingness on the part of poor and rural residents to access health insurance, accompanied by their low demand for health ser-vices compared to the wealthy, the inference is that many of them will seek health services based only on severity of illness. Chronic illnesses are such that non-medical practitioners should not interpret when conditions are serious and warrant health care assistance. It is this culture underpinning that accounts for the premature mortality and not the poverty or illness, as those with chronic health conditions in Jamaica are able to access public he-alth care despite their reluctance to access public health insurance coverage. With not having health insurance coverage, poverty and illness are likely to become a burden to individuals and family, and when those social agents are unable to assist with the costing of medical treatment, it will then beco-me the responsibility of the state.

510



This paper did not examine nutrition and heal-th, but a study by Khetarpal and Kochar [22] fo-und a statistical relationship between nutrition and health in rural women, which offers some expla-nation for the great health disparities in geograp-hical areas of residence. Another study by Foster [23] on low-income rural areas concurs with Khe-tarpal and Kochar [22] that nutrition accounts for health or ill-health, as the body requires particular nutrients. It can be extrapolated from the afore-mentioned studies, to that of the current one, that great disparities in health status among the diffe-rent geographical areas in Jamaica can be expla-ined by the nutritional intake (or lack of intake) based on where people dwell in this nation. There is a question which must be addressed in order to provide some explanation for the seemingly low nutritional intake of rural uninsured residents: Are rural residents less likely to intake the required nu-trients compared to residents in other geographi-cal areas in Jamaica? The answer is clearly yes as more of the uninsured ill Jamaicans are poor, and this means that they will be less concerned about the required nutrient intake than food consump-tion and mere survivability. Poverty is therefore more a factor in insurance, illness, lower health status and health care-seeking behaviour than the geographical area of residence, but what about the general health status of the uninsured ill, and is it lower than that of the population of Jamaica?

Almost 78 out of every 100 uninsured ill Ja-maicans claimed to have at least good health sta-tus. A study by Bourne [24] found that 82 in every 100 Jamaicans reported at least good health sta-tus, which is greater than that for the uninsured ill people. Furthermore, 3.3 times more Jamaicans indicated very good health compared to the unin-sured ill Jamaicans. The health disparities were not only between the good and very good health status of Jamaicans and uninsured ill Jamaicans, but were also evident for poor health status. Com-paratively, 4.4 times more uninsured ill Jamaicans claimed at least poor health as compared to the general population (i.e. 4.9%), and 3 times more uninsured chronically ill Jamaicans reported at least poor health status compared to those with acute health conditions. The current study concurs with (1) Reed and Tu’s work [25] that uninsured chronically ill people in America reported lower

health status (or worse health) and (2) Bourne and McGrowder [26] which stated that 25.3% of chro-nically ill Jamaicans reported at least poor health. Reed and Tu went on to state that the majority of uninsured people with chronic illnesses delay he-alth care utilisation owing to cost, which explains an aspect of this study, that although 43.2% of the uninsured ill people were living in poverty (i.e. poorest 20% and second poor income quintile), 39% did not seek medical care.

Faced with poverty, no health insurance cove-rage and chronic illness, uninsured ill Jamaicans are highly likely to face all kinds of life challenges such as material deprivation, dietary and nutriti-onal deficiencies, high risk of health complicati-ons, high out-of-pocket medical bills, disruptions in family life, future vulnerabilities and premature mortality. When this burden becomes untenable for the individual, family and wider community, it will then become the responsibility of the state [27]. This justifies the need to expand public he-alth insurance to protect the poor, the chronically ill and the vulnerable in a society [28], as chronic illness can erode the economic livelihood of an individual and therefore delay needed health care [29]. One study stated that uninsured househol-ds are one illness away from financial catastrophe [30], indicating that if a household was already in poverty this will become the burden of the state or may lead to premature mortality, as the individual will be unable to access needed health care owing to his/her inability to afford medical care. This implies that poverty encapsulates powerlessness, physical weakness, illness, chronic illness, prema-ture mortality, lack of productive assets, emotional distress, constricted freedom and future impove-rishment due to the aforementioned conditions, if they are not addressed by policy makers.

While impoverishment in urban areas is highly visible in the form of squalor, dilapidated edifices, zinc fencing, improper sanitation, squatting and vi-olence, rural poverty is less easily identifiable and may be overlooked by the naked eye. Clearly, using health disparities between area of residence and the socioeconomic strata, rural poverty in Jamaica is showing signs of depleting the human capital more than urban poverty. According to Harpham and Reichenheim [31], on the disaggregating of rural and urban health indicators, the latter ‘appe-



ar’ to have better health status. This study dispels the notion of ‘appearance’ and goes to the reality of the health differential using self-reported health among urban, semi-urban and rural uninsured ill Jamaicans. The discipline of public health cannot only use external findings to carry out its mandate, or divorce itself from the realities which emerge from the current study; poverty is destroying the human capabilities and resilience of the Jamaican people and more so in the case of rural uninsured ill people. Because poverty is strongly associa-ted with illness, and illness can result in poverty [32-34], those who are presently uninsured, ill and poor are highly vulnerable to ill-health and pre-mature mortality, which argues for an immediate health campaign to address the challenges among the socioeconomic strata and area of residence, as these were not alleviated with the introduction of the National Health Fund – NHF [35].

The NHF is a statutory company which was established by the NHF Act (2003) with a Chair-man and Board of Management appointed by the Minister of Health. It was established in 2003 to provide direct assistance to patients with chronic conditions, to purchase drugs and fund support to private and public companies for approved pro-jects [35]. The NHF is a social health insurance which is geared towards alleviating out-of-poc-ket payments for medication for those who suffer from chronic illnesses. Fourteen chronic illnesses are covered by the NHF, with respect to pharma-ceutical benefits in direct assistance to ill indi-viduals. The chronic health conditions that are covered by the NHF are hypertension, diabetes mellitus, breast cancer, prostate cancer, glaucoma, arthritis, asthma, high cholesterol, rheumatic heart disease, major depression, epilepsy, psychosis, is-chemia and vascular diseases. The NHF became operational in August 2003, and has undoubted-ly aided many chronically ill, non-poor and poor Jamaicans. With all the investment, the NHF has not failed to have a major coverage of chronically ill respondents using the Fund. The individuals are mostly rural residents, poor, under 60 years of age, and female. Such a reality speaks to the ad-ministrative and operational failure of the NHF to improve the lives of its intended population owing to the centralization of its operations in Kingston, which is an urban area in Jamaica. The verdict is

in, that merely instituting an agency to carry out a particular task (which is to distribute benefits evenly across the socioeconomic strata, area of re-sidence and sex) will not provide solutions to the inequalities and inequities in health between the particular groups in Jamaica. This study concurs with one in Finland [36] showing that the poor are more vulnerable to illnesses, and research conduc-ted in the United Kingdom [37] found that those in the lower socioeconomic stratum were more likely to die prematurely than those in the upper income groups. Embedded in those findings is the fact that any equitable distribution of NHF bene-fits to those in the different socioeconomic strata will show further unfairness and injustices in the health outcomes which already exist, owing to in-come inequalities.

Conclusion

Two-thirds of uninsured ill Jamaicans are chro-nically ill. The uninsured ill are mostly within the dependent age cohort (children and elderly), they are female and are rural respondents who are ge-nerally poor people. With one half of the uninsu-red ill respondents utilising the public health care system, and only 2 in every 10 of them purchasing medications, there are serious future challenges for public health in Jamaica. There is an inverse rela-tionship between the health status of uninsured ill Jamaicans and those in socioeconomic strata. The findings of this study highlight the likely challen-ge of the state in assisting uninsured ill Jamaicans. Despite the fact that health insurance coverage is freely accessible to those who are chronically ill in Jamaica, there are still many such people who are without health insurance coverage, and some are not even seeking medical care. Another rea-lity which emerged from this paper is that altho-ugh health care utilisation is free in Jamaica for children 18 years and younger, 45 out of every 100 of those uninsured and ill did not seek me-dical care, emphasizing people’s interpretation of illnesses that require medical attention, and how this retards health care demand. The task of public health specialists and policy makers, therefore, is to fashion public education and intervention pro-grammes that will address many of the realities

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which emerged in this research. The great health disparity between the lower socioeconomic strata and those in the upper strata, as well as those who reside in rural areas, cannot be left to resolve itself, as clearly it has not happened in the past and the situation cannot be allowed to continue indefini-tely in the future.

The Way Forward

The variations in health status and health care-seeking behaviour within and between the socio-economic strata who are uninsured ill people, cle-arly present information that reveals public health concerns, and highlights many challenges which are still unresolved in Jamaica. The current study did not examine the emotional distress and morta-lity patterns of uninsured ill respondents, and this should be the subject of some future study, as it would provide needed information about these in-dividuals. Despite the investments in health, the health sector and poverty alleviation programmes in Latin America and the Caribbean, there is still a need to study the heterogeneity in health outcome between the socioeconomic strata and area of resi-dence, as health disparity between and within co-untries is still great and not in keeping with health inequality eradication in the region. Another unre-solved issue stemming from the present research is how much of the cognitive dimension explains the health differential between the socioeconomic strata and the area of residence. In order to under-stand how to address policy intervention and he-alth education programmes for people in Jamaica, studies need to examine the breadth and scope of cognitive dimensions in explaining health inequ-alities. This will allow public health technocrats to understand why 70.3% of those who were ill in Jamaica in 2007 did not have health insuran-ce, and some of the chronically ill people, despite having access to public health insurance, did not possess such insurance, and did not seek medical care. A critical issue which needs to be addressed in the future is the structure of the National Heal-th Fund (the NHF is accessible to, and provides public health insurance coverage for, those expe-riencing chronic illnesses). Barrett and Lalta [32] wrote that “The National Health Fund dealt with

these issues by treating the non-poor and the poor as part of the same target beneficiary. Survey data and health officials indicate that the poor suffer as much from chronic diseases as the rich, but are less likely to seek treatment, or are only able to pay for part of their prescription drugs by reducing out-of-pocket payment …” This study is 4 years after the operational establishment of the NHF, and new findings are coming in, which show that the NHF cannot treat different socioeconomic strata in the same way, neither can it deal equitably with those who reside in different geographical areas. The health disparities will not be addressed by merely offering equal benefits to all within the context of the current findings, as these will only perpetuate health inequalities and inequities. The NHF the-refore needs to be restructured in order to provide definitions based on socioeconomic class and area of residence, so as to effectively alleviate some of the challenges which emerged from this research.

Disclaimer

The researcher would like to note that while this study used secondary data from the Jamaica Survey of Living Conditions, 2007, none of the errors that are within this paper should be ascribed to the Planning Institute of Jamaica or the Statisti-cal Institute of Jamaica as they are not theirs, but are instead owing to the researcher.



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Corresponding author Paul Andrew Bourne, Dept. of Community Health Statistics, Faculty of Medical Sciences, University of the West Indies, Jamaica, E-mail: [email protected]



Abstract

Background: Artemisinin (ART) is a potent antimalarial drug, practically has a low aqueous solubility thereby exhibits poor and low bioava-ilability after oral administration. Therefore, the improvement of artemisinin solubility and disso-lution from its oral solid dosage forms is an im-portant issue for enhancing its bioavailability and therapeutic efficiency.

Objectives: The present study was carried out to investigate the utilization of Gelucire 44/14 for preparing solid dispersion of artemisinin with aim of increasing its aqueous solubility.

Methods: Solid dispersion, with Gelucire 44/14 at ratios ranging from 1:10 to 8:10 was pre-pared using the melt (fusion) method. Scanning electron microscopy (SEM), solubility determi-nation and diferential scanning calorimetry (DSC) were used to investigate the physicochemical cha-racteristics of the preparations.

Results: The solubility of artemisinin from the solid dispersion preparations increased linearly with increased the ratio of Gelucire 44/14; howe-ver the increase in artemisinin solubility was not very significant. The data obtained from SEM and DSC studies revealed that the solid dispersions with Gelucire 44/14 were not capable of increa-sing the solubility of ART significantly through increasing the ratio of Gelucire 44/14.

Conclusion These preparations did not result in increasing the aqueous solubility of artemisinin significantly, so it can be concluded that the solid dispersion led to reduce the drug crystal as compa-red to pure artemisinin.

Keywords: Artemisinin, Solid dispersions, Gelucire 44/14, Solubility, DSC

Introduction

Artemisinin (ART) is a potent antimalarial drug isolated from the traditional Chinese medi-cinal herb Artemisia annua (Klayman, 1985; Luo & Shen, 1987). It is one of the few antimalarial that remain effective against multidrug-resistant strains of Plasmodium falciparum (WHO, 1981).

However, artemisinin has low aqueous so-lubility, resulting in poor and erratic absorption upon oral administration. This together with its short half-life and high first past-metabolism mi-ght lead to incomplete clearance of the parasites resulting in recrudescence (Titulaer et al., 1991). Many approaches have been used to improve the solubility of the poorly soluble drugs; Wong and Yuen (2001) have demonstrated that artemisinin solubility could be increased through inclusion complexation with β-cyclodextrin

Among the other approaches which have been utilized to enhance the drug solubility is solid dis-persion. The use of solid dispersion prepared from self-emulsifying material such as Gelucire 44/14 have shown a good potential to enhance the disso-lution and absorption of many poor water-soluble drugs (Sethia and Squillante, 2002;Yűksel et al., 2003; Tashtoush et al., 2004; Choy et al., 2005; Karatas et al., 2005). Barker et al. (2003) succe-ssfully prepared a solid dispersion of α-tocopherol with Gelucire 44/14, in which they proved that the application of solid dispersion technology as a means of incorporating pharmaceuticals and nu-traceuticals into a solid dosage form and also im-proving the bioavailability of the drugs.

Preparation and physicochemical characterization of artemisinin-gelucire 44/14 solid dispersions Gamal Osman Elhassan, Kah Hay Yuen, Jia Woei Wong, Jiyauddin Khan, Samer Al-Dhalli

School of Pharmaceutical Sciences,Universiti Sains Malaysia, Malaysia.

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Materials and Method

Materials

Artemisinin was obtained from Kuming Phar-maceutical Corporation (Kuming, China) and Ge-lucire 44/14 was obtained from Gattefossé (Fran-ce).Disodium hydrogen phosphate and methanol was obtained from BDH (Dorest, England) and Merck (Darmstadt, Germany) respectively. Ace-tonitrile (ACN) and glacial acetic acid were pur-chased from J.T. Baker ( Phillpsburg, NJ, USA). All chemicals or solvents used were either of anal-ytical or HPLC grades.

Preparation of Artemisinin-Gelucire 44/14 Solid Dispersions

Solid dispersions with Gelucire 44/14 at ratios ranging from 1:10 to 8:10 Table(1) were prepared by heating Gelucire 44/14 at 64 - 68°C for an hour with stirring to ensure that all the crystallization clusters were fully melted. Artemisinin was then added gradually. The resulting molten mixture was stirring for another 25 minutes. The heater was then turned off and stirrer was continued. The molten mixture was filled into hard gelatin capsu-les by Pasteur pipette when the temperature dro-pped at 65°C, 45°C and room temperature.

All the capsules were subsequently, stored in airtight amher bottle containing silica gel at ambi-ent temperature for 24 hours prior to characteriza-tion by solubility determination, differential sca-nning calorimetry (DSC) and scanning electron microscopy studies.

Determination of Artemisinin Solubility in Artemisinin-Gelucire 44/14 Solid Dispersions

The solubility of artemisinin in ART-Gelucire 44/14 solid dispersions was determined according to the method of Higuchi and Connors (1965). An excess amount of pure artemisinin and ART-Gelu-cire 44/14 solid dispersions were separately added into flask containing 20 ml of water. All the sam-ples were shaken vigorously at 30°C for 24 hours.

Samples (5 ml) were collected after 24 hours and filtered through a 0.2 µm nylon membrane with propylene housing (Puradisc, Whatman, USA). The filtrate were then suitably diluted and trea-ted prior to analysis by high-performance liquid chromatography (HPLC). The HPLC method was based on the method reported by Zhao & Zeng (1985) using UV detector operated at a wavelen-gth 260 nm. The chromatographic separation was performed using a Genesis C18 Column (150 x 4.6 nm) (Genesis, UK). The mobile phase composed of a mixture of acetonitrile and 0.01M disodium hydrogen phosphate adjusted to pH 6.5.

Scanning Electron Microscopy (SEM)

The images of artemisinin and ART-Gelucire 44/14 solid dispersions at different ratios were obtained by SEM (leica Cambridge 5-360, Leica, UK). The pictures were taken at magnification of 400X.

Differential Scanning Calorimetry (DSC)

The thermal studies were conducted using di-fferential scanning calorimetry, TA instrument DSC 210 (DE, USA). Each sample of approxi-mately 10mg was accurately weighed in hermetic aluminum pan. The pans were heated at a rate of 5°C/minute and scanned between 5°C to 200°C. The data was analyzed using the Universal Anal-ysis Software (TA Instrument, USA). All the sam-ples were run in triplicate.

Results and Discussion

The undissolved drug in the molten mixture of Gelucire 44/14 made the mixture opaque, indica-ting that the solid dispersion had been produced. All mixtures had sufficiently low viscosity in the molten state to allow them be filled into hard gela-tin capsules. The capsules showed no leakage after solidification of the mixture.

Figure (1) showed the solubility of pure arte-misinin and artemisinin in different ratios of ART-Gelucire 44/14 solid dispersions. From this figure



it has been observed that, the aqueous solubility of artemisinin in Gelucire 44/14 solid dispersions increased with an increase in the amount of Gelu-cire 44/14 in the solid dispersion. The solubility of artemisinin in Gelucire 44/14 at a ratio of 1:10 was approximately 3 times higher than the solubility of pure artemisinin. Overall this can be ascribed to solublizing properties of Gelucire 44/14, as non io-nic surfactant with a hydrophilic-lipophillic balan-ce (HLB) value of 14. Gelucire 44/14 reduces the interfacial tension between artemisinin particles and water, decreases the contact angles and hence promotes the wetting of artemisinin particles and decreases their aggregation. A similar observation was given by Abdul Fattah and Bhargava (2002) in the preparation of solid dispersions of halofan-trine and Gelucire 44/14.

Figure (2) showed the micrographs of pure ar-temisinin and the solid dispersions prepared at di-fferent ratios of ART-Gelucire 44/14. It was obvi-ous that, solid dispersion at a ratio of 1:10 showed the smallest drug crystals compare to other rati-os. It can be concluded that the solid dispersion showed much smaller drug crystal as compared to pure artemisinin. Moreover, the crystal size also appeared to be smaller when the ratio of artemisi-nin to carrier was decreased.

Figure (3) showed typical DSC thermal profi-les for pure artemisinin, Gelucire 44/14 and solid dispersion of ART-Gelucire at a ratio of 1:1o whi-ch was prepared at different filling temperature, 60°C, 45°C and room temperature. This ratio was selected for the study because the high content of Gelucire 44/14 in the solid dispersion would greatly influence the drug particle size. From the figure (3) it has been observed that the melting endotherm of the carrier was still intact while the melting endotherm of the drug was completely disappeared. As evident from the DSC thermal profiles it can be concluded that there was a decre-ase in the heat of fusion of Gelucire 44/14 as there was decrease in the filling temperature of the pre-pared solid dispersion as reflected in Table-1. So, the filling temperature at room temperature was selected for filling ART-Gelucire solid dispersions into capsules, as its heat of fusion was the lowest (99.85 J/G) among the temperature studied. Lower the heat of fusion more is the probability of drug completely melting in the carrier system, which in

turn may reduce crystallization of the drug during preparation. Similar findings have been reported by Yang et al. (2007) in the study of griesofulvin solid dispersions with Gelucire 44/14.

An explanation of this paradox is that as the temperature increased during the DSC scan (0-200°C), the molten carrier began to dissolve the drug crystals and as a result, no drug crystals re-mained when the temperature reached the melting point of the drug. This may explain why the drug melting peak disappeared. The phenomenon was observed in solid dispersion prepared by using po-lymers with low melting points as carriers, such as the dispersions of ciprofloxacin in polyethyle-ne glycol (PEG) 6000 (Frances et al., 1991) and paracetamol in polyethylene glycol (PEG) 4000 (Lloyd et al., 1997). In such systems, DSC tech-niques must be used in combination with hot stage microscopy otherwise, the possibility of misinter-pretation of DSC curves, leading to misunderstan-ding of the physical nature of solid dispersions, could occur (Khan & Craig, 2003; Bikiaris et al., 2005).

Figure (4) showed the DSC thermal profiles for the different ratios of ART-Gelucire 44/14 solid dispersions, pure artemisinin and Gelucire 44/14. It is evident from this figure that the DSC thermo-gram of solid dispersion of ART-Gelucire at a ratio of 1:10, there was a single melting endotherm for the carrier and the drug melting endotherm com-pletely disappeared. As the ratio of ART was in-creases the DSC curves showed a drift in the base line within the range of 60-75°C. The disappea-ring of the drug melting peak in the thermogram has been considered to be the evidence of the solid solution (Lloyd et al., 1997).

However, the microscopic study showed that the drug was still in the crystalline form in all solid dispersion ratios prepared.

Conclusion

From the results of the in vitro evaluation of ART-Gelucire 44/14 solid dispersion prepared by melt or fusion method, it can be conclude that this method was not capable of increasing the solubili-ty of artemisinin significantly. DSC studies indica-ted that the presence of the drug had a marginal ef-

518



fect on the melting profile of the carrier, indicating limited miscibility between the two components. So this preparation may just reduce the crystalli-zation of the artemisinin rather than forming pure amorphous phase. This finding was supported by the scanning electron microscopy studies.

Table Legends

Table 1 Formula for preparing different ratios of ART-Gelucire 44/14 solid dispersions.

Table 2 Total heats of fusion and temperatu-re for Gelucire 44/14 alone or incorporated into ART-Gelucire 44/14 solid dispersion at a ratio of 1:10 using different filling temperature (60°C, 45°C and room temperature) (mean ± SD).

Figure Legends

Figure 1 Solubility of artemisinin in pure ar-temisinin and in different ratios of Art-Gelucire 44/14 solid dispersions.

Figure 2 SEM micrographs of pure artemisi-nin and different ratios of Art-Gelucire 44/14 solid dispersions.

Figure 3 Thermal profiles of pure artemisinin and Art-Gelucire 44/14 solid dispersion at a ratio of 1:10 using different filling temperature (60°C, 45°C and room temperature).

Figure 4 Thermal profiles of pure artemisinin, Gelucire 44/14 and different ratios of ART-Gelu-cire 44/14 solid dispersions using filling tempera-ture at room temperature.

References

1. Abdul Fattah, A.M. & Bhargava, H.N. (2002). Pre-paration and in vitro evaluation of solid dispersions of halofantrine. Int J Pharm 235(17-33), 17-33.

2. Barker, S. A., Yap, S. P., Yuen, K. H., McCoy, C.P., Murphy, J.R. & Craig, D.Q.M. (2003). An investi-gation into the structure and bioavaibility of alpha-tocopherol dispersions in Gelucire 44/14. J Control Release 91(3), 477-488.

3. Bikiaris D., Papageorgian, G., Sterigiou A., Par-lidone, E., Karavas, F. & Georgarkis, M. (2005). Physicochemical studies on solid dispersions of po-orly water- soluble drugs : Evaluations of capabi-lities and limitation of thermal analysis techniques: Thermochim Acta 439 (1-2), 58-67.

4. Choy, Y.W., Khan, N. & Yuen, K.H., (2005). Signifi-cance of lipid matrix aging on in vitro release and in vivo bioavailability. Int J Pharm 299, 55-64.

5. Frances, C., Veiga, M.D., Espanol, O.M. & Cador-niga, R. (1991). Preparation, characterization and dissolution of ciprofloxalin / PEG 6000 binary sy-stems. Int J Pharm 77 (2-3), 193-198.

6. Higuchi, T. & Connors, K.A. (1965). Phase solubi-lity techniques. Adv Anal Chem Instru 4, 117-212.

7. Karatas, A., Yűksel, N & Baykara, T. (2005). Im-proved solubility and dissolution rate of piroxam using gelucire 44/14 and labrasol. IL. Farmaco 60, 771-782.

8. Khan, N. & Craig D.Q.M. (2003). The influences of drug incorporation on the structure and release properties of solid dispersions in liquid matrices. J Control Release 93 (3), 355-368.

9. Klayman, D.L. (1985). Qinghasou (Artemisinin): An antimalaria drug from China. Science 228, 1049-1055.

10. Lloyd, G.R., Craig, D.Q.M. & Smith, A. (1997). An investigation into the melting behaviour of bi-nary mixtures and solid dispersions of paraceta-mol and PEG 4000 J Pharm Sci 86(9), 991-996.

11. Luo, X. D. & Shen, C. C. (1987). The chemistry, pharmacology and clinical applications of Qing-hasou (artemisinin) and it’s derivatives. Med Res Rev 7 (1), 29-52

12. Sethia, S. & Squillante, E. (2002). Physicoche-mical characterization of solid dispersion of carbamazepine by super critical carbon dioxide and conventional solvent evaporation method. J Pharm Sci 91(9), 1948-1957.



13. Tashtoush, B.M., Al-Qashi, Z.S. & Najib, N.M. (2004). In vitro and in vivo evaluation of gliben-clamide in solid dispersion systems. Drug Dev Ind Pharm 30(6), 601-607.

14. Titulaer, H.A.C., Zuidema, J. & Lugt, C.B. (1991). Formulation and pharmacokinetics of artemisinin and its derivatives. Int J Pharm 69, 83-92.

15. WHO, 1981. The development of qinghaosu and its derivatives as antimalarial drugs. Fourth mee-ting of the sceintific working group on chemothe-rapy of malaria, Beijing, People’s of China

16. Wong, J.W. & Yuen, K.H. (2001). Improved oral bioavailability of artemisinin via inclusion com-plexation with cyclodextrin. Int J Pharm 227,177-185.

17. Yang, D., Kulkarni, R., Behme, R.J. & Kotiyam, P.N. (2007). Effect of the melt granulation tech-nique on the dissolution characteristics of griseo-fulvin. Int J Pharm 329(1-2), 72-78.

18. Yŭksel, N., Karatas, A., Õzkan, Y., Savaser, A., Õzkan, S. & Baykara, T. (2003). Enhanced bi-oavaibility of piroxicam using gelucire 44/14 and Labrosol : In vitro and in vivo evaluation. Eur J Pharm Biopharm 56, 453-459.

19. Zhao, S.S. & Zeng, M.Y. (1985). High- performan-ce liquid chromatography Determination of arte-misinin (qinghaosu). Planta Medica 3, 223-237.

Corresponding author Gamal Osman Elhassan School of Pharmaceutical Sciences, Universiti Sains Malaysia (USM), Malaysia, E-mail: [email protected]

Table 1. Formula for preparing different ratios of ART-Gelucire 44/14 solid dispersions

RatioDrug : Carrier

Weight (mg)Artemisinin Gelucire 44/14

1:102:103:104:105:106:107:108:10

100200300400500600700800

10001000100010001000100010001000

Table 2. Total heats of fusion and temperature for Gelucire 44/14 alone or incorporated into ART-Ge-lucire 44/14 solid dispersion at a ratio of 1:10 using different filling temperature (60°C, 45°C and room temperature) (mean ± SD)

Preparations Total Heats of Fusion (J/g)

Temperature(°C)

Gelucire 44/14 96.65 ± 0.03 42.94 ± 0.03ART-Gelucire 44/14 (1:10) at room temperature 99.85 ± 0.04 39.25 ± 0.03ART-Gelucire 44/14 (1:10) at 45°C 104.45 ± 0.03 38.55 ± 0.03ART-Gelucire 44/14 (1:10) at 60°C 107.55 ± 0.03 37.60 ± 0.03

520



Figure 1. Solubility of artemisinin in pure artemi-sinin and in different ratios of Art-Gelucire 44/14 solid dispersions

Figure 2. SEM micrographs of pure artemisinin and different ratios of Art-Gelucire 44/14 solid dispersions

Artemisinin

Ratio 1:

Ratio 2:10

Ratio 3:10

Ratio 4:10



Ratio 6:10

Ratio 8:10

Figure 3. Thermal profiles of pure artemisinin and Art-Gelucire 44/14 solid dispersion at ratio of 1:10 using different filling temperature (60°C, 45°C and room temperature)

Figure 4. Thermal profiles of pure artemisinin, Gelucire 44/14 and different ratios ART-Gelucire 44/14 solid dispersions using filling temperature at room temperature

522



Abstract

Introduction: Recurrent aphthous ulcers (RAU) are one of the most common ulcers in oral cavity. Several studies have shown conflicting variations for inflammatory cytokines and other biologic mar-kers in RAU.

Aim: The aim of the present study was to de-termine the level of IL-8 and IL-6 in the serum of patients with RAU.

Materials and method: In this case – control study, serum levels of IL-8 and IL-6 levels were measured in patients with RAU (n=40) and in he-althy control subjects (n=40). The cytokines levels were measured in serum by ELISA. The SPSS software was used to analyze the results.

Results: The mean value of IL-8 and IL-6 in patients (52.24 pg/ml and 9.39) was 10 and 4 fold higher than control group (5.01 pg/ml and 2.09 pg/ml), respectively. The difference was statistically significant ( t-test p=0.01).

Conclusion: The results of our study showed that serum levels of IL-8 and IL-6 were signifi-cantly higher in patients compared to controls.

Key words: Interleukin-8, Interleukin-6, Re-current aphthous ulcers

Introduction

Recurrent aphthous ulcerations are the most co-mmon oral mucosal ulcers. The aphthous lesions

present as recurrent painful ulcers on non-kerati-nized mucosa.1,2 So far many investigations have been performed on serum immunoglobulin levels,3 circulating immune complexes,4 T lymphocytes subsets5 and natural killer cells activity6 in patients with aphthous lesions. Interleukin 6 is a cytokine with different actions involved in inflammatory and immune responses.7 Yamamoto8 and Sun9 re-ported high level of IL-6 in patients with aphthous ulcerations. They also reported marked reduction of serum level of IL-6 after treatment. These findin-gs put forward the hypothesis of the role of IL-6 as a novel biomarker for the treatment of aphthous lesions.10 IL-8 is considered as an essential medi-ator in tissue injuries and inflammation and also as chemo tactic factor for basophil and neutrop-hil activation.11 IL-8 is produced by monocytes, T lymphocytes ,neutrophils and endothelial cells in pathologic conditions.12 The level of IL-8 increases in response to the rising level of pro-inflammatory cytokines like Interferon alpha and cellular stress 13 in conditions such as Behçet disease.14 Of note, the increasing of serum level of IL-8 is related to pre-sence of ophthous ulcerations.14,15 Neutrophils acti-vated by IL-8 can produce different enzymes which cause tissue destruction and ulcer formation.11 IL-8 is produced by inflammatory cells such as mono-cytes, macrophages, T lymphocytes, neutrophils, endothelial cells, fibroblasts and keratinocytes through pro-inflammatory cytokine stimulation.9 Therefore, it might have a role in pathogenesis of RAU. Currently, there are no serum markers, which

Evaluation of serum levels of interleukin-6 and interleukin-8 in patients with recurrent aphthous ulcerationsBehnoush Bakhtiari1, Pejman Bakianian Vaziri2, Mehrdad Hajilooi3, Hamed Mortazavi2

1 Dental School, Hamedan University of Medical Sciences, Hamedan, Iran,2 Dept of Oral Medicine, Dental School, Hamedan University of Medical Sciences, Hamedan, Iran,3 Research Center of Dental School, Hamedan University of Medical Sciences, Hamedan, Iran)



could be used to early diagnosis and predict the pro-gression of aphthous lesions. Such markers could be used as diagnostic and therapeutic purposes in patients suffering from aphthous lesions. This study was performed to measure the serum levels of IL-6 and IL-8 in early stages of aphthous lesions.

Material and Methods

A one-year (2005-2006) descriptive analytical study was performed in 40 patients with the dia-gnosis of new onset (less than 48 hours) recurrent aphthous ulcers referred to the dental school, Ha-medan University of Medical Sciences, Hamedan, Iran and 40 matched healthy individuals.Confir-mation of the aphthous diagnosis based on clinical features was made by two experts in oral medi-cine. The clinical characteristics for the diagnosis consisted of a symmetric circular, yellowish-white ulcer with erythematous halo and less than 1 cen-timeter in diameter. Only patients with recurrent lesions (recurrence 3 times per year) enrolled the study. Any history of systemic diseases such as Reiter’s syndrome, Behçet syndrome, crohn dise-ase, bowel diseases, gingival and periodontal dise-ases, evidence of pregnancy, and history of taking any medicine at least one month before entering the study were considered as exclusion criteria. Forty age and sex matched healthy individuals en-tered the study as the normal controls.

Blood samples (5ml whole blood) were taken from patients and controls. After coagulation the serums were separated and transported to transparent plastic sterilized tubules and were kept in -20 centigrade. Serum levels of IL-8 and IL-6 were determined by diagnostic kits (Bender Med System, serial number BMS2131NST, USA) and ELISA technique. The sensitivity of kits was 2pg/ml with the Coefficient Variation (CV) Intra-assay: 3.6-3.8% and CV (In-terassay): 5.2-7.4%. According to the Reference Value of the used kit, a value of 0-9.6 pg/ml was adopted as the normal range for IL-8. As for IL-6 the amount between 0-5 pg/ml was considered as normal value.

Data tabulation and statistical analyses were performed using the SPSS package (SPSS Inc, Chicago, IL). Student t-test was used to compare the differences.

Results

Of 40 patients with the diagnosis of RAU 19 patients were males and 21 patients were females (age range: 11-43 years, mean=25.8 years).The control group consisted of 20 males and 20 female healthy individuals (age range: 30-40 years, mean =33.6 years). Both groups were matched in terms of their age and sex (p-value>0.05).In thirty-five out of 40 patients high level of IL-8 was detected (Table-1). In control group 37 subjects had a se-rum level of IL-8 within the normal limit and the mean serum level of IL-8 was 5.01 pg/ml. the se-rum IL-8 level was significantly higher in patients compared to controls (p=0.01).Table 1. Serum level of IL-8 in RAS and healthy subjects

Study groups n

Serum level of IL-8 (pg/ml)SD ± Mean

Confidence interval 95%

RAS 40 52.24± 31.56

33.91~52.60Healthy group 40 5.01± 4.35

The serum level of IL-6 in the patients` group was higher than the normal limit in 19 cases (10 males) with 9.39 pg/ml (Table-2) whereas the ab-normal serum level of IL-6 was only reported in 3 cases (Figure 1). Statistically significant differen-ces were observed between two cases and controls concerning the IL-6 level (p=0.01).Table 2. Serum level of IL-6 in RAS and healthy subjects

Study groups n

Serum level of IL-6 (pg/ml)SD ± Mean

Confidence interval 95%

RAS 40 9.84± 9.39

4.138~10.46Healthy group 40 2.09± 2.04

524



Figure 1

Discussion

So far many studies have been done on inflam-matory cytokines and different biological markers in different diseases including RAU and conflic-ting results have been reported.16 The aim of this study was to determine serum levels of two infla-mmatory cytokines (IL-6, IL-8) in Iranian patients suffering from RAU. The findings of our study showed that the mean serum level of IL-6 in new onset aphthous lesions (less than 48 hours) is mar-kedly higher than controls. These findings were consistent with the previous studies.9,17

In the present study, the serum level of IL-6 in patients with RAU increased 4- fold comparing to controls. Yamamoto8 and Sun18 reported higher serum level of IL-6 by 3 and 2 folds, respectively. In Sun’s study18, the mean age of the patients was 14 years older than our study. On the other hand, in the studies of Yamamoto and Sun, the blood samples were taken after ulcer formation where-as in the present study blood samples were drawn 48 hours after the ulcers appearing to coincidence with production of highest level of inflammatory mediators.19,20 In Sun’s study18 ,in 25% of the pati-ents the serum level of IL-6 was higher. However, in our study, increased serum level of IL-6 was fo-und in 47.5% of the patients.

In the current study, the serum level of IL-8 in patients with RAU markedly increased .Many pre-vious studies showed the presence of CD4, CD8 and active T and B lymphocytes in patients with RAU.21,22 They also demonstrated T lymphocytes in peripheral blood in patients with RAU. Incre-ased serum level of TNF-alpha and its products in patients with RAU23 results in increasing serum level of IL-8 in location and peripheral blood.24

In our study, serum level of IL-8 in patients with RAU increased by 10 -fold .In the study of Sun et al. it increased by 7- fold. In Sun’s study18, the serum level of IL-8 and IL-6 were higher in 60% and 25% of the patients, respectively, and were more sensitive to monitoring comparing to serum level of IL-6 in RAU patients. In the pre-sent study, these figures were 87.5% and 47.5%, respectively which were similar to Sun’s findings.

In Conclusion, the present study showed that the serum levels of IL-6 and IL-8 in patients with RAU were significantly higher compared to the control group. These findings emerge the possible effect of using anti-inflammatory drugs with the potent ca-pability on the cytokines for the treatment of RAU.

Acknowledgements

This study was carried out in the University of Hamedan, Iran. The authors would like to thank Mr. Khosro Mani Kashani for data analysis, and Dr. S. Irani and Dr. M. Farshchian for their kind assistance. Also authors would like to thank De-puty of Research, Hamedan University of Medical Sciences for supporting towards the publication of this paper through grant No: 48932.

References

1. McCullough MJ, Abdel-Hafeth S,Scully C. Re-current aphthous stomatitis revisited; clini-cal features, associations, and new associati-on with infant feeding practices?J Oral Pathol Med.2007;36(10):615-20.

2. Scully C. Recurrent aphthous stomatitis: New En-gland journal of Medicine. 2006; 355: 165- 72.

3. Porter S. Scully C. Hematology status in recurrent aphthous ulcers compaired with other oral disea-ses. Triple O; 1998; 6: 41-4.

4. Roger S. Recurrent aphthous stomatitis, clinical, Characteristics and evidence for immune patho-genesis. J of investigative dermatology 1997;69: 499-509.

5. Landesberg R. Altered T4/ T8 ratio in a patients with severe recurrent aphthous ulcer. J Maxillo fa-cial surg. 1999; 25: 480- 82.



6. Sun A. Mechanism of depressed natural killer cell activity in recurrent aphthous ulcer. Clinico immu-no Pathol. 1998: 60: 83- 92.

7. Sun A, Chia JS, Chang YF, Chiang CP. Serum IL-6 level is a useful marker in evaluating therapeutic effects of levamisol and chines medicinal herps on patients with oral lichen planus. J Oral Pathol Med. 2002; 31: 196- 203.

8. Yamamoto T, Yoneda K, Ueta E, Osaki T. Serum cytokines, interleukin-2 receptor and soluble inter-cellular adhsionmolecule-1 in Oral disorders. Oral Surg Oral Med Oral Pathol 1994; 78: 727-35.

9. Sun A, Chia JS, chang YF, Chiang CP. Levamisole and chines medicinal herb can modulate the serum interleukin-6 level in patients with recurrent apht-hous ulcerations. J Oral Pathol Med 2003; 32: 206-14.

10. al- Dalaan A, al-Sedairy S, al Balaa S. Enhanced interleukin-8 Secretion in circulation of patients with Behcet’s disease. J Rheumatol 1995; 22: 904- 7.

11. Peichl P, Ceska M, Broell H, Effenberger F, Lin-dley IJ. Human neutrophil activating peptide/ in-terleukin-8 acts as an autoantigen in rheumatoid arthritis. Ann Rheum Dis 1992; 51: 19-22.

12. Ozoran K, Aydintug O, Tokgoz G, Duzgum N, Tut-kak H, Gurler A. Serum level of interleukin-8 in patients with Bechcet’s disease. Ann Rheum Dis 1995; 54: 610.

13. Hoffmann E, Dittrich- Breiolz O, Holtmann H, Kracht M. Multiple Control of interleukin-8 gen expression. J Leuk Biol 2002; 72: 847-55.

14. Katsantonis j, Adler Y, Orfanos CE, Zouboulis CC. Adamantiades- Behcet’s disease: serum IL-8 in a more reliable marker for disease activity than C- reactive protein and erythrocyte sedimentation rate. Dermatol 2000; 201: 37-9.

15. Zouboulis CC, Katsantonis J, Ketteler R, Treud-ler R, Kaklamani E, Hornemann S, Kaklamanis P, Orfanos CE. Adamantiades-Behçet’s disease: in-terleukin-8 is increased in serum of patients with active oral and neurological manifestations and is secreted by small vessel endothelial cells. Arch Dermatol Res. 2000;292:279-84.

16. Buno IJ, Huff JC, Weston WL, Cook DT, Brice SL. Elevated levels of interferon gamma, tumor necrosis factor α, interleukins 2,4 and 5,but not interleukin 10,are present in RAU. Arch Dermatol 1998;134:827-31.

17. Bazrafshani MR, Hajeer AH, Ollier WE, Thornhi-ll MH. IL-1β and IL-6 gene polymorphisms enco-de significant risk for the development of recurrent aphthous slomatitis(RAS). Genes Immun 2002; 3: 302-5.

18. Sun A, Chang YF, Chia JS, Chiang CP. Serum IL-8level is a more sensitive marker than serum IL-6 level in monitoring the diseases activity of RAS.J Oral Pathol Med 2004;33:133-39.

19. Sun A, Chiang CP, Chio PS, Wang JT, Liu BY, WU YC. Immunomodulation by levamisole in patients with recurrent aphthous ulcers or oral lichen pla-nus. J Oral Pathol Med 1994;23:172-7.

20. Savage NW, Seymour GJ, Kruger BJ. T- lympho-cyte subset changes in recurrent aphthous stoma-titis. Oral Surg Oral Med Oral Pathol 1985; 60: 175-81.

21. Hayrinen- Immonen R, Nordstrom D, Malmstrom M, Hietanen J, Konttinen YT. Immune- inflamma-tory cells in recurrent oral ulcers. Scand J Dent Res. 1991; 99: 510-8.

22. Hayrinen- Immonen R. Immune- activating in re-current oral ulcers. Scand j Dent Res. 1992; 100: 222-7.

23. Natah SS, Hayrinen-Immonen R, Hietanen j, Malmstrom M, Konttinen YT. Immunolocalizati-on of tumor necrosis factor-α expressing cells in recurrent aphthous ulcers lesions. J Oral Pathol Med 2000; 29: 19-25.

24. Taylor LJ, Bagg J, Walker DM, Peters TJ. Incre-ased production of tumor necrosis factor by pe-ripheral blood leukocytes in patients with recu-rrent oral aphthous ulceration. J Oral Pathol Med 1992; 21: 21-5.

Corresponding author Behnoush Bakhtiari Dental School, Hamedan University of Medical Sciences Iran E-mail : [email protected]

526



Abstract

Introduction: The self-perception of weight appropriateness is an important element of eating and weight behaviors.

Objectives: To compare objective and self-perceived weight status, as well as to analyze association between overweight (BMI≥25 kg/m2) and socio-demographic characteristics of adult population.

Materials and methods: A cross-sectional study of the adult population in Province of Vojvo-dina was carried out in 2006. The study involved 3627 participants aged 20 years and over (1685 men and 1942 women). Individuals were asked to classify their body weight (underweight, normal weight or overweight). Responses to this questi-on were compared with objective weight status. Logistic regression model was used to assess the predictive effects of various socio-demographic factors on overweight.

Results: Overall, 2.5% of adult population was underweight, 40.0% of normal weight and 55.7% overweight. The results of this study indicated that underweight women (33.8%) and men (36.8%) thought that they were of normal weight. Normal weight men (23.1%) and women (17.3%) consi-dered themselves “underweight”, while 0.5% of normal weight men and 4.5% of normal weight women classified themselves “overweight”. More than half overweight population (56.3% women, 71.9% men) classified themselves as “underwei-ght” or “normal weight”. In women and men, the

chance for overweight is increased with aging. Hi-gher education of women was followed with de-creased chance for overweight while in men with higher chance for overweight. Single women and men had lower odds for overweight. Men who live in rural settlement had higher odds for overweight. Income was significantly associated with overwei-ght in men.

Conclusion: Differences were found between objective weight status and self-perception of we-ight in adult population. Obtained data requires development of educational programs aimed to in-crease knowledge about risk factors of overweight and importance of recognizing overweight.

Key words: weight status, self-perception, overweight

Sažetak

Uvod: Adekvatna procena telesne mase je zna-čajan element pravilnih navika u ishrani i održava-nju normalne telesne mase.

Cilj rada: Uporediti distribuciju stvarne i per-cipirane telesne mase, kao i analiza povezanosti prekomerne telesne mase (BMI≥25kg/m2) i socio-demografskih karakteristika odraslog stanovništva.

Materijal i metodologija: U 2006. godini je sprovedeno istraživanje zdravstvenog stanja odra-slog stanovništva Vojvodine u vidu studije preseka. Istraživanje je obuhvatilo 3627 ispitanika starosti 20 i više godina (1685 muškaraca i 1942 žena). Ispitanici su klasifikovali svoju telesnu masu (pot-

Objective and self-perceived weight status in Province of VojvodinaObjEkTIVnA I sAmOPrOCEnjEnA uHrAnjEnOsT sTAnOVnIkA VOjVOdInEGrujic Vera¹, Dragnic Natasa², Harhaji Sanja², Cankovic Sonja¹, Radic Ivana², Cankovic Dusan3

¹ Center for analysis, planning and organization, Institute of Public Health of Vojvodina, Novi Sad, Serbia² Center for informatics and biostatistics in health care, Institute of Public Health of Vojvodina, Novi Sad, Serbia³ Center for health promotion, Institute of Public Health of Vojvodina, Novi Sad, Serbia



hranjenost, normalna telesna masa ili prekomerna telesna masa). Percipirana telesna masa je upore-đena sa stvarnom telesnom masom ispitanika. Da bi se utvrdio uticaj različitih socio-demografskih faktora na postojanje prekomerne talesne mase korišćen je model logističke regresije.

Rezultati: Ukupno posmatrano, 2.5% odraslih je bilo pothranjeno, 40.0% je imalo normalnu i 55.7% prekomernu telesnu masu. Rezultati ovog istraživanja su pokazali da je 33.8% pothranjenih žena i 36.8% pothranjenih muškaraca ocenilo da imaju normalnu telesnu masu. Muškarci (23.1%) i žene (17.3%) normalne telesne mase su sebe sma-trali “pothranjenim”, dok je 0.5% normalno uhran-jenih muškaraca i 4.5% normalno uhranjenih žena ocenilo da imaju prekomernu telesnu masu. Više od polovine ispitanika sa prekomernom telesnom masom (56.3% žena, 71.9% muškaraca) klasifiko-valo je sebe kao “pothranjeni” ili “normalne teles-ne mase”. Kod oba pola, starenjem se povećava šansa postojanja prekomerne telesne mase. Viši nivo obrazovanja kod žena je bio praćen smanjen-jem šanse za prekomernu telesnu masu dok je kod muškaraca povećana. Muškarci i žene koji nisu u braku su imali manje šanse za prekomernu telesnu masu. Muškarci iz seoskih domaćinstava su imali veće šanse za prekomernu telesnu masu. Prihodi su bili značajno povezani sa prekomernom teles-nom masom kod muškaraca.

Zaključak: Utvrđeno je postojanje razlike između stvarne i percipirane telesne mase kod od-raslog stanovništva. Dobijeni rezultati zahtevaju ra-zvoj edukativnih programa sa ciljem da se poveća znanje o faktorima rizika vezanim za prekomernu telesnu masu i značaju njenog prepoznavanja.

Ključne reči: telesna masa, percepcija, preko-merna telesna masa

Introduction

The World Health Organization (WHO) emp-hasizes that the world is in the grip of a global epidemic, and it is estimated that by 2020 obesity will be the single biggest killer on the planet (1). The prevalence of obesity has steadily increased over time within both developed and developing world, with obesity being described as most pre-valent among those who are socioeconomically

and locationally disadvantaged (2). The propor-tion of obese adult population in most European countries is between 15 to 20%. If overweight is also taken into account than these percentages re-aching more than 50% (3).

The prevalence of overweight and obesity in Province of Vojvodina (northern part of Serbia with 2031992 inhabitants, population census 2002) in 70-ties of 20th century was 51.6% (4). A study carri-ed out in 2000 reported 58.5% adults with overwei-ght and obesity, 23% of them were obese (5).

Inaccurate recognition of weight status is a thre-at to health weight control. Many studies showed that a considerable proportion of overweight adults, men especially, do not recognize that their body weight is too high (6, 7, 8, 9, 10, 11). Many studies showed that there is misperception of weight sta-tus for adults compared with measured body mass index (BMI). Men and women generally overesti-mate their height and underestimate their weight, particularly if they are obese, BMI tends to be unde-restimated (9, 10). Some studies found that women report a lower BMI as ideal (12,13,14) and are more likely than men to perceive themselves as overwei-ght even if they are of normal weight, whereas men are more likely to consider themselves as normal weight even if they are overweight (8, 15). Previo-us work indicates that there is a strong association between self-perceived weight status and weight control behavior. Self–perceived weight appropria-teness may be important point of focus for the desi-gn and implementation of clinical and public health initiatives (8). A number of factors have been linked to obesity, including age, gender and socio-econo-mic status. In developed countries body weight is an increase with ageing. Gender differences are seen in most countries with more women than men be-ing obese. In developed countries level of obesity is higher in the lower socio-economic groups (16). In developed countries there is evidence of an inverse association between different measures of socio-economic status, including income and educational level, and risk of women’s obesity, whereas weaker and more variable association with socio-economic status characterizes obesity in men (17).

This study aims to compare objective and self perceived weight status, as well as to analyze associ-ation between overweight (BMI≥25kg/m2) and so-cio-demographic characteristics of adult population.

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Methods

Ministry of Health of the Republic of Serbia carried out The 2006 National Survey for the po-pulation of Serbia (without data on Kosovo and Metohija). The study was the follow up of the ba-seline study conducted in 2000 and according to its methodology this investigation was conducted. Target population was the age group of 20 and abo-ve who are members of household (persons from particular institutions were not included – old peo-ple houses, social institutions, prisons, psychiatric institutions). Sample frame encompassess all hou-seholds within Census for 2002 in Serbia.

A stratified two-stage sample of the populati-on was used. In Serbia, six geographic areas were identified as main stratum in the sample-Vojvodina, Belgrade, Central, West, Estearn and South East Serbia. It was conducted in the way that in the first stage were distinguished census circles selected by Propability Proportional Sampling. In the second stage were selected households by Simple Random Sampling Without Replacement.The survey con-ducted in the Province of Vojvodina covered 1810 households (1060 in town and 750 in other settle-ments) when 3627 adults were examined.

Measures

For a measure of self-perceived weight status the participants were asked to estimate their own body weight from the following list: underweight, about the right weight or overweight. Objective weight status was based on measured participant’s BMI and the following cut-points for weight classifica-tion: overweight (BMI ≥25kg/m²), normal (BMI 18.5-24.9 kg/m²) and underweight (BMI <18.5 kg/m²) (18, 19). With respect to overweight the vari-ables of interest were age, sex, type of settlement, marital status, educational level, income. The sam-ple characteristics are presented in Table 1.

Educational level, total years of education re-ceived, was categorized as primary (elementary complete and incomplete school), secondary (se-condary completed) and postsecondary (any co-llege or university), marital status as unmarried, married, divorced or widowed, while types of settlements as urban (town) and rural (other set-

tlements). Household income according to the wealth index was divided into five levels - poo-rest (first level), poor (second level), middle class (third level), wealthy (fourth level) and the weal-thiest (fifth level).Table 1. Socio-demographic characteristic of adult population of Vojvodina

Women (%) Men (%)Marital statusunmarried 10.8 20.6married 61.8 70.0divorced 4.5 3.1widowed 22.9 6.3Type of settlementurban 56.9 57.3rural 43.1 42.7Educational level primary 46.0 30.4secondary 44.2 57.3postsecondary 9.8 12.2Incomepoorest 20.3 20.5poor 25.1 25.6middle 20.6 19.3wealthy 21.2 20.8wealthiest 12.8 13.7Age20-34 21.2 24.335-44 15.9 18.445-54 18.1 20.255-64 18.1 16.665+ 26.7 20.5Body mass index (mean) 26.4 27.6

Statistical Analysis

The obtained data were statistically processed using SPSS version 14.0. Results are given as mean ± SD and proportion. Multivariate logistic regression was used in construction of model to associate overweight and potential risk factors (sex, age, type of settlement, marital status, edu-cation level, income). Model fit was analyzed by using classification table (cut-off 0.50), for calcu-lation sensitivity and specificity.



Results

Overall, 2.5% of adult population was underwe-ight, 40% of normal weight and 55.7% overweight. Total number of participants with objective and self perceived weight status was 3441 (94.9%). Among women 3.7% was underweight, 42.3%

normal weight and 54% overweight. Among men 1.2% was underweight, 37.2% normal weight and 61.6% overweight.

It was found that underweight women (33.8%) and men (36.8%) thought they were of normal weight. Normal weight men (23.1%) and women (17.3%) considered themselves “underweight”,

Table 2. Objective and self perceived weight status in adult population of VojvodinaSelf perceived weight status

Objective status Underweight About right Overweight Can not estimate TotalWomen (n =1822)

Underweight 64.7 33.8 0.0 1.5 3.7Normal 17.3 75.8 4.5 2.3 42.3

Overweight 2.9 53.4 39.6 4.1 54.0Total 11.3 62.1 23.3 3.2 100.0

Men (n =997)Underweight 63.2 36.8 0.0 0.0 1.2

Normal 23.1 73.3 0.5 3.2 37.2Overweight 2.8 69.1 24.1 4.0 61.6

Total 11.1 70.3 15.0 3.6 100.0

Table 3. Factors associated with overweight (BMI≥25kg/m2) in adult population of Vojvodina

Explanatory variablesWomen Men

OR 95%CI OR 95%CIAge20-34 1.00 1.0035-44 2.15** 1.53-3.03 2.30** 1.64-3.2145-54 4.34** 3.12-6.05 2.26** 1.62-3.1355-64 11.14** 7.71-16.08 2.54** 1.77-3.6265+ 7.23** 5.04-10.34 2.36** 1.68-3.30Type of settlementUrban 1.00 1.00Rural 1.22 0.97-1.52 1.40** 1.12-1.76Marital statusMarried 1.00 1.00Single 0.57** 0.45-0.73 0.57** 0.45-0.73Educational levelPrimary 1.00 1.00Secondary 0.74* 0.56-0.97 1.50** 1.15-1.94Postsecondary 0.53** 0.35-0.81 1.43 0.97-2.12Household incomePoorest 1.00 1.00Poor 1.14 0.96-1.81 1.21 0.89-1.66Middle 1.54* 1.10-2.16 1.66** 1.17-2.36Wealthy 1.43* 1.00-2.02 1.55* 1.08-2.22The wealthiest 1.46 0.96-2.21 2.29** 1.50-3.48

* p<0.05** p<0.01

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while 0.5% of normal weight men and 4.5% of normal weight women classified themselves as “overweight”. More than half overweight populati-on (56.3% women, 71.9% men) classified themsel-ves as “underweight” or “normal weight” (table 2).

Age, marital status and educational level were significant associated with overweight of women and men. With aging the odds for overweight was increased. The highest odds for overweight was in the age group of 55-64 year when it was 2.5 times higher in men (95%CI: 1.77-3.62) compared with 20-34 year person, while the odds was 11.1 times higher (95%CI: 7.71-16.08) for women. Higher education of women decreased odds for overweight (47% lower odds for postsecondary level of educa-tion regarding to primary level of education), whi-le the odds for overweight was higher in men with higher level of education (50% higher in men with secondary level of education compared to primary educational level). Single women and men had 43% lower odds for overweight. Men who live in rural settlement had 40% higher odds for overweight. Income is significantly associated with overweight in men, i.e. higher income is followed with higher chance for overweight (table 3).

Sensitivity for Logistic Model was 85% in sam-ple of men, and 76.6% in sample of women. Spe-cificity was 36.7% in sample of men, and 57.9% in a sample of women.

Discussion

Survey carried out in the Province of Vojvodi-na, 2006 indicated that 2.5% of adults were un-derweight, 40.0% of normal weight and 55.7% overweight.

Self-perception of body weight showed that two thirds of both men and women with normal weight status properly assess their weight, while about one fifth of those under assess their weight. It is well known that men and women underestimate their weight, especially if they are obese (9, 10). Wo-men more often perceive themselves as overweight even if they are with normal weight while men are more likely to consider themselves as normal body weight, even if they are overweight (8,15,11). Our results indicate that the majority of overweight par-ticipants (56.3% of women and 71.9% of man) vi-

ewed themselves as “underweight” or “about right weight”. Men were more likely than women to un-der assess their weight. The reason for this, found in literature, is that women are more dissatisfied with their bodies than men, and therefore more likely to be aware of their weight status (20, 21, 22, 23).

In Vojvodina, it was found that with age odds to become overweight is increased. The greatest odds to become overweight was in the age group 55-64 years for both men and women. Martinez Ross also reported that the highest prevalence is reached in the age group 55-60 years (24). Group of the aut-hors discussed the influence of age on both overwe-ight and obesity, and offered the possible explana-tion as people in older age live more sedentary life-style (25). There are a number of factors attributed to this occurrence, including less physical activity and increased accessibility of food (26, 27, 28). Therefore, effort must be put on promotion of he-althy lifestyle, balanced nutrition with low energy density and increased physical activity (29).

Higher educational attainment through incre-ased knowledge enables an individual to make healthy choices regarding diet and physical acti-vity. Therefore women in Vojvodina with higher educational level have lower odds for overweight. On the other hand, increase in educational level in males was followed with higher odds for overwei-ght. While other studies have found for both sexes, a low educational attainment was the strongest predictor of overweight (30, 31). Some authors argued that the highest level of education is esta-blished at young adulthood and for most people it will remain the same all their lives. Therefore, education is an easy and reliable indicator but it does not capture changes in social status that take place when the schooling process finishes (32, 33).

One of the examined factors was the type of set-tlement. In Vojvodina, people from the rural areas had a higher probability for overweight than peo-ple from the urban areas (women had 1.22 odds ratios, while men’s odds ratios was even more si-gnificant (1.40)). Our results were consistent with previous study conducted in United States (34). On the other hand, there was no difference in the prevalence of overweight and obesity in rural and urban areas of ten European countries – Switzer-land, France, Denmark, Sweden, The Netherlan-ds, Germany, Italy, Austria, Spain, Greece in indi-



viduals aged 50-79 years (35). Such data requires deeper research in this field.

Results of this study indicate that an increa-se in income was followed with higher odds for overweight in men. The wealthiest men had more than two times higher odds for overweight regar-ding to the poorest population. The similar results were obtained in other studies, too (36, 37).

Single men and women in Vojvodina had a lower probability for overweight than married one. We confirmed results from other studies (38, 39, 40). Prior studies have found that married wo-men weight more than those who have never been married (41). Divorce/separation was associated with weight loss among women, and being never married, divorced/separated, or widowed was re-lated to weight loss among men (42).

Differences between objective and self-perce-ived weight in adult population exist. Findings reflect relationship between age, marital status, type of settlement, educational level, income and odds for being overweight. Therefore, multilevel interventions are needed, especially through deve-lopment of educational programs aimed to increa-se awareness about risk factors of overweight and importance of recognizing it, in order to prevent the growing negative consequences.

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Corresponding author Vera Grujic, Institute of Public Health, Futoska 121, 21000 Novi Sad, Republic of Serbia E-mail: [email protected]



Abstract

Background: Rhabdomyosarcomas are the most common soft tissue sarcomas in pediatric age and rare in adults.

Case Report: A 37-year-old male admitted with abdominal discomfort and mass during the previous 9 months. The patient was hospitalized for the diagnosis of abdominal mass. Initial he-modynamic parameters were stabile. During di-agnosis course, hemoglobin level decreased and hemodynamic instability occurred. Abdominal computed tomography revealed a 30x20 cm mass in left retroperitoneal space. The physical exami-nation revealed the enlargement of the mass. The surgical resection of tumor and the adjacent ab-dominal wall resection were performed. Alveolar rhabdomyosarcoma was diagnosed. The patient received adjuvant chemotherapy and radiothera-py. The patient had 8 mouths follow up without signs of recurrence.

Conclusion: The clinical presentation of this tumor with haemodynamic instability due to in-tra tumoral hemorrhage is a challenging course for physicians and this rare entity must be kept in mind for accurate and urgent treatment.

Key words: Rhabdomyosarcoma, Alveolar su-btype, Intratumoral Hemorrhage, Adult, Surgery

Introduction

Rhabdomyosarcomas (RMSs) are the most co-mmon types of malignancy in children and young adults. The clinical experience in adults is limi-ted. Alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma represent the two main histologic patterns (1). Alveolar rhabdomyo-sarcoma is an uncommon high-grade malignant soft tissue tumor of mesenchymal origin (2). The clinical manifestations of retroperitoneal rhab-domyosarcomas are related to extrinsic compre-ssion or invasion to the adjacent visceral organs. Palpable abdominal mass and tenderness are not rare symptoms and intratumoral hemorrhage lea-ding to hemodynamic instability is an extremely rare condition. Here in, we reported a case of an adult alveolar rhabdomyosarcoma originated from retroperitoneal space, which was operated in the urgent course for hemodynamic instability due to intratumoral hemorrhage.

Case Report

A 37-year-old male admitted to the surgery cli-nic with persistent left abdominal flank discom-fort, palpable abdominal mass, and weight loss

CASE REPORT

Hemodynamic Instability due to Intratumoral Hemorrhage in retroperitoneal Alveolar rhabdomyosarcoma in AdultAtakan Sezer1, Mehmet Ali Yagcı1, Ahmet Rahmi Hatipoglu1, Irfan Coskun1, Fedai Calta1, Irfan Cicin2, Ufuk Usta3, Osman Temizoz4, Teyfik Aktoz5

1 Department of General Surgery, Trakya University, Faculty of Medicine, Edirne, Turkey2 Department of Medical Oncology, Trakya University, Faculty of Medicine, Edirne, Turkey3 Department of Pathology, Trakya University, Faculty of Medicine, Edirne, Turkey4 Department of Radiology, Trakya University, Faculty of Medicine, Edirne, Turkey5 Department of Urology, Trakya University, Faculty of Medicine, Edirne, Turkey

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previous nine months. The medical history was unremarkable. The patient was hospitalized for the diagnosis of abdominal mass. Results of la-boratory examinations were within normal limits and initial hemodynamic parameters were stabile. During the diagnosis course, the patient had cool skin, hypotension, tachycardia, and weak periphe-ral pulses with hemodynamic instability. Hemo-globin level decreased to 8,7gr/dl. Blood pressure was 90/40 mmHg, and heart rate of 142/m. Plain x-ray graphy demonstrated an opacity in left qu-adrant and colonic gas images snookered to the right upper quadrant. Abdominal computed to-mography (CT) revealed a large enhancing left retroperitoneal mass which involves the left iliop-soas muscle. The mass was 30x20cm with internal septation and semisolid property (Figure 1). The physical examination revealed the enlargement of the mass. The patient was resuscitated with IV flu-id and blood and urgent operation was performed. On exploration a giant mass with 30x30cm in size originated from left psoas muscle was observed. Complete tumor and adjacent abdominal wall re-section were performed. Pathologic examination revealed alveolar rhabdomyosarcoma with CD99, vimentin, myoglobin positive reaction (Figure 2). The postoperative course uneventful and the pati-ent was discharged on the 19th postoperative day. Adjuvant chemotherapy with adriamycin, vincri-stine, and cyclophosphamide were administered, and radiotherapy was delivered to the operation site. The patient had 8 mouths follow up without signs of recurrence.

Figure 1. (A) CT scan showing the tumor mass in left retroperitoneal space fully filled whole

abdomen. (B) Plain abdominal x-ray showed di-lated large bowel with air fluid levels and no air in the rectum. The opacity shows the margins of the mass.

Figure 2. Atypical tumor cells with round to oval nuclei and scant cytoplasm. (A) Forming somew-hat alveolar like pattern. (B) Showing prominent coagulative necrosis (HEx200). (C) Immuno-histochemically positivity in dot pattern in the nuclei of the tumor cells for myoglobin antibody (Myoglobinx400)

Discussion

We report a retroperitoneal alveolar rhabdo-myosarcoma in an adult patient presented with enlarging abdominal mass and hemodynamic in-stability due to intratumoral hemorrhage. Rhabdo-myosarcoma is a morphologically and clinically heterogeneous family of malignant soft tissue tumors related to myogenic lineage. They are the most common types of malignancy in children and young adults (1). 50% of soft tissue sarcomas ari-sing in children 0 to 14 years of age and approxi-mately %80 of patients are less than 20 years of age. RMSs in older adults are distinctly unusual. Pleomorphic, spindle cell, embryonal, botryoid, ARMSs are the main subtypes. ARMS are most frequent in adolescents and in young adults (3). The most effected regions are head and neck, lim-bs, trunk. Skin, spermatic cord, testis, perineum, uterus, and breast are rare origin sites. Clinical presentation varies by site of presentation. Becau-se of the relative paucity of vital structures and the



abundance of loose connective tissue of retrope-ritoneum, alveolar retroperitoneal rhabdomyosar-comas have a generally late clinical presentation as large space occupying lesions. Symptoms tend to be related to gastrointestinal, urinary or vascu-lar compromise (4, 5). Intratumoral hemorrhage which leads to hemodynamic instability is an un-common clinical manifestation of retroperitoneal rhabdomyosarcomas. To our knowledge, this is believed to be the first patient with the intratumo-ral hemorrhage leading to hemodynamic instabi-lity. ARMS is positive for CD99, vimentin, myo-globin in immunohistochemical staining (2, 5, 6). RMSs of the retroperitoneum in adults are often large, invade adjacent structures and encase ma-jor vessels and highly aggressive in nature. The-se tumors have early and widespread metastasis involving regional and distant lymph nodes, soft tissues and bone marrow. Primary tumor site, me-tastatic disease, tumor invasiveness, tumor size, lymph node involvement is important prognostic factors in the survival rate. Also alveolar histology is an independent predictor of the worse outcome. A 5-year survival rate of <30% has been reported in children with metastatic ARMS (2, 5, 7). An accepted multimodal treatment combination con-sists of surgery, chemotherapy, and radiation the-rapy to the tumor site is necessary to achieve long-term survival. Although new adjuvant or surgical therapeutic regimes exist long-term survival rates still remain poor in the range of 35–45% (7, 8).

Conclusion

The late occurring of physical findings and in-sidious presentation of the retroperitoneal rhabdo-myosarcomas, the diagnosis is generally delayed until the tumor is widely enlarged. The intratumo-ral hemorrhage causing hemodynamic instability is an unusual clinical presentation of these tu-mors. Oncology emergencies due to intratumoral hemorrhage are rare but life treating conditions. Early diagnose, accurate resuscitation, and exact timing may reduce morbidity and mortality in hemodynamic instability due to intratumoral he-morrhage in retroperitoneal rhabdomyosarcomas.

References

1. Yasuda T, Perry KD, Nelson M, Bui MM, Nasir A, Goldschmidt R, Gnepp DR, Bridge JA. Alveolar rhab-domyosarcoma of the head and neck region in older adults: genetic characterization and a review of the literature. Hum Pathol 2009; 40: 341-348.

2. Enzinger FM, Shiraki M. Alveolar rhabdomyosarco-ma. An analysis of 110 cases. Cancer 1969; 24: 18-31.

3. Hawkins WG, Hoos A, Antonescu CR, Urist MJ, Le-ung DH, Gold JS, Woodruff JM, Lewis JJ, Brennan MF. Clinicopathologic analysis of patients with adult rhabdomyosarcoma. Cancer 2001; 91: 794-803.

4. Raney RB, Stoner JA, Walterhouse DO, Andrassy RJ, Donaldson SS, Laurie F, Meyer WH, Qualman SJ, Crist WM; Intergroup Rhabdomyosarcoma Study-IV, 1991-1997. Results of treatment of fifty-six patients with localized retroperitoneal and pelvic rhabdomyo-sarcoma: a report from The Intergroup Rhabdomyo-sarcoma Study-IV, 1991-1997. Pediatr Blood Cancer 2004; 42: 618-625

5. Van Roggen JF, Hogendoorn PC. Soft tissue tumours of the retroperitoneum. Sarcoma 2000; 4: 17-26.

6. Paner GP, Gasilionis V, Hammadeh R. A retroperito-neal mass in an elderly woman. Pleomorphic rhab-domyosarcoma, classic variant, with reactive osteoc-last-like giant cells. Arch Pathol Lab Med 2005; 129: 703-705.

7. Blakely ML, Andrassy RJ, Raney RB, Anderson JR, Wiener ES, Rodeberg DA, Paidas CN, Lobe TE, Crist WM; Intergroup Rhabdomyosarcoma Studies I throu-gh IV. Prognostic factors and surgical treatment gu-idelines for children with rhabdomyosarcoma of the perineum or anus: a report of Intergroup Rhabdomyo-sarcoma Studies I through IV, 1972 through 1997. J Pediatr Surg 2003; 38: 347-353.

8. Cecchetto G, Bisogno G, Treuner J, Ferrari A, Matt-ke A, Casanova M, Dall’Igna P, Zanetti I, Volpato S, Siracusa F, Scarzello G, Boglino C, Carli M; Italian and German Soft Tissue Cooperative Groups Studies. Role of surgery for nonmetastatic abdominal rhab-domyosarcomas: a report from the Italian and Ger-man Soft Tissue Cooperative Groups Studies. Cancer 2003; 97: 1974-1980.

Corresponding author Atakan Sezer, Trakya University, Faculty of Medicine, Department of General Surgery, Turkey, E-mail: [email protected]

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Abstract

Objective: To evaluate the prevalence and risk factors of anti-tuberculosis drug induced hepatitis in the state of Penang, Malaysia.

Materials and Methods: Penang General Hospital is the referral center for all the tuberculo-sis patients in the State of Penang. Patient records were reviewed retrospectively and prospectively to identify patients with confirmed diagnosis of drug induced hepatitis (DIH) through chemothe-rapy of tuberculosis from January 2006 to October 2008. Data were analyzed using SPSS version 16 The values are expressed as median and variables are compared by using student‘t’ test and χ2 tests..

Results: A total of 1542 patients diagnosed of active tuberculosis infection with normal pre-treatment liver function were followed up both clinically and biochemically during the course of the study. Their data were collected on Performa and patients were treated with anti-tubercular drugs. Anti-TB-DIH developed in 126 (8.2%) out of 1542 patients within 15 days (median) com-mencement of therapy. Almost 60% of patient had normalization of their LFT within 2 weeks of cessation of anti TB therapy. No recurrence of anti-TB-DIH was observed after reintroduction of therapy. There is significant difference between males and females [75(59.5%) vs. 51(40.5), P <0.05]. Age more than 35 years and concomitant use of alcohol were found to be independent pre disposing factors (P < 0.05) of anti-TB-DIH.

Conclusion: Hepatotoxicity was severe in pulmonary tuberculosis, and numbers of factors like age, gender, pre existing hepatic abnormali-ties were significantly associated with TB chemo-therapy.

Key words: Hepatotoxicity, Risk factors, Tu-berculosis, Anti-tubercular chemotherapy, Drug induced hepatotoxicity

Introduction

Tuberculosis continues to be a major threat to the health of the world, with an estimated 8-10 million new cases and 3 million deaths annually1. An effective control has been achieved by the wi-despread use of anti tuberculosis drugs. However, despite their efficacy, superadded problems have to be faced in terms of long duration of treatment, emergence of multi drug resistant (MDR) strains and certain adverse effects ascribed to these dru-gs. Among these adverse effects hepatotoxicity is a well known complication of Anti Tuberculose Therapy (ATT) 2-4. The severity ranges from al-teration in liver enzymes, chronic active hepatitis and picture of acute hepatitis, occasionally com-plicated by acute liver failure carrying very high chance of mortality unless transplanted. It is co-mmon with Isoniazid especially when given in combination with Rifampicin and Pyrazinamide. Fifteen to twenty percent of patients receiving Iso-niazid as a single agent for prophylaxis against tu-berculosis may have increased serum alanine and aspartate aminotransferase levels, but only 1 per-cent have severe hepatic necrosis that require the withdrawal of the drug5. The clinical, biochemical and histopathological features of drug induced he-patotoxicity (DIH) are indistinguishable from that of viral hepatitis6. The DIH incidence is higher in the developing countries with rates ranging from 8% to 39%, compared to developed countries at 3%–4%, despite similar regimens used7,8-10. A hi-

Anti-Tubercular Chemotherapy and drug Induced HepatitisAmer Hayat Khan1*, Syed Azhar Syed Sulaiman1, Mohamed Azmi Hassali 1, A. Razzak Muttalif2

1 School of Pharmaceutical Sciences, Universiti Sains Malaysia, Malaysia,2 Respiratory Clinic, Penang General Hospital, Malaysia



gher risk of 11.5% and 9.7% has been reported in Indian and Malaysian patient’s respectively2, 11. Teleman MD, Chee CB et al. (2002) from Singa-pore has reported anti Tuberculose drug induced hepatitis a incidence of 5.3%12. In Malaysia, it has been reported that tuberculosis is among the top 5 communicable diseases, and the trend of tubercu-losis notification rates has been constantly increa-sing from 60/100,000 population in the year 1991 to 66/100,000 in the end of year 200113.

The contribution of Pyrazinamide to the deve-lopment of drug-induced hepatotoxicity during treatment of TB appeared to be inconclusive in earlier reports.2, 14 However; later studies showed that Pyrazinamide has higher hepatotoxicity po-tential compared to other drugs in the short course regimen and also found that from developing co-untries revealed that re-introduction of a regimen including Pyrazinamide was more likely to result in recurrence of hepatotoxicity than that without. 12, 14-18

The clinical, biochemical and histopathologi-cal features of drug induced hepatotoxicity (DIH) are indistinguishable from that of viral hepati-tis.17 Early identification and modification of tre-atment regimen are required for patients who are at increased risk of anti tuberculosis induced he-patotoxicity and hence reducing the morbidity and mortality.18 DIH due to anti-TB drugs can cause significant morbidity and compromise treatment regimen for TB. 19 The reasons for this higher rate of hepatotoxicity are not clear. Reported risk fac-tors for hepatotoxicity includes older age, gender variation (female are at a higher risk), malnutriti-on, high alcohol intake, pre-existing liver disease, hepatitis B carriage, increased prevalence of viral hepatitis in developing countries, hypoalbumine-mia, advanced TB, inappropriate use of drugs and acetylated status.15,18,20-22.

There are controversies related to prevention and management of hepatotoxicity as a result of Anti-tuberculosis therapy (ATT). Data on monito-ring of liver enzymes and their usefulness and the time for withdrawal of ATT, drug which is to be withdrawn first and duration of withdrawal befo-re reintroduction, Data on the modified regimen if ATT is restarted is insufficient or rather incon-clusive. This study was aimed to evaluate the risk factors associated with the development of Anti-

TB-DIH and also to study the association between use of PZA in the treatment regimen and hepato-toxicity.

Methodology

Study type: A retrospective and prospective cross sectional observation study was conducted among the tuberculosis patients.

Study site: The current study was carried out in the Respiratory clinic of Penang Hospital. This centre is a tertiary level reference centre for respi-ratory diseases in the state of Penang, Malaysia. Any person with a respiratory problem in the state of Penang can attend this centre without a physi-cian referral.

Study duration: The study was carried out from January 2006 to October 2008, and all regi-stered cases at present and or past were reviewed.

Study design: Patients suspected to have TB will undergo a series of tests. These tests inclu-de serial sputum direct smear (three samples), full blood counts (FBC), erythrocyte sedimentation rate (ESR), urea and electrolytes (U+E), baseli-ne liver function tests (LFT), HIV and Hepatitis screening. Once the diagnosis is confirmed or the decision to treat has been made, TB treatment is commenced after reviewing the blood results. The first line anti-TB treatment regimens were either a combination of streptomycin, isoniazid, rifampi-cin and pyrazinamide (SHRZ), or a combination of ethambuthol, isoniazid, rifampicin and pyrazi-namide (EHRZ). Any abnormality noted would necessitate adjustment to the treatment regime or further tests being done to ascertain the cau-se. In the case of hepatitis, viral serology screen (Hepatitis A, B and C) and ultra-sonography were performed. Directly observed therapy short cour-se (DOTS) was implemented almost universally, unless this was not physically or socially possible.

All TB patients were reviewed at two weeks upon treatment and thereafter every two months until the treatment was completed. During the re-view, inquiries were made about compliance, drug side effects, tolerability as well as any worsening symptoms to indicate other diagnoses. If drug-in-duced hepatitis was suspected, a repeat LFT was performed. However, at the study centre, repeat

538



LFTs were done routinely at one to two weeks after the commencement of therapy in most of the patients though they were asymptomatic. The duration of treatment varied from a minimum of six months to one year in some cases. All cases of hepatitis fulfilling the criteria outlined below were treated with treatment cessation. Biweekly LFTs guided the doctor on the progress of the hepatitis. Once the LFT results had returned to normal, drug challenge was commenced until an acceptable treatment combination was reached. However, in most patients the previous regimen consisting of the primary drugs could be restarting safely.

Inclusion criteria: 1. Normal LFT prior to starting anti-TB drugs

regimen.2. Drugs were given in standard doses

(isoniazid: 5–8 mg/kg/day; rifampicin: 10–15 mg/kg/day; pyrazinamide: 20–40 mg/kg/day), alone or in combination for at least five days prior to the development of hepatitis.

3. Aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) increased to ≥ 120 IU/ L (normal ≤ 40 IU/L) and/or an increase in total bilirubin to > 1.5 mg/dL (25 μmol/L)(normal < 1.5 mg/dL or < 25 μmol/L), at least five days after starting anti-TB drugs with no other apparent causes of abnormal LFT.

Exclusion criteria:1. Patients with hepatitis meeting the inclusion

criteria above prior to commencement of anti-TB medications.

2. Receiving a higher dosage of anti-TB drugs than the recommended dosage according to body weight.

3. Inadequate medical records to allow complete analysis.

4. Receiving other potentially hepatotoxic medications concurrent with the anti-TB treatment.

Drug regimen:Doses were fixed according to the total body

weight of the patient. Doses were as follows: Iso-niazid (I) 5mg / kg, Rifampicin (R) 10 mg / kg,

Pyrazinamide (Z): 25 mg / kg, Ethambutol (E): 15 mg / kg and streptomycin(S) 15 mg / kg per day. The patients received at least two weeks of inpatient care. Laboratory evaluations including ALT, AST, bilirubin and albumin were performed twice a week during hospitalization and every two weeks for the first two months of treatment. Then their treatment was supervised monthly until end of treatment.

Diagnosing criteria for drug-induced hepatotoxicity:2,3,8-10

Normalization of liver enzymes level and re-solution of signs and symptoms of hepatotoxicity after withdrawal of all anti-TB drugs, and presen-ce of at least one of the following criteria: a rise to five or greater than five times the normal level of ALT and/or AST; a rise in the level of serum total bilirubin over 2 mg/dl. Normal maximum value in the laboratory is 35 IU/L for ALT and for AST it is 40 IU/L. For ALP normal upper limit is 120 IU/L. In addition to laboratory changes, drug-in-duced hepatotoxicity was accepted as a cause of temporary discontinuation of therapy when the following criteria were observed: (1) occurrence of nausea, vomiting, anorexia and vague abdomi-nal pain particularly at right upper quadrant, (2) occurrence of jaundice with or without abdominal signs and symptoms. It is important to notice that asymptomatic jaundice with no evidence of hepa-titis is probably due to rifampin.2 However, it sho-uld be considered that the clinical diagnosis of this situation is difficult and if diagnosed by liver fun-ction tests then over-diagnosis could result, since Rifampicin can cause cholestatic jaundice which is not that clinically significant.2, 23

Management of anti TB DIH:After discontinuation of the hepatotoxic drugs

(IRZ) an alternative regimen including ethambutol (15 mg kg-1), ciprofloxacin (500-750 mg, b.i.d.) and an aminoglycoside antibiotic (Streptomycin 5 mg kg-1) were substituted temporarily. Alterna-tive regimen was continued until normalization of serum transaminases levels. The anti-TB drugs in-cluding isoniazid, rifampicin, pyrazinamide and et-hambutol were reintroduced gradually in a stepwise manner to all patients. After normalization of serum transaminases, small dose of isoniazid (50 mg) was



reintroduced and gradually increased to recommen-ded daily dose over one week. This procedure was repeated by adding rifampicin and pyrazinamide, respectively. In fact reintroductions of these dru-gs were possible for all patients over three weeks. Reintroduction of anti-TB drugs is an accepted method.12,24 Also information regarding the drugs, dosage and duration of treatment were noted. Nutri-tional status was estimated according to body mass index (BMI). BMI of > 19 kg/m2 was considered to denote normal nutritional status.

Ethical Consideration

The study protocol was approved by Clinical Research Centre (CRC), Penang Hospital and Mi-nistry of Health, Malaysia.


The data collected in the study was analysed by using the Statistical Package for Social Sciences version 16 (SPSS Inc, Chicago, IL, USA). Cate-gorical variables, such as patients’ gender, hepati-tis B virus carrier status, HIV infection, sites of TB and treatment regimens, were expressed in frequ-ency, percentage and statistical tests were applied, whereas numerical variables such as patients’ age, BMI, serum albumin, serum globulin, serum ALT, serum AST and serum bilirubin were expressed in means and standard deviation. The various risk factors in the study population were analysed with independent t-test and chi-square test to evaluate their association with the development of anti-TB drug-induced hepatitis. A p-value < 0.05 was con-sidered to be statistically significant.

Results

Among 1542 registered cases 126 (8.7%) confir-med TB cases were suffering from anti-tubercular drug induced hepatitis. Table 1, shows that male 75 (59.5%) were more affected then female 51 (40.5%) with mean body weight of 41.85kg. There was a si-gnificance difference between males and females (p = 0.002). Age of the patients was divided into two

groups as shown in table 1, 24 (19.0%) were found in age group of ≤ 35 while 102 (81.0%) were from the age group of > 35 and mean age was 43.71 ye-ars. There is significance difference between both groups (p < 0.001). The occurrence of DIH with anti-tubercular drugs in the three major races in Malaysia is as follows, Malay 41 (32.5 %), Chinese 69 (54.8%), Indian 13 (10.3%) and 3 (2.4%) were others, which shows non-significant difference (p = 0.160). Majority of cases were newly registered 106 (84.1%) and 20 (15.9%) were found relapsed cases which shows significant difference (p = 0.001). In prison 19 (15.1%) cases were found, the remaining majority of cases were reported from hospital 107 (84.9%) and the table 1 shows that there is differen-ce (p < 0.001). There was a significant difference in the drug induced hepatitis B (p < 0.001). The hi-story of Diabetes Mellitus and HIV/AIDS shows 12 (9.5%), 35 (27.8%) respectively and both group are statistically significant (p < 0.001). Smoking habit was found in 58 (46.0%) patients while 18 (14.3%) cases were ex-smoker and 50 (39.7%) were non-smoker, statistically smoking habit was in-signi-ficant (p = 0.126). History of alcohol intake was highly significant (p < 0.001), as 58 (46.0%) were actively habitual and 64 (50.8%) were non habitual and only 4 (2.2%) cases were found to be ex-drin-kers. 27 (21.4%) cases were found to be intraveno-us drug user (IVDU) and only 8 (6.3%) cases were Ex-IVDU’s. 90 (72.2%) cases were noon-users and table 1, shows highly significant value (p < 0.001). The majority of cases 70 (55.6%) had pulmonary TB (PTB), another 51 (40.5%) had extrapulmonary TB (EPTB), while the remaining 5 (4.0%) had both (PTB with EPTB). It was analyzed that hepatotoxi-city was induced in maximum number of patients 70 (55.2%) out of 126 by Isoniazid, which is si-gnificantly higher as compared to Pyrazinamide (p < 0.001). Rifampicin was second to follow in 43 (34.3%) patients and Pyrazinamide was found to cause hepatotoxicity in 13 (10.4%) patients.

As shown in Table 2, EPTB was reported in the lymph node in 11 patients (21.6%), bones and joints in 8 patients (15.7%), Genitourinary TB in 8 (15.7%) cases, TB meningitis in 7 patients (13.7%), Miliary TB in 6 (11.8%), Pleura TB in 5 (9.8%) , Intestinal TB in 3 (5.8%) and other sites in the remaining 3 patients (5.8%).

540



Table 1. Socio-Demographic Status of TB - Drug Induced Hepatitis Patients Variables TB-D.I.Hepatitis N. (%) X2 df P-value (%)

Age group Mean Age (Years) 43.71 ≤35 24 (19.0) 12. 195 1 < 0.001 >35 102 (81.0)Body Weight (Kg)Mean Weight 41.85Gender Male 75 (59.5) 9.952 1 0.002 Female 51(40.5)Race Malay 41 (32.5)

5.172 3 0.160 Chinese 69 (54.8) Indian 13 (10.3) Others 3 (2.4)TB Case

New TB Case 106 (84.1) 13.261 2 0.001 Relapse TB Case 20 (15.9)Place Hospital 107 (84.9) 34.276 1 < 0.001 Prison 19 (15.1)Hepatitis B Carrier

Yes 11 (8.7) 14.781 1 < 0.001No 115 (91.3)TB DM

Yes 12 (9.5) 15.980 1 < 0.001No 114 (90.5)TB HIV/AIDS

Yes 35 (27.8) 58.900 1 < 0.001No 91 (72.2)Smoking Habit

Yes 58 (46.0)5.712 3 0.126No 50 (39.7)

Ex-Drinker 18 (14.3)Alcohol Drinking Habit

Yes 58 (46.0)1.272 3 < 0.001No 64 (50.8)

Ex-Drinker 4 (2.2)Intravenous drug user (IVDU) Yes 27 (21.4)

83.439 3 < 0.001 No 91 (72.2) Ex-IVDU 8 (6.3)Use of Pyrazinamide Yes 90 (71.7) 9.157 2 < 0.001 No 36 (29.3) TB TypesPTB 70 (55.6)

49.093 2 < 0.001Extra PTB 51 (40.5)Both (PTB with Extra TB) 5 (4.0)



Table 2. Hepatotoxicity among patients with diffe-rent site of action of tuberculosis

SITE OF EPTBHepatotoxicity

Number Percentage TB Lymphadenopathy 11 21.6Bone & Joint TB 8 15.7Genitourinary TB 8 15.7TB meningitis 7 13.7Miliary TB 6 11.8Pleura TB 5 9.8Intestinal TB 3 5.8Others 3 5.8

During the study period, anti-TB-DIH was de-tected clinically and confirmed biochemically in 126 patients (8.2%). The time interval from onset of therapy and the detection of hepatotoxicity ran-ged from 2 to 120 days (Median: 15 days). 3 pati-ents (26.6%) had anti TB-DIH within 2 weeks of starting of therapy. 76 (60.0%) patients developed anti-TB-DIH within 30 days and only 17 (13.3%) patients developed anti TB-DIH after 30 days. The clinical manifestations of anti-TB DIH were nau-sea 86 (68.2%), anorexia in 80 (63.5%), vomiting in 72 (57.1%), loss of weight in 71 (56.3%), fever in 56 (44.4%), fatigue in 51 (40.5%) and abdomi-nal pain in 26 (20.6%) patients (Table 3).Table 3. Clinical presentations of DIH Patients

Variable Patients with antiTB DIH

(n = 45)

Number Percentage Anorexia 80 63.5 Loss of weight 71 56.3Nausea 86 68.2 Vomiting 72 57.1 Fever 56 44.4 Fatigue 51 40.5 Abdominal pain 26 20.6

In all the patients developing anti-TB DIH (n=126) a significant rise in ALT and AST levels was observed. The AST levels of 55 (43.3%) pati-ents were found to be up to 5 times the normal value, while in 44 (35.1%) patients levels were between 5-10 times the normal. In 27 patients (21.6%) AST levels were above 10 times the normal value (range 37-1024 IU/L). ALT levels of 70 (55.6%) patients

were found to be up to 5 times the normal value while in 42 (33.1%) patients it was in between 5-10 times the normal value and in 14 patients (11.3%) the values was above 10 times the normal value (range 83-1791 IU/L). Both AST and ALT levels showed significant rise compared to the baseline (p < 0.001). A rise in bilirubin level was detected in all 126 patients (range 0.4 - 25), 90 (71.4 %) of them had an increase of up to 5 mg/dL and 19 (15.4 %) patients had a rise between 5-10 mg/dL and anot-her 17 (13.2%) patients had more than 10 times the normal level of bilirubin (Table 4). Maximum level of enzyme elevation was 1400 IU/L and maximum rise in bilirubin was 25 mg/dL.Table 4. Alteration in liver function test in the pa-tients who developed anti TB DIH

Variable Patients with DIH (n = 126)

n Percentage

ALT ≤ 5 times ULN 5- 10 times > 10 times

70 4214

55.6 33.1 11.3

AST ≤ 5 times ULN 5- 10 times > 10 times

55 4427

43.335.121.6

ALP ≤ 5 times ULN 5- 10 times > 10 times

110124

87.3 9.8 2.9

Bilirubin ≤ 5 times ULN 5- 10 times > 10 times

9019 17

71.4 15.413.2

ULN: Upper limit of normal, N: Number, ALT: Alanine Aminotransferase, AST: Aspartate Aminotransferase ALP: Alkaline Phosphatase.

Discussion

The use of multidrug regimen for the treatment of TB, such as the combination of INH, RIF and PZA has been associated with an increased inci-dence of hepatotoxicity when compared with INH monotherapy used for antiTB prophylaxis.2,14 This study was conducted to assess the role of concu-

542



rrent risk factors that are contributing to the hepa-totoxicity. In our study, 8.2% of patients developed antiTB DIH, an incidence lesser than those reports available from India (8.0–19.8%) 2,26,22 and more than those from the West (4.3%).28,38 The variati-on in the incidence of antiTB DIH observed in this study may be due to differences in patients’ charac-teristics, regimens used, type of monitoring, and the diagnostic criteria defining hepatotoxicity.39,40

Anorexia, nausea, vomiting and fever within 15 days of initiation of therapy were the manife-stations of antiTB DIH observed in the study pati-ents. Similar observations were reported by other authors where antiTB DIH was reported within two weeks of initiation of therapy.6 This empha-sizes the need for close and more frequent moni-toring of patients in the first one month of anti-TB therapy. In the present study, the interval from the onset of hepatotoxicity to LFTs normalization was 2-120 days (Median: 15 days). In almost two third of patients, their LFTs normalized within 2 weeks of anti-TB therapy withdrawal, and similar obser-vations were reported by other author’s .25

Several studies suggested that increasing age is a potential risk factor for antiTB DIH.25,29-33 One study reported that the rate of antiTB DIH ranges from 2 to 8% as age increases, with an average of 5%.33 Other studies reported that hepatotoxicity ranges from 22 to 33% in patients older than 35 years compared with a range from 8 to 17% in the-se younger patients.29,33 Similarly in this study, 102 (81.0%) patients aged 35 and above developed an-ti-TB DIH compared to 24 (19.0%) patients below 35 years of age. Gender differences were highly observed in many studies and it has been reported that females are at increased risk of hepatotoxici-ty compared to males.30,31, 34 However study was conducted by Marzuki et al. (2008) in which they explained hepatotoxicity as a higher risk factor in male than female. In this study there is a signifi-cant (p=0.002) difference in the occurrence of he-patotoxicity between male and female gender. The results reflex that the incidence is higher in male which is in correlation to that reported by Marzuki et al. (2008). This variation between may be due to the fact that high percentage of patients in the present study were male (59.5%).

In some reported studies it has been reported that the incidence of PZA-induced hepatotoxicity

was significantly higher than that observed with the other first-line drugs for treatment of TB.27,25 However some studies have reported that the use of Pyrazinamide was not associated with incre-ased incidence of anti-TB DIH. 36 In the present study the use of Pyrazinamide significantly incre-ased anti-TB DIH in the study population. (p < 0.001). This may be attributed to difference in the study population or dosage regimen of Pyrazina-mide used in this study.

History of alcohol consumption was found to be significant contributory factor for the develo-pment of anti-TB DIH (p< 0.001), as 58 (46.0%) patients who developed anti-TB DIH had a history of alcohol compared to 64 (50.0 %) patients who did not have the history. In a study reported by Khalid M et al. (2007), 25% of alcoholics deve-loped anti-TB DIH whereas only 19.6 % of non-alcoholics developed anti-TB DIH.

HIV statuses of patients were assessed to study the effect of it on the development of anti-TB DIH. Marzuki et al. (2008) proved that HIV co-morbid condition is an independent risk factor for develo-ping anti-TB drug induced hepatitis. It was found that HIV status have significant effect on anti-TB DIH. Although number of patients with HIV was less than those without it (35 vs 91).

Many studies proved that potential risk factors for antituberculosis drug-related hepatic dysfun-ction included: 1) heroin addiction; 2) long-term steroid administration; 3) heavy alcohol consump-tion (>60 g·day-1); 4) hepatitis B carrier status (HBsAg positive); 5) diabetes mellitus; 6) eleva-ted serum urea (>6.7 mmol / L); 7) elevated serum creatinine (>120 μmol·L-1); 8) decreased serum albumin (<35 g / L); 9) decreased lymphocyte co-unt (<1.0×109·L); 10) elevated baseline ALT (>38 IU / L); and 11) elevated baseline bilirubin (>17 μmol /L).37,38

Similar to the result of other studies,28 it has been observed that patients with pre-existing liver dise-ase were at higher risk of antiTB DIH. Indeed, an increased risk of hepatotoxicity during treatment of TB was observed in patients with abnormal baseli-ne transaminases.25 In the current study 11 (8.7%) patients with pre existing liver function abnormali-ty developed hepatotoxicity. One Malaysian study concluded that EPTB was a significant risk factor for anti-TB drug induced hepatitis.11



Conclusion

In conclusion, anti-TB DIH is a fairly common among treated patients. The incidence of anti-TB DIH observed in this study is similar to those re-ported from western countries. Number of factors like age, gender, nutritional status and pre-existing hepatic disease were found to be significantly associated with anti-TB DIH. It has been observed that inclusion of PZA has significantly increased the rate of DIH in patients. Intensive monitoring of patients on anti-TB drugs with risk factors was identified in this study especially during the first month of treatment which might help to identify potential hepatotoxicity in time and to withdraw drugs at the right time to prevent severe hepato-toxicity which may be mortal.

Acknowledgements

The author (Amer Hayat Khan) would like to thank Institute of Post Graduate Studies, Univer-siti Sains Malaysia for providing USM fellowship to carry out the project successfully.

References

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2. Parasarthy, Raghupati, Sharma RG, Janardanam B, Ramachandran P, Santha T, et al. Hepatic toxi-city in South Indian patient during treatment of TB with short course regimen containing INH, RMP and PZA. Tubercle 1986;67:69-108.

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Corresponding author Amer Hayat Khan, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Malaysia, E-mail: [email protected]



Abstract

Purpose: The object of this study is to design a simple and sensitive enzyme immune assay met-hod for detecting Helicobacter pylori antigens in stool.

Materials/Methods: Stool samples were co-llected from 116 adults who were undergoing en-doscopic examinations and stomach biopsies and for whom histology and rapid urease tests were performed. A monoclonal antibody was used as the capturing antibody and a polyclonal antibody of rabbit origin conjugated with a peroxidase en-zyme as the tracer.

Results: In the histology and rapid urease tests, 21 of the 116 patients (18.1%) had positive results. H. pylori antigens were detected by the designed method in 19 of 21 cases (a sensitivity of 91%). Also, all of the 95 cases with negative results in the histology and rapid urease tests were negative for the stool antigen test (a specificity of 100%). For comparison, the sensitivity and specificity of the rapid immuno-chromatography test by the Certest Company were 95% and 99%, respecti-vely. The total correlations between the results of the designed ELISA test with the results of the ra-

pid test and the ELISA test of the Astra Company were 96% and 80%, respectively.

Conclusions: This non-invasive and economi-cal method for the detection of H. pylori antigens in stool can be considered as an alternative test that provides comparable reliability and validity to the histology and rapid urease tests for the de-tection of H. pylori infections.

Key words: Helicobacter pylori; Enzyme lin-ked immune sorbent assay (ELISA); Stool

Introduction

H. pylori is one of the most prevalent bacterial pathogens in humans. The incidence of infection in various countries ranges from 10% to 80%, and the average is 50%. Infection of the digestive tract by H. pylori is among the most widespread, chro-nic infections in humans (1), and the relationship between H. pylori infection and stomach ulcers, duodenal ulcers, and stomach adenocarcinoma has been established (2). Identification of this in-fection is performed by invasive and non-invasive methods. The invasive method involves endoscopy, after which other tests, such as rapid urease, histo-

diagnosis of helicobacter pylori infection by ELIsA stool antigen and comparison with the other diagnostic methods Maryam Razaghi1, Seyyed Mehdi Boutorabi2, Ali Mirjalili3, Shirin Norolahi4, Masoumeh Hashemi5, Mehrdad Jalalian6,7

1 Faculty of basic sciences, Islamic Azad University of Iran, Iran,2 Director of Research Lab., Pishtaz Teb Inc. Iran,3 Razi Vaccine and Serum Research Institute, Iran,4 Faculty of basic sciences, Islamic Azad University of Iran, Iran,5 Faculty of basic sciences, Payam e Nour University of Tehran, Iran,6 Research Center of Iranian Blood Transfusion Organization, Khorasan-e Razavi Blood Center, Iran,7 Department of Community Health, faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor D.E., Malaysia

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pathology, culturing, and Polymerase Chane Re-action (PCR), are used for identification purposes. The non-invasive methods include the respiratory urea test and serologic tests for detecting antibodies and antigens in feces (3). The utilization of invasive methods and endoscopy is limited in some patients, such as children and old patients. The respiratory urea test may be not applicable for children and pregnant women (4, 5). Therefore, it is essential to develop a simple and sensitive method that can be applied to all age groups with results comparable to those of the conventional techniques. The pre-sent study is devoted to developing a method for detecting H. pylori antigens in stool by the means of monoclonal and polyclonal antibodies.

Materials and Methods

Samples

The samples used in this study consisted of 116 stool samples that were collected from patients who were undergoing endoscopic examinations at the Digestion Research Center at the Shariati Hos-pital in Tehran. Histological and rapid urease tests were performed for each of the samples.

Stool Antigen Test for Helicobacter pylori

In order to investigate the bands of H. pylori antigens, they were studied applying sodium do-decyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method, and the bands were com-pared with a molecular weight marker. A 12% gel with a diameter of 0.75 mm was subjected to elec-trophoresis in a vertical manner with a constant voltage of 60 volts for two hours.

Immuno-blotting for studying the best Antibody Pair

Immuno-blotting was conducted using the extracted antigens by the acid-glycine method. The SDS-PAGE method was performed on a gel with a 10% concentration. 20 micrograms of anti-gen were added to each well and electrophoresis

was performed. The antigen was then transferred to nitrocellulose paper and kept frozen at -20 oC until the test time.

For immuno-blotting, the antibody with a con-centration of 5 µg/ml was diluted with a saline phosphate buffer solution that was supplemen-ted with 1% Bovine Serum Albumin (BSA), and, then, each antibody solution was added to a strip of nitrocellulose paper. In order to control the band presence, a mixture of several sera (pooled sera) that contained the antibody against H. pylori was diluted by phosphate buffer at a ratio of 1:100. Af-ter two hours of incubation at 37 oC, washing was performed using a saline phosphate buffer soluti-on with a 0.05% concentration of Polysorbate 20 (Tween 20). The utilization of enzyme conjugate as a tracer was dependent on the type of antibody.

Stool Antigen Test

Coating was conducted using mouse monoclo-nal antibody number 2 at different concentrations in a carbonate buffer with pH = 9.6 at 4 oC and at room temperature for 24 hours. Subsequently, the plates were washed with phosphate buffer contai-ning Tween 20, and the wells were blocked for one hour at room temperature by a blocker solution containing 1% BSA and carbohydrate. Afterwar-ds, the contents of the well were decanted and kept at room temperature for six hours until they were completely dry. These plates were maintained at 2-8oC in a foil that contained a desiccant until the experiment was to be performed. Rabbit polyclo-nal antibody number 4 was utilized as a tracer.

Sample Preparation

The collected stool samples were kept frozen at -20 oC. Before the test, the samples were thawed and thoroughly mixed so that the probable antigens could locate all over the stool sample constantly. In the next stage, 200 µl of the triple buffers, i.e., 0.05 M saline phosphate buffer, saline phosphate buffer containing 0.1% triton X-100, and 1.5 M glycine buffer with pH = 7.2 as a diluent, were added to a 5 mm diameter piece of completely premixed stool that was further mixed thoroughly



using a vortex mixer. Subsequently, the samples were centrifuged at 5000 rpm for 10 minutes. The supernatant was transferred to a 1.5-ml Eppendorf tube that was then used for the ELISA test. This sample was kept in the freezer at -20 oC until it was needed in the experiment.

Performing the Test

Six 100-µl volumes that contained different concentrations of the H. pylori antigenic soup of the supernatant of stool samples diluted by extrac-ting solutions were prepared. The concentrations of them were 0, 0.1, 0.5, 1, 2.5, and 5 µg/ml, and these solutions were added to each of the wells co-ated with monoclonal antibody.

Then, 50 µl of the anti-Helicobacter pylori polyclonal antibody conjugated with peroxiada-se enzyme were added to each well. The mixtu-re was incubated for two hours in a water bath at 37 oC. After incubution, the contents of the we-lls were removed, washed five times with 300 µl of washing buffer and then removed completely. Subsequently, 100 µl of a substrate chromogen solution containing tetramethyl benzidin and hy-drogen peroxide was added to all the wells, and they were incubated at room temperature for 15 minutes. The reaction was terminated using 100 µl of 1 N HCl, followed by reading the Optical Density by Asys ELISA reader at wavelengths of 450 nm and 630 nm as reference filters.

Investigation of fecal inhibitive effect

To study the fecal inhibitive effect, a certain amount of antigen was added to the feces super-natant of several negative and positive samples, in which the final concentration of antigen was 5.2 µg/ml. The resultant samples were tested along with the negative and positive samples without adding antigen, for antigen concentration.

Study of Cross-reaction

A microbial suspension with a concentration of 1*108 per ml containing the following bacteria

was added to the negative and positive samples; then, the samples were analyzed and compared to the samples prior to adding the bacterial suspension. The bacteria included: Klebsiella pneumoniae, Sal-monells group B, Shigella flexneri, Shigella sonnei, Citrobacter freundii, Citrobacter diversus, Escheris-hia coli, Pseudomona aeroginosa, Proteus mirabilis, Proteus vulgaris, Staphylococcus aureus, Staphylo-coccus epidermidis, and Enterococcus faecalis.

Analytic Sensitivity of the Assay

Using serial dilution of the antigen with phosp-hate buffer, the standard curve was drawn, and the amount of antigen that had an optical density (OD) greater than the cut-off value was defined as the assay detection limit.

Investigation of Assay Imprecision

Assaying was conducted several times on ne-gative, weak positive, and strong positive samples. Averages, standard deviations, and coefficients of variation (CV%) were calculated.

Comparative Test

In order to compare the results of the new method with other methods, we used an immu-no-chromatography kit from the Certest and the ELISA kit from the Astra Companies. Both were used according to the manufacturers’ instructions.

Results

The results of the SDS-PAGE method on the antigenic soup and staining with Coomassie Bri-lliant Blue revealed that this antigenic soup conta-ins most of the bacterial antigens.

The results of immuno-blotting of existing an-tibodies indicated that only two antibodies, i.e., number 2 and number 4, can identify most signi-ficant antigens of the H.pylori. Antibody number 5 only identified antigens in the range of 60 to 70 kD; it is noticeable that most antigens of H. pylori

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that have cross-reactions are located in this region. Furthermore, antibodies number 1 and number 3 can identify a few antigens.

Fig. 1. Immuno-blotting of anti-Helicobacter pylori antibodies

Row 1: anti-H. pylori serum; 2: monoclonal antibody number 1; 3: monoclonal antibody num-ber 2; 4: polyclonal antibody number 3 of rabbit origin; 5: polyclonal antibody number 4 of rabbit origin; Row 6: monoclonal antibody number 5.

Investigating the appropriate buffer for the extraction of antigens from stool

There is no advantage to use the phosphate buffer containing triton X-100 and glycine buffer compared to phosphate buffer alone. (Table 1).

Investigating the inhibitive effect of fecal material on assays

The data indicated that no interfering inhibitor existed in the samples to result in false negative values (Table 2). This was obtained by adding some amount of antigen to stool so that the final concentration of antigen in the supernatant of the stool samples was 2.5 µg/ml, comparing the OD antigen with the same concentration in the phosp-hate buffer as the diluent for the feces sample, and calculating the recovery level of antigen.

Investigating the Cross-reaction

After addition of the microbial suspension, no increase was observed in the ODs of the negative samples. Furthermore, no interfering effect was noticed in the positive samples subsequent to the addition of the microbial suspension.

Table 1. Results of tests of different buffers for extracting antigens from fecesGlycine buffer

OD unitPhosphate buffer containing

triton OD unitPhosphate buffer

OD unitPositive sample 1.7 1.9 2.2Negative sample 0.05 0.02 0.03Negative sample containing 5 µg of antigen 1.6 1.7 2.0

Antigen sample in buffers 2.3 2.4 2.7Only buffers 0.04 0.02 0.03

Table 2. Results of the investigating the interfering effect of stool composition on antigen assay

Recovery mean (%)

OD unit of stool samples after adding

2.5 µg antigen

OD unit of phosphate buffer after adding 2.5 µg

antigen

OD unit of native samples

Negative sample 1 93.5 1.31 1.4 0.03Negative sample 2 91.4 1.28 1.4 0.025Negative sample 3 95.7 1.34 1.4 0.017



The mean OD of the samples that were negati-ve in the histology tests and the rapid urease tests was 0.04 with standard deviation of 0.02. The cut-off OD was calculated adding three SD to the mean OD of the negative samples, which yielded the value of 0.10. All samples with OD values gre-ater than 0.10 were considered positive.

Fig. 2. Comparison of the ODs of negative and positive samples using the designed ELISA met-hod

Among 116 samples obtained from patients un-dergoing endoscopic examinations, 21 cases had positive results in the histology and rapid urease tests. Nineteen of these 21 samples (90%) were also positive in the stool antigen test. Among the 95 samples with negative results in the histology and rapid urease tests, all (100%) were negative in the stool antigen test. According these results, the sensitivity and specificity of the designed assay method were calculatedly 90 and 100%, respecti-vely. The positive and negative declarative values of the designed assay were 100% and 98%, respec-tively. The sensitivity and specificity of the rapid kit produced by the Certest Company were 95% and 99%, respectively. Furthermore, the correlati-ons of the designed assay for all samples with the rapid kit and with the ELISA kit were 96% and 80% respectively (Table 3).

Analytical Sensitivity of Assay

Using serial dilutions of antigen in phosphate buffer and according to the cut-off value, the lowest recognizable concentration using this assay was de-termined to be less than 0.1 µg/ml of antigen.

Fig. 3. Standard curve for antigen assay in stool by the ELISA method

According to the results obtained, this assay has an acceptable level of imprecision that is com-parable to levels observed with other existing kits (Table 3).

Discussion

Regarding the wide-spread incidence of Heli-cobacter pylori infections in developing countries, including Iran, the correct and proper identificati-on of this infection and the extermination of this bacterium can change the normal period of the di-sease through decreasing the rate of its recurrence in treated patients.

Histological evaluation is considered to be the gold standard for identifying H. pylori bacteria. Nevertheless, it suffers from limitations, such as need for an endoscopic examination, which is unpleasant for patients; in addition, biopsy sam-

Table 3. Results of imprecision tests of the antigen assay in feces by the ELISA methodMean OD Standard deviation Coefficient of Variation (CV), %

Negative sample 0.07 0.009 12.8Weak positive sample 0.56 0.004 7.8Strong positive sample 1.92 0.8 4.2

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ples may not be adequate for identification pur-poses. Culturing is another invasive method that has a limited role in the identification of infecti-ons due to difficulty in growing the bacteria and their slow rate of growth in the culture medium (8). Polymerase Chain Reaction (PCR) is consi-dered as a method with desirable sensitivity and specificity. However, it is expensive and may not be used in all laboratories. It is not applicable in case of treatment, and also produces false posi-tive results sometimes. The rapid urease test, on the other hand, is a fast and available technique that is applied to biopsy samples; nevertheless, it has low specificity, and the results it produces are influenced by antibiotics, bismuth compounds, or proton-pump inhibitors. Among the non-invasive methods, serology is a simple and fast option that is also inexpensive. It is practical for use in regi-ons with low incidences of H. pylori infections, and it should be performed and considered as the second priority of diagnosis tests, due to its low predictive value (9).

A non-invasive, simple, inexpensive, and easi-ly applicable method will be very useful in sol-ving the existing problem of identifying H. pylori infection. Taking advantage of assaying H. pylo-ri antigens in stool by the ELISA method is an appropriate solution for this problem (10).

In 2003, Andrews et al. studied 111 adult dyspepsia patients under endoscopy and post-biopsy, and they compared the results regarding the presence of H. pylori antigens in stool sam-ples using three types of ELISA kits. Cultures, hi-stopathological tests, and rapid urease tests were conducted as the gold standard for investigating infection status. The three kits used were Premier Platinum, Femtolab, and Diapro. In the first kit, a polyclonal antibody is used, while the other two kits make use of monoclonal antibody. The three mentioned kits had sensitivity values of 63.6%, 88%, and 56%, respectively. Also, their specificity values were 92.6%, 97.6%, and 97.6%, respecti-vely (11). We acquired better results by using a monoclonal antibody with an incubation time of two hours at 37 oC.

In 2004, A.S. Chisholm et al. surveyed 112 pa-tients using the Premier Platinum HpSA ELISA kit and obtained sensitivity and specificity values of 91% and 96%, respectively. Additionally, in the-

ir research, they utilized the ImmunoCard STAT! HpSA kit and reported sensitivity and specificity values of 91% and 95%, respectively. They con-sidered histological methods and culturing as the gold standard (12).

In 2005, E.M. Gulcan and his colleagues per-formed a study on 80 patients under endoscopy by the HpSA ELISA method (Premier Platinum kit) and obtained sensitivity and specificity values of 99% and 96%, respectively (13).

In our research, the sensitivity and specificity of the designed kit were determined to be 90% and 100% and the test is accomplished in 2.25 hours. We compared our assay with ImmunoCard HpSA kit from the Certest Company and the ELISA kit from the Astra Company. For the ImmunoCard kit, the resulting sensitivity and specificity valu-es were 95% and 99% respectively; whereas the values for the ELISA kit were 85% and 90%, res-pectively. It should also be noted that the histo-logical and rapid urease tests were considered as gold standard in the present study.

Conclusions

This non-invasive and economical method for the detection of H. pylori antigens in stool can be considered as an alternative test that provides comparable reliability and validity to the histology and rapid urease tests for the detection of H. pylori infections.



Reference

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3. Hardin. F. j. wright. R.A. (2002) Helicobacter pylo-ri: Review and update. Haspital physician: 23-31

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6. Weingart. V , Russmann, H , Koletzko. S et al (2004). Sensitivity of a novel Stool Antigen test for Detection of Helicobacter Pylori in Adult outpati-ents before and after Eradication Therapy. J clini-cal microbiology; 42 (3) : 1319-1321

7. Barden. B, Posselt. H-G, Ahrens. P et al (2000). New immunoassay in stool provides an accurate no-nivasive diagnostic method for Helicobacter pylori screening in children. Pediatrics (106) 115-117

8. Mitcehll and F. Megraund Epidmiology and digno-sis of Helicobacter pylori infection. Helicobacter. 2002. 7 Suppl 1:8-16

9. N. Vakil and D.Varia Nonivasive test for the Dia-gnosis of H.pylori Infection. Reviews In gastrologi-cal Disorders 2004. 4: 1-6

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Corresponing author Maryam Razzaghi, Faculty of basic sciences, Islamic Azad University of Iran, Iran, E-mail: [email protected]

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Abstract

Aims: To study the clinical characteristics of acquired immune deficiency syndrome (AIDS) patients with mycobacterium tuberculosis (MTB) or nontuberculosis mycobacterium (NTB) co-in-fections.

Methods: Mycobacterium screening was con-ducted by examining the sputum smear, bacteri-al sputum cultures, and bacterial blood cultures in 683 AIDS patients. The patients’ clinical data were then analyzed.

Results: Out of 683 patients, 380 qualified (380/683, 55.6 %) for the study by passing the sputum smear examination, and the mycobac-terium-positive rate was 5.8 % (22/380). A total of 392 and 40 patients (392/683, 57.4%; 40/683, 5.9%) qualified by passing the examination for bacterial cultures of sputum and blood, respective-ly. The combined MTB-positive percentage was 15.5% (67/432), and the NTM-isolation rate was 30.2% (29/96). There were statistically significant differences (χ2 = 53.29, P > 0.05). AIDS patients with MTB/NTM associated pulmonary diseases presented mainly with fever, cough, expectorati-on, and dyspnea. However, there were no obvi-ous differences between patients with MTB and NTM infections. The percentage of patients with abnormal chest X-ray radiographs was 43.8% (299/683). However, 32 out of 310 patients with normal chest radiographs were mycobacterium-positive via bacterial culture of sputum or blood. The percent of mycobacterium-positive cultures was as high as 63.5% (61/96) in patients whose CD4+ T lymphocyte counts were <50 cells/μL.

Conclusions: AIDS patients with MTB/NTM infections are difficult to diagnose, as they have complicated clinical presentations, low detectable rate for sputum examination, and atypical chest ra-diographs. Moreover, there is an increasing trend of NTM co-infection in AIDS patients.

Key words: AIDS, Mycobacterium tuberculo-sis, Nontuberculosis mycobacterium

Introduction

Acquired immune deficiency syndrome (AIDS) with tuberculosis (TB) has been a public health issue world-wide, which posing a major risk to human life. At the end of 2007, there were abo-ut 700,000 living human immunodeficiency virus (HIV)/AIDS patients in China, comprising an in-fection rate of 0.05% among the total population in the country. Among which, there are 85,000 existing AIDS patients, 50,000 new HIV infection patients, and 20,000 patients who have died from AIDS in 2007 alone (1).

With respect to the epidemiology of tuberculo-sis, there are a total of two billion mycobacterium tuberculosis-infected individuals, as well as 20 million active tuberculosis patients, with eight mi-llion new cases every year. Furthermore, three mi-llion patients die of tuberculosis across the world every year. In China, TB presents with a high in-fection rate, high morbidity rate, high mortality rate, high drug resistance rate, and a high HIV/TB mixed infection rate. There are a total of 550 milli-on TB-infected Chinese patients, and the infection rate is increasing up to 0.72% of the total popula-

Characteristics of AIds Patients with mycobacterium Co-Infection: A Pilot Clinical TrialRuichao Lu1*, Yong Zhang1, Weiping Cai 2, Hongzhou Lu3

1 Department of Infectious Diseases, Guangxi Longtan Hospital, China2 Department of Infectious Diseases, Guangzhou Eighth People’s Hospital, China3 Shanghai Public Health Clinical Center Affiliated to Fudan University, China



tion in China. About five million patients have ac-tive TB (367 per 10 million), and 1.5 million (160 per 10 million) patients are TB-positive via sputum smear examinations. Moreover, 130,000 patients die from TB every year, and among whom 18.5% carry initial signs of drug resistance and 46.5% have already acquired drug resistance. About 1/3 of AIDS patients are complicated by TB, which is one of the most common opportunistic infections in AIDS patients, and TB has resulted in the death of about 1/3 of AIDS patients. TB has further re-duced the survival rate of AIDS patients by 50%.

Mycobacterium infection is one of the most common opportunistic infections among AIDS patients, and attention has been paid to AIDS with mycobacterium co-infection in various countries. Results from a nationwide tuberculosis epidemi-ological survey in China in 2000 indicated that there was an increasing trend of NTM infections. The infection rate was as high as 11.1% for some isolated strains (2). However, there are seldom lar-ge-sample surveys on the epidemiology and clini-cal characteristics of mycobacterium infection in AIDS population in China. Therefore, we report this pilot clinical trial.


Inclusion and exclusion criteria

The inclusion criteria were as follows: male or female patients 18-69 years of age who were HIV-1 antibody-positive via ELISA and western blotting and who were positive for TB (MTB-po-sitive via sputum smear examination or bacterial sputum culture) without having undergone any anti-TB therapy, or who presented with NTM-po-sitive indications via bacterial culture of sputum. All patients gave informed consent and agreed to follow-up studies.

AIDS patients with other opportunistic compli-cations besides TB (such as HIV lymphoma), pre-gnant or lactating patients, and patients suffering from severe mental or neurological diseases were excluded from this study.

Diagnostic criteria

The AIDS diagnostic criteria abided by Gui-delines for diagnosis and treatment of HIV/AIDS in China (2005) (3). The TB diagnostic criteria complied with the Tuberculosis diagnosis and treatment in Ref (4), which defined the diagnosis of TB as (1) MTB positive via sputum and pleu-ral effusion examinations or diagnosis via lymph node and pulmonary examinations; (2) obvious TB toxic symptom; (3) at least moderately posi-tive for anti-TB antibody or PPD; (4) typical TB chest radiograph; and (5) achieving therapeutic ef-fect via anti-TB therapy. TB patients were diagno-sed if they met item (1) or three of the other four items. The NTM diagnostic criteria obeyed for di-agnosis and treatment of NTM (5), which defined the diagnosis of NTM as (1) performance of pul-monary symptoms, tubercles or cavity shadows in chest radiograph, or multifocal bronchiectasis with small nodules; (2) duly excluding other dia-gnosis; (3) at least two independent sputum cultu-res or one bronchial brushing, or bronchoalveolar lavage fluid cultures presenting with positives; and (4) diagnosis via pathological examination.

Screening

The confirmed AIDS patients underwent re-gular chest x-ray radiography, mycobacterium sputum smear examination (if they had produc-tive coughs), mycobacterium culture of sputum (if sputum smear was negative for mycobacteria), mycobacterium culture of blood (if the patient had no sputum), pathological examination (if the pa-tient had co-morbid indications), and cultures of secretion and other types of specimens.

Cultures and tests

The sputum smear was treated by using Zie-hl-Neelsen staining and then observed under a microscope. The isolation, culture and characte-rization of MTB or NTM from sputum or blood samples were performed in accordance with pro-cedures of Tuberculosis diagnosis (6). A 3D au-tomatic microbiological cultivator (BioMérieux

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Bact/ALERT, France; relevant reagents from BD, USA) was used in the TB reference laboratory of our hospital. Positive mycobacterium was classi-fied into MTB and NTM, but we did not conduct further serotyping on the positive NTM samples due to limitations in our test conditions.

Cases and clinical data

The 683 HIV patients in our study came from different parts of Guangxi province. There was a total of 417 males and 266 females, and the pati-ents’ age ranged from 18-69 years old (mean age 31.4 ± 8.3). Based on the patients’ accounts, 355 (52%) patients contracted HIV through intraveno-us drug, 307 (45%) through sexual transmission, and 21 (3%) through uncertain pathways. Each pa-tient underwent sputum smear examination three times (night, morning, and immediate sputum), and mycobacterium cultures of sputum or blood once. The sputum specimens were collected in the following schedule: night, morning, and immedia-te sputum specimens (total three copies) were co-llected and examined. If the patients had no night sputum, the sputum specimen was collected again within 2-3 hours after the morning collection, or otherwise two copies of immediate sputum were collected. During the follow-up period, morning and night sputum samples (total two copies) were examined. Qualified sputum specimens should present with pyoid, caseous or pyomucoid appea-rances and the amount of expectoration fell within 3-5 mL. The container for sputum collection was an international plastic case or wax coated sputum bottle with approximately 4 cm in diameter and 2 cm in height. Detailed patient clinical information was then recorded.

Statistics analysis

Enumeration data underwent χ2-test, and the software SPSS 13.0 were used in all statistical analysis.

Results

Screening results

Among the 683 patients, 380 patients (55.6%, 380/683) qualified for the sputum smear examina-tion, and of whom 22 (5.8%, 22/380) were myco-bacterium-positive. Next, the sputum samples from smear-negative patients were cultured. The culture results showed that 392 patients (57.4%, 392/683) qualified, and of whom 91 (23.2%, 91/392) were mycobacterium-positive. This in-cluded 62 strains of MTB and 29 strains of NTM. Meanwhile, patients negative for sputum cultures were further assessed with blood cultures. This test resulted in 40 qualified patients (5.9%, 40/683), of whom 5 (12.5%, 5/40) carried MTB-positive stra-ins. Hence, there were a total of 96 strains of iso-lated mycobacteria, including 67 strains of MTB [the positive rate was 15.5%, (62 + 5) / (392 + 40)] and 29 strains of NTM (the isolation rate was 30.2%, 29/96). The differences were significant (χ2 = 53.29, P < 0.05). The characteristics of the sputum smear samples are shown in Table 1, and the results of the MTB and NTM sputum or blood cultures are shown in Table 2.Table 1. Sputum smear case number and its pro-portion (%)Sputum sample No. of Cases Proportion (%)Smear + 22 22/380 (5.8)Smear- 302 302/380 (79.5)Smear – but culture+ 56 56/302 (18.5)

Table 2. The results of bacterial cultures of sputum and blood for MTB and NTM (strain number/%)

Sample (case No.) MTB NTM In total

Positive No. (%) Positive No. (%) Positive No. (%)Sputum culture (392) 62 (15.8) 29 (7.4) 91 (23.2)Blood culture (40) 5 (12.5) 0 5 (12.5)



Clinical data

There were a total of 96 mycobacterium-infec-ted cases. Among the 67 patients with MTB, 51 were males and 16 were females, all within 29-57 years of age. Among the 29 patients with NTM, 21 were males and 8 were females, all within 21-63 years of age. The results from NTM isolation among the different age groups are shown in Table 3. Through a statistical test, we found no statisti-cally significant differences in the NTM separati-on rate among the different age groups (χ2 = 2.46, P > 0.05). Furthermore, patients from both groups presented with fever, cough, expectoration, dys-pnea, chest pain, and a reduction in physical qu-ality in various degrees. The detailed clinical pre-sentations of the two groups are shown in Table 4 for comparison.

Chest radiographs

All 683 patients underwent chest x-ray ra-diography. Results indicated that 384 (56.2%, 384/683) patients showed normal chest radio-graph, and of whom 310 further underwent myco-bacterium culture of sputum or blood. This resul-ted in the identification of 32 positive cases (the positive rate was 10.3%, 32/310). There were 299 (43.8%) patients with an abnormal chest radio-graph, and of whom 63 (21.1%, 63/299) showed patterns typical of a TB positive chest radiograph, and 236 (78.9%, 236/299) more patients were

suspected of having TB. Among patients with ab-normal chest radiographs, 106 of them presented with isolated, patchy and poorly defined density in the bilateral inferior lobes of the lung. Others also presented with a high lesion center density as well as haziness in ambient density. There were 74 patients with patchy haze shadows in the right inferior lobe of the lung, 29 patients with bilate-ral superior lobes lesions, 27 with bilateral hilar diffusive ground-glass opacity, 23 with miliary nodular opacity, 17 with either an enlargement of the hilar shadows or widened superior mediastinal shadows, and 23 with pleural effusion.

T lymphocyte subpopulation

Among the 683 patients, there were 262 pa-tients whose CD4+ T lymphocyte counts were < 50 cells/μL, and of whom 61 (61/96, 63.5%) pre-sented with positive MTB/NTM cultures. A total of 235 other patients showed CD4+ T lymphocyte counts within 50-100 cells/μL, and of whom 24 (24/96, 25%) presented with positive MTB/NTM cultures. Finally, 186 patients showed CD4+ T lymphocyte counts >100 cells/μL, and of whom 11 (11/96, 11.5%) presented with positive MTB/NTM cultures. Detailed proportions of the MTB/NTM cases with respect to the various values of CD4+ T lymphocyte counts are shown in Table 5.

Table 3. Isolation of NTM among different age groupsAge (year) No. of strains characterization No. of NTM isolation Isolation rate (%)

0- 0 0 0.015- 19 7 36.830- 23 8 34.845- 26 9 34.660- 28 5 17.9

Total 96 29 30.2

Table 4. Clinical symptoms of 96 mycobacterium pulmonary disease cases (No. %)Group/case No.

Fever No. (%) Cough No.(%) Expectoration

No.(%)Dyspnea No.(%)

Chest painNo.(%)

MTB/67 61/91.0 63/94.0 52/77.6 34/50.7 36/53.7NTM/29 23/79.3 25/86.2 27/93.1 19/65.5 16/55.2

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Discussions

There is an increasing number of HIV/AIDS patients with mycobacterium co-infections, whi-ch presents a significant challenge for effective diagnosis and treatment. The key to a successful treatment is early diagnosis. Thus, mycobacterium testing is an important diagnostic method for HIV/AIDS patients. By analyzing 683 patients, we fo-und that only 22 (5.8%, 22/380) HIV/TB patients were mycobacterium-positive through sputum smear examination. We speculated that the low positive rate was related to the newly acquired acid resistance of the MTB strains trapped inside the macrophage. In addition, there were no differen-ces in clinical presentations between patients with MTB and NTM infections, and the MTB/NTM associated intrapulmonary and extrapulmonary diseases presented mainly with cough, low grade fever, asthenia, and weight loss without other pa-thological changes. Since TB is a latent infection, latent TB infection (LTBI) in HIV/AIDS patients is very easy to convert to overt TB. This further increases the difficulty of early TB diagnosis in AIDS patients. Hence, the laboratory capacity of mycobacterium detection should be improved as soon as possible. For example, in comparison with the traditional testing method, enzyme-linked immunospot assay (ELISPOT) can detect specific cellular immune responses to the MTB-specific antigens EAST-6 and CFP-10. This may improve the accuracy and sensitivity of early diagnosis of TB in AIDS patients.

Our obtained data showed 67 MTB positive sputum cultures (67/432, positive rate 15.5%) and 29 positive NTM sputum cultures (29/96, isolati-on rate 30.2%). The percentage of positive myco-bacterium sputum cultures was as high as 63.5% (61/96) in patients whose CD4+T lymphocytes were < 50 cells/μL, indicating that AIDS patients

whose CD4+T lymphocytes are < 50 cells/μL sho-uld undergo conventional mycobacterium culture regardless of whether the chest radiograph shows any abnormality. Meanwhile, our data also de-monstrated that the positive rate for MTB sputum culture (15.5%) has a higher sensitivity rate than that for the sputum smear examination (5.8%). Some reports (4, 6) have indicated that a positi-ve result may come from as little as 10-100 living bacilli cultures, while at least 5000-1000 acid-fast bacilli per ml were needed to produce positive results in a smear examination. In addition, the isolated strains from the cultures could undergo mycobacterium strain identification, drug sensi-tive and molecular epidemiological testing. The only caveat is that mycobacterium cultures requ-ire a long incubation period. Thus, mycobacteri-um culture plays a very important role in guiding clinical diagnosis and treatment. This finding was similar to that obtained from a multi-center study in Latin Africa (7).

The rate of mycobacterium infections among HIV/AIDS patients is sharply rising, but AIDS patients with mycobacterium co-infection are ra-rely reported by literatures in China or abroad. Therefore, we performed a large-sample scree-ning for TB among AIDS patients for the first time in China. We identified 29 (29/96) positive NTM cultures, which gave us an isolation rate of 30.2%. This was higher than the 11.1% reported in a nationwide TB epidemiological survey conduc-ted in China in 2000. A previous study by the tu-bercle bacillus reference laboratory of our hospital showed that HIV(-) patients had an extremely low NTM detection rate, which may have been falsely low due to a previous lack of understanding and limitations in strain identification. In addition, it has also been shown that the rising trend of NTM infection was mainly due to the AIDS epidemic. Some reports have further indicated that the ge-

Table 5. Proportion of MTB/NTM cases referred to the segment values of different CD4+ T lymphocytes (No./%)

T lymphocyte subpopulation (cells/μL)MTB NTM

Case No.% Case No. %CD4+ T lymphocyte <50 46 75.4 (46/61) 15 24.6 (15/61)50< CD4+ T lymphocyte <100 15 62.5 (15/24) 9 37.5 (9/24)CD4+ T lymphocyte >100 6 54.5 (6/11) 5 45.5 (5/11)



neral trend of NTM infection is higher in the So-uthern of China than in the Northern regions. The infection rate is also higher in the coastal regions, as well as in more moderate climate regions. The Chinese nationwide TB epidemiological survey result in 2000 showed that the drug resistant rate of NTM was 95.9% and the multidrug resistance rate was 83.7%. Both results indicated that NTM was highly resistant to traditional anti-tuberculo-sis medications, a conclusion that should be taken very seriously (2, 8).

The chest radiographs of AIDS patients with TB were very atypical, but there were no cha-racteristic differences in clinical presentations between patients with normal and abnormal chest radiographs. Conventionally, patients with normal chest radiographs are diagnosed via sputum sme-ar examinations and bacterial cultures. Our study showed that 56.2% of patients showed normal chest radiographs and 43.78% showed abnormal radiographs. Among the patients with normal chest radiographs, we identified 32 positive sputum cul-tures for TB. However, among patients with ab-normal chest radiographs, we identified TB ima-ging changes in only 21.1% of the patients. The chest radiographs of AIDS patients with TB often presented with early HIV-infection pulmonary tu-berculosis. The pulmonary lesions were common-ly located in the superior lobe of the lungs. The lesions often showed bilateral infiltration, cavity formation, as well as lung collapse and shrinkage that were associated with pulmonary fibrosis. The clinical presentations changed further when the HIV infection started to suppress the body’s im-mune function. For instance, x-ray images showed atypical changes mainly in the mid inferior lobe of the lungs, and there were less cavity formations and more pleural effusions as well as mediastinal lymph node enlargements (8).

In general, AIDS with MTB/NTM co-infecti-ons result in complicated clinical presentations, and often elude diagnosis due to its lack of spe-cificity in symptom presentation (9, 10). Therefo-re, TB patients with poor treatment effects should undergo CD4+ T lymphocyte counts as well as se-rological testing for anti-HIV antibodies to make a definite diagnosis and begin treatment as ear-ly as possible. Furthermore, treatment for AIDS patients with MTB/NTM co-infections is often

accompanied by anti-retroviral therapies. These are multi-class medications that can bring about more toxic side effects, which is an additional po-int of consideration that should be carefully asse-ssed by the clinician.

Acknowledgments

This work was supported by “Eleventh Five-Year” National Science and Technology Major Project (No. 2008ZX10001-008).

References

1. State Council AIDS Working Committee Office; United Nations Theme Group on HIV/AIDS in China; A Joint Assessment of HIV/AIDS Preventi-on, Treatment and Care in China. 2007;

2. Technique Guide Team for National Tuberculosis Epidemiological Investigation; National Tubercu-losis Epidemiological investigation Report in 2000. Chin J Anti-Tuberculosis Assoc. 2002;24:102.

3. Guidelines for diagnosis and treatment of HIV/AIDS in China (2005). Chin Med J (Engl). 2006;119:1589-608.

4. National Collaborating Centre for Chronic Condi-tions. Tuberculosis: clinical diagnosis and manage-ment of tuberculosis, and measures for its preven-tion and control. London (UK): Royal College of Physicians. 2006:215.

5. Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, Holland SM, Horsburgh R, Huitt G, Iademarco MF, Iseman M, Olivier K, Ruoss S, von Reyn CF, Wallace RJ, Jr. , Winthrop K. An of-ficial ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial disea-ses. Am J Respir Crit Care Med. 2007;175:367-416.

6. American Thoracic Society, CDC, and Infectious Diseases Society of America. Treatment of Tubercu-losis. Am J Respir Crit Care Med. 2003;67:603-62.

7. Robledo JA, Mejia GI, Morcillo N, Chacon L, Ca-macho M, Luna J, Zurita J, Bodon A, Velasco M, Palomino JC, Martin A , Portaels F. Evaluation of a rapid culture method for tuberculosis diagnosis: a Latin American multi-center study. Int J Tuberc Lung Dis. 2006;10:613-9.

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8. Xiang XP, Li XW , Xu KQ. HIV Treatment [M]. He-fei: Anhui Science and technology Press; 2007.

9. Benson CA, Kaplan JE, Masur H, Pau A , Holmes KK. Treating opportunistic infections among HIV-infected adults and adolescents: recommendati-ons from CDC, the National Institutes of Health, and the HIV Medicine Association/Infectious Di-seases Society of America. MMWR Recomm Rep. 2004;53:1-112.

10. Havlir DV, Getahun H, Sanne I , Nunn P. Opportu-nities and challenges for HIV care in overlapping HIV and TB epidemics. JAMA. 2008;300:423-30.

11. 11. Gordana Cavrić, Klara Jurić, Josip Vincelj, Duško Kardum, Dubravka Bartolek, Snježana Fi-ličić, Romana Katalinić, Hyponatriemia in patient with Aids, HealthMED 2008; 2: 108-110

Corresponding author Ruichao Lu, Department of Infectious Diseases, Guangxi Longtan Hospital, China, E-mail: [email protected]



Abstract

The purpose of research was to ascertain the impact of diet and physical activity (a moderate walk exercise) in the treatment of metabolic syn-drome during the six month period.

We analyzed, 422 patients previously diagno-sed with metabolic syndrome (341 males and 81 females). Diagnostic criteria used for diagnosing the metabolic syndrome were applied in accor-dance with NCEP ATP III (Third Adult Treatment Panel), such as: waistline perimeter, BMI (body mass index), triglyceride level, HDL-C choleste-rol level, systolic and diastolic arterial pressure, and serum glucose level.

During the 6 month’s treatment with integra-ted diet intake that consisted of all necessary com-ponents, we have observed the energy intake and consumption necessary for the daily reduction of 200 cal, followed in parallel with physical activity (moderate walking 1 hour a day, 5 times a week) for each subject of the research that has lasted du-ring the period 2003 – 2008 years.

Descriptive and discriminative analysis of re-sults has indicated that treatment with diet and physical activity has had an impact in: waistline decrease by 6,05 cm or 5,50% in males, and 4,92 cm or 5,10% in females; body mass index (BMI) decrease by 1.78 or 6.20% in males, and 2.3 or 8,16% in females; serum triglyceride level decre-ase by 0,35 mmol/L or 16,28% in males, and 0,27 mmol/L or 13,30% in females; increase of serum HDL-C cholesterol by 0,48 mmol/L or 34,78% in males, and 0,06 mmol/L or 4,28% in females;

systolic arterial pressure decreased by 15 mmHg or 10,18%, and diastolic arterial pressure by 8,74 mmHg or 9,47% in males, and systolic arterial pre-ssure decreased by 7,39 mmHg or 5,17%, and di-astolic arterial pressure decreased by 5,18 mmHg or 5,75% in females; the level of blood glucose decreased by 0,45 mmol/L or 7,04% in males, and by 0,64 mmol/L or 9,92% decreased in females.

In order to improve symptoms of metabolic syndrome, it is necessary to keep on with healthy diet and physical exercise that means the change of lifestyle.

Key words: metabolic syndrome, diet, physi-cal activity, walking.

Introduction

Main factors that influence the onset of meta-bolic syndrome are: genetic susceptibility, poor physical activity, and wrong dietary habits.

All experts don’t agree nor with the name, nei-ther with the definition of metabolic syndrome, even though a cluster of metabolic abnormalities such as: abdominal fat, insulin resistance, glucose intolerance, lipid disorders, and hypertension are conditions present in the body and make up the traits of this syndrome.

Clinical manifestations and disorders that deri-ve from this syndrome are: vascular inflammation, hyper coagulation, hyperuricemia and microalbu-minuria. Several names have been used for this syndrome, such as: “insulin resistance syndrome”, “syndrome X”, “plurimetabolic syndrome” and

Treatment benefits on metabolic syndrome with diet and physical activityGani Dragusha1, Abdulla Elezi², Shpend Dragusha4, Daut Gorani1, Luljeta Begolli³, Valton Sahiti4 1 Internal Medicine Clinic, Cardiology Department, University Clinical Center Prishtina, Kosovo,² The Faculty of Physical Culture, Kosovo,³ Biochemistry Institute, University Clinical Center Prishtina, Kosovo,4 Kosovo Research Project on Metabolic Syndrome, Kosovo.

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recently often used name is “cardio metabolic di-sease”(1)

Crepaldi has noticed that overweight, lipid dis-orders, glucose intolerance and hypertension are present at the same time among many people, that has lead German researchers to dub this condition as metabolic syndrome (2)

It is necessary to distinguish the metabolic syn-drome as diagnosing category from the metabo-lic syndrome as pathologic entity, the latter being common denomination for the similar metabolic abnormalities (3, 4).

During the last 25 years various studies have offered evidences proving that insulin resistance at the muscles level and fatty tissue are common disorders which increase the probability for deve-lopment and onset of not only diabetes mellitus type 2, but also the cardiovascular diseases among population. (5).

The World Health Organization (WHO) in 1998 has approved the official name and definiti-on for the metabolic syndrome (6).

The metabolic syndrome is a genuine multi-component disorder developed due to the lifestyle and impact of environment, but also the genetic susceptibility has important role in this disorder. National Cholesterol Education Program – Adult Treatment Panel III, NCEP-ATPIII held in 2001 has recognized and defined the metabolic syndro-me as group of abnormal conditions that increases the risk of cardiovascular diseases and diabetes mellitus type 2 (7)

The NCEP-ATP III program also underlined the impact of waistline size in the manifestation of metabolic syndrome.

Very significant derangements of metabolic syndrome are glucose intolerance and insulin resi-stance. The latter is considered as one of the most important factors causing the metabolic syndrome. Causes of manifestations of insulin resistance are very complex. There are numerous hereditary and environmental factors that contribute the develo-pment of this condition. But today, with full plau-sibility it is accepted that insulin resistance is fully expressed among overweight individuals, those who consume excessive calories, individuals who have specific lifestyle and little physical activities.

Fatty tissue has specific correlation to insu-lin resistance; namely, the abdominal fatty tissue

reacts differently in comparison with fatty tissue distributed in the extremities. Adipocytes in the abdomen have different action in comparison to adipocytes in other parts of the body. These adipo-cytes excrete inflammatory cytokines that aggra-vate the insulin resistance (8, 9).

Researches have revealed that insulin resistan-ce along with increased levels of insulin in the blood lead to many other disorders that may act as risk factors for the onset of the heart and blood vessels disease. As a consequence, disorders in the lipid metabolism are reported, namely triglyceri-des serum levels are increased, whereas the serum HDL C cholesterol levels are decreased.

High insulin concentrations have direct da-maging impact on the cells of vesel endothelium, because it leads to deranged balance between the blood clotting process and thrombolysis in favor of the first, which subsequently favors the throm-botic episodes (10)

Hyperinsulinemia has an impact in the elevati-on of blood pressure, which is by itself an impor-tant factor in the development of cardiovascular diseases.

Purpose of work

Metabolic syndrome means the presence of nu-merous disorders at the same time, whereby each of them by itself increases the risk of developing the cardiac and vascular diseases. The more deran-ging component factors of syndrome are present, the more risk for the onset of disease is multiplied.

Thus, problem tackled in this paper is as impor-tant and actual, because there is an increased ne-cessity in identification of exogenous factors, par-ticularly diet and physical exercise, therefore, in a safe and efficient manner we might determine the diagnose, set out the program, and subsequently directly lead and monitor the processes aiming to prevent and treat the metabolic syndrome.

The purpose of research was to determine the effects of diet and physical activity in the trea-tment of metabolic syndrome during the six mon-th period.



Sample and methods

In the research, 460 patients with metabolic syndrome have been included, but for the purpose of research results of 422 patients have been used. Out of total number of patients 341 were male and 81 female.

Diagnostic criteria applied for diagnosing of metabolic syndrome were in accordance with NCEP ATP III and the diagnoses of metabolic syndrome is ascertained if at least three of below mentioned symptoms were present (6): Waistline perimeter: > 102 cm males, > 88 cm females; tri-glycerides > 1,7 mmol/L HDL (high density lipo-protein, “good” cholesterol) < 1,03 mmol/L ma-les, < 1,29 mmol/L females respectively; blood pressure > 130/85mmHg; fasting blood glucose > 5,6 mmol/L.

For the purpose of research the following para-meters of metabolic syndrome were applied:

Waistline perimeter is measured in centimeters (cm). BMI – Body mass index, or body fat tissue index indicates proportion of fat in the total body mass. The World Health Organization (WHO) pre-fers the body mass index as most adequate para-meter for assessment of body fat tissue. Proportion of body fat mass is measured through calculation of body height and weight, so that body height is divided with body weight and obtained index indi-cates the level of body fat. Body mass index (BMI) = kg/m2 = m/v2. Underweight < 18,5; Normal body weight 18,5-24,9; Overweight 25-29,9; and obesity 30 and over.

Values of triglycerides are expressed with mmol/L; Values of cholesterol HDLC (High-le-vel Data Link Control) in mmol/L; Systolic blo-

od pressure in mmHg; Diastolic blood pressure in mmHg; fasting Glucose mmol/L.

Before the start of diet and physical exercise, patients pledged their free will consent to take part in the research. Timeframe of research lasted during the period 2003 – 2008 years, while indi-vidual patients underwent the diet treatment and physical exercise during the six month period. Diet consisted of reducing the caloric intake with food for each patient in amount of approximately 200 calories per day. Patients were informed on their daily caloric needs, and each consumed com-ponent of diet is expressed in calories and grams.

Physical activity consisted of one walking hour in moderate intensity (4-4,5 km/h), five days a week. Obtained outcomes from calculating the above mentioned parameters are processed

with the descriptive and discriminative statisti-cal analysis for testing the variations between two different time periods. For all parameters were calculated arithmetic mean values (X) and stan-dard deviations (DS). Variations between testing periods of time were proved through the student’s t-test (difference between arithmetic mean values) and also were tested with the level of likelihood of p<0.05.

Results

Table 1 and Graph 1 demonstrate basic stati-stical parameters for males before and 6 month after the treatment with diet and physical activi-ty, as well as the difference between these results. Average of waistline prior to diet and physical activity was 109,13 cm, while the standard de-

Table 1. Changes in the symptoms of metabolic syndrome prior and after the 6 month treatment with diet and physical activity among males

In start In finish Probability

Variable N Mean S. Dev N Mean S. Dev t df Sig.Abdominal P. 341 109.13 4.13 341 103.08 3.88 19.94 340 .000BMI 341 28.55 2.01 341 26.78 1.95 18.57 340 .000Triglyceride 341 2.15 .22 341 1.80 .13 38.87 340 .000HDLC 341 .90 .13 341 1.38 .39 -24.88 340 .000Systolic-BP 341 147.30 17.75 341 132.30 15.10 46.62 340 .000Diastolic-BP 341 92.31 7.09 341 83.57 8.57 35.06 340 .000Glucose 341 6.39 .71 341 5.94 .31 17.86 340 .000

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viation of arithmetic mean value was 4,13. After the 6 month period waistline mean value dropped to 103,08cm, while standard deviation was 3,88. Arithmetic mean values of waistline on males in the beginning and 6 month after the application of diet and physical activity indicate the significant statistical difference (p<0,000).

Graph 1. Graphic demonstration of changes in the parameters of the metabolic syndrome among males before and after the 6 month treatment with diet and physical activity

Results of body mass index - BMI prior to tre-atment with diet and physical activity indicate that arithmetic mean value was 28.55, standard devi-ation was 2.01, while after 6 month of treatment arithmetic mean value was reduced to 26.78 while standard deviation dropped to 1.95. Differences in the arithmetic mean values obtained before and after the treatment with diet and physical activity indicate that there is a significant statistical diffe-rence in the body mass index – BMI (p<0,000).

As for the biochemical parameters, prior to tre-atment the mean value for triglycerides was 2,15 mmol/L and standard deviation was 0,22 whereas after the diet and physical activity the mean value for triglycerides dropped to 1,80 mmol/L while standard deviation was 0,13 which is a significant statistical difference (p<0,000).

Mean value for cholesterol (HDL-C) in the ta-ble is 0,90 mmol/L and standard deviation is 0,13 while at the end of treatment with diet and physi-cal exercise the mean value for HDL-C decreased to 1,38 mmol/L while standard deviation changed to 0,39. These differences in the mean arithmetic values indicate the significant statistical difference (p<0,000).

Systolic arterial pressure (systolic BP) in the beginning have had mean value 147.30 mmHg, the standard deviation value was 17,75. At the end of treatment with diet and physical activity the mean value for systolic arterial pressure was 132,30 mmHg, while standard deviation has

changed to 15,10. Obtained differences in the mean arithmetic values indicate the significant sta-tistical difference (p<0,000).

Mean value for diastolic arterial pressure (di-astolic BP) prior to treatment was 92,31 mmHg, standard deviation was 7,09, while after the 6 mon-th treatment with diet and physical activity the dia-stolic pressure reduced to 83,57 mmHg, while stan-dard deviation changed to 8,57, which indicates that there is a significant statistical difference (p<0,000) between these two mean arithmetic values.

Mean value for blood glucose level prior to examination was 6,39 mmol/L, standard deviation 0,71, whereas after the six month treatment with diet and physical activity the level of blood gluco-se dropped to 5,94 and standard deviation reduced to 0,31. These differences in the mean arithmetic values indicate the significant statistical difference (p<0,000). Table 1.

Results of basic statistical parameters for fema-les before and 6 month after treatment with diet and physical activity, as well as the difference

Table 2. Changes in the symptoms of metabolic syndrome prior and after the 6 month treatment with diet and physical activity among females

In start In finish Probability

Variable N Mean S. Dev N Mean S. Dev t df Sig.Abdominal P. 81 97.04 5.92 81 92.12 7.02 4.561 80 .000BMI 81 28.20 3.24 81 25.90 2.49 6.257 80 .000Triglyceride 81 2.03 .34 81 1.76 .28 6.849 80 .000HDLC 81 1.25 .23 81 1.31 .18 -2.220 80 .029Systolic-BP 81 142.93 11.87 81 135.54 10.45 5.281 80 .000Diastolic-BP 81 90.06 7.72 81 84.88 9.25 5.292 80 .000Glucose 81 6.45 .87 81 5.81 .63 5.940 80 .000



between the results of these parameters are indi-cated in the table 2 and graph 2. Parameter indi-cating the obesity, namely the waistline in the be-ginning was 97,04 cm, the standard deviation of arithmetic mean value was 5,92 while after the 6 month treatment the mean value of waistline re-duced to 92,7 cm whereas the standard deviation change to 7,02. Differences between arithmetic mean values of waistline among females, respec-tively the difference of mean arithmetic value in the beginning and after 6 months of treatment with diet and physical activity indicate that there is a significant statistical difference (p<0,000) of para-meters before and after the treatment.

Graph 2. Graphic demonstration of changes in the parameters of the metabolic syndrome among females before and after the 6 month treatment with diet and physical activity

Body mass index - BMI prior to treatment with diet and physical activity indicates that arithme-tic mean value was 28.20, standard deviation was 3,24, whereas after the 6 month treatment with diet and physical activity mean arithmetic value for body weight dropped to 25.90 while the standard deviation to 2,49. Differences of mean arithmetic values for the body mass index (BMI) before and after treatment with diet and physical activity indi-cate that there are significant statistical differences (p<0,000).

Mean triglyceride level in the blood was 2,03 mmol/L with standard deviation value 0,34 prior to treatment, and subsequently after the 6 month trea-tment with diet and physical activity the mean value reduced to 1,76 mmol/L with the standard deviation value 0,28. Obtained results indicate a significant statistical difference (p<0,000).

Mean cholesterol blood values (HDL-C) are in-dicated in the table and initially were 1,25 mmol/L with standard deviation 0,23, while at the end of tre-

atment with diet and physical activity the mean cho-lesterol values (HDL-C) changed to 1,31 mmol/L while standard deviation reduced to 0,18. Differen-ces between these arithmetic mean values indicate a significant statistical difference (p<0,029).

Mean systolic blood pressure (BP) level pri-or to treatment was 142,93 mmHg, with standard deviation 11,87, while at the end of treatment with diet and physical activity mean systolic BP reduced to 135,54 mmHg, with standard deviation 10,45. Obtained differences in mean arithmetic values in-dicate an important statistical difference (p<0,000).

Mean diastolic blood pressure (BP) value prior to treatment was 90,06 mmHg, with standard de-viation 7,72, whereas 6 months after the treatment with diet and physical activity the diastolic BP reduced to 84,88 mmHg, with standard deviation 9,25, which indicates an important statistical diffe-rence (p<0,000).

Mean glucose level in the blood initially was 6,45 mmol/L, with standard deviation of 0,87, whi-le at the end of 6 months treatment with diet and physical activity blood glucose level dropped to 5,81 with standard deviation 0,63. Obtained diffe-rences among mean arithmetic values indicate an important statistical difference (p<0,000). Table 2.

Discussion

Regarding the health care, healthy lifestyle habits are of utmost importance that consists on care about the healthy diet, as well as the necessi-ty for physical activities, namely walking. Human body is constructed in such a way that for its vital functions it is very important the engagement on physical activities. Therefore, physical activity as a part of recreation has become an important part of life for many people around the globe and mo-reover, it is becoming the part of lifestyle.

With application of contemporary drugs in our research, theoretically it is possible to cure each component of the metabolic syndrome, which can lead to healing of each individual symptom of me-tabolic syndrome. However, starting point in our efforts was focused in employment of methods that will improve several symptoms of the meta-bolic syndrome at the same time. Application of two methods in this research, namely diet treatment

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and physical activity (walking) that aimed to im-prove the symptoms of metabolic syndrome have been proved to be the best method for improving the parameters of research. Also other authors have confirmed these methods to the best ones (11). High values of waistline and body mass index – BMI are main parameters that describe human overweight and are the most important indicators for identifica-tion and diagnosing the metabolic syndrome (12). Obtained results from the 6 month treatment with diet and physical activity (walking) have resulted in reduction of all parameters related to symptoms of the metabolic syndrome. Decrease of waistline by 6,05 cm among males indicate that treatment with diet and physical activity (walking) during the 6 month period have had an impact in the decrease of abdominal fat by 5,50%. Dietary treatment along with physical activity among females have had an impact in the decrease of waistline by 4,92 cm or 5,10% lower. Obtained values of waistline indicate that in order to achieve the ideal waistline value it is necessary to exercise a longer dietary treatment and physical activity. Namely, the excessive wei-ght have been gained over the long period of time, therefore we have to be patient because we cannot eliminate it quickly. Reduction of intra abdominal fat tissue and of adipocytes (which are today consi-dered active endocrine cells) in the levels obtained in our research, diminishes the cardio metabolic risk such as: lipid profile (it is a source of free fatty acids), insulin resistance, glucose intolerance, and deranged inflammatory reaction and hemostasis (12).

Reduction of abdominal subcutaneous fatty tissue after the treatment with diet and physical activity (walking) has impacted in the reduction of body mass index(BMI) value by 1.78 or 6.20% on males, and by 2.3 or 8,16% on females. When talking about fats of metabolic syndrome, namely regarding the triglycerides and cholesterol HDLC, it would be more accurate to use the phrase dysli-pidemia, because it describes more plausibly the complexity of this disorder. Dyslipidemia means increase in levels of triglycerides (hipertriglyce-ridemia), decreased level of cholesterol HDLC, increase in levels of cholesterol LDL, decrease in LDL/HDLC ratio, increased levels of free fa-tty acids (13). Treatment with diet and physical activity have impacted in the reduction of level

of triglycerides in the blood by 0,35 mmol/L or 16,28% on males, and by 0,27 mmol/L or 13,30% on females. In contrast, treatment with diet and physical activity has impacted in increasing the le-vels of HDLC cholesterol in the blood. Levels of cholesterol in the blood among males have recor-ded an increase by 0,48 mmol/L or 34,78% and by 0,06 mmol/L or 4,28% on females. Results obta-ined in the research are approximately the same with similar researches in this field (12, 13).

During the physical activity cardiovascular sy-stem is experiencing various changes, such as incre-ase in cardiac output which is in close relationship with the dilation of blood vessels in muscles during the physical exercise. Except this direct link in the metabolic events, we must also take into account the reflexive activation of sympathetic nerves in relation with heart, vascular resistance and circula-tion capacity. Based on these facts, results obtained from systolic BP and diastolic BP in males and fe-males indicate that after 6 months of treatment with diet and physical activity these parameters eventu-ally were reduced. Reduction in systolic BP by 15 mmHg or 10,18%, and diastolic BP by 8,74 mmHg or 9,47% in males, as well as reduction of systolic BP by 7,39 mmHg or 5,17%, and reduction of dia-stolic BP by 5,18 mmHg or 5,75% in females clear-ly indicate the positive impact of diet and physical activity in reduction of blood pressure.

There is no doubt that diet and physical activity might impact in curing the metabolic syndrome. The impact of diet and physical activity is obvious in insulin resistance, which is the key feature of this disorder (11). Our results from treatment with diet and physical activity show reduced levels of blood glucose by 0,45 mmol/L or 7,04% in males, and by 0,64 mmol/L or 9,92% in females.

As mentioned above, the largest number of pe-ople with metabolic syndrome is overweight and has contemporary lifestyle (insufficient physical activity). Changes in the lifestyle are the main ac-tion to be undertaken among people with metabo-lic syndrome. Reduction of body weight alongsi-de the specific dietary actions are necessary steps, and physical activity has to be shaped based on individual needs. Walking exercise monitored in intensity, duration and frequency may be succe-ssful and without risks applied in all individuals affected by metabolic syndrome.



Conclusion

Total of 460 patients with metabolic syndrome were included in the research, but for the purpose of research, results from 422 patients have been used. Out of total number involved in the research, 341 were males and 81 females respectively.

For diagnosis of metabolic syndrome, criteri-ons were applied in accordance with NCEP ATP III (Third Adult Treatment Panel). The purpose of research was to determine the impact of diet and physical activity (moderate intensity walking) in the treatment of metabolic syndrome during the six month period. Obtained results indicate that:

- Diet and moderate intensity walking have impacted the reduction in waistline and body mass index (BMI).

- Dyslipidemia has been improved; reduction of serum triglyceride level and increase in HDLC cholesterol level has been recorded.

- Reduction in systolic and diastolic BP was recorded.

- Serum glucose level improvement was evident in our results.

Treatment of metabolic syndrome should start at the moment of onset, and the patient should be informed about its consequences. The doctor may prescribe drugs, but it is necessary as a part of tre-atment the application of diet, physical activity, and changes in lifestyle.

In this regard, we must consider the definition of World Health Organization (WHO) on the quality of life which takes into account relations of each individual with his environment. “Quality of Life as an individual’s perception of their position in life in the context of the culture and value systems in whi-ch they live and in relation to their goals, expecta-tions, standards and concerns. It is a broad ranging concept affected in a complex way by the person’s physical health, psychological state, personal beli-efs, social relationships and their relationship to sa-lient features of their environment”.

References

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4. Reaven G.M. Insulin resistance, cardiovascular di-sease, and the metabolic syndrome: How well do the emperor’s clothes fit?. Diabetes Care, 2004, 27(4): 1011-2.

5. American College of Endocrinology. Insulin re-sistance syndrome: Position Statement. Endocr Pract, 2003, 9(Suppl 2), str. 9-21

6. World Health Organization (1999) Definition, dia-gnosis and classification of diabetes mellitus and its complications, Part 1: Diagnosis and classification of diabetes mellitus. Geneva: Department of Non-communicable Disease Surveillance.

7. Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Eva-luation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) nal report Cir-culation 2002, 106; 3143-421.

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13. Chen C-Y, Blumberg JB. Oxidative Stress Status in Humans with Metabolic Syndrome. In: Obesity: Epidemiology, Pathophysiology, and Prevention. Bagchi D, Preuss HG (eds.) CRC Press/Taylor & Francis Group, Boca Raton, FL 2007;123-37

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14. National Diabetes Program of Kosova, G. Drag-usha, D.Mekuli, M.Spahiu, E.Sefedini.

15. Edina Kuduzovic, Sabina Nuhbegovic, Farid Lju-ca, Sanela Imamovic, Importance of physical ac-tivity in the patients with Diabetes Dellitus type 2; HealthMED 2009; 3: 296-302

16. Budimka Novakovic, At all. Medical nutrition pre-vention and medical nutrition therapy of lipid me-tabolism disorder, HealthMED 2009; 3: 235-244

Corresponding author Gani Dragusha, Head of the National Diabetes Program in Kosovo, Internal Medicine Clinic, Department of Cardiology, University Clinical Center of Kosovo, Kosovo, e-mail: [email protected]



Abstract

Introduction: Emergency care must be un-derstood as a complete system with independent components. Considering the high statistics of the cardiovascular diseases and unintentional events in Iran, the pre-hospital emergency system in the country needs fundamental changes. The aim of this study is to identify the determinants influen-tial in performance of the pre-hospital emergency system of Iran and analysis of the relationships among them.

Methodology: This research is of the com-parative-descriptive studies type, and it has been accomplished by using cross –sectional design during the first half of the year 2009. The resear-ch population included 10 pre-hospital emergency experts, and special forms, together with Nominal Group Technique (NGT), were used as data gathe-ring tools. Also the DEMATEL approach and MA-TLAB software were used for analysis of the data.

Findings: The most important determinants influential in the performance of the Pre-hospital emergency system of Iran include the “organiza-tion,” “transportation,” “communications,” “acce-ssibility,” “care model,” “manpower combinati-on,” “regulations,” and the “training,” among whi-ch the “organization” and the “care model” have been identified as the most influential and the most influenced determinants respectively.

Discussion: Although the prep-hospital emer-gency system of Iran has grown quantitatively, to develop fundamentally from both the qualitative and the quantitative point of view, it needs struc-tural modifications which will establish an inde-pendent emergency medical care organization, and integrated management of pre-hospital and hospital emergency systems. Compiling the regulations and standards of reviewing the specialized training co-urses while training the people are also of conside-rable importance for implementing reforms.

Key words: Pre –hospital Emergency, Deter-minants, DEMATEL Method

Introduction

Emergency care must be realized as a comple-te system with independent components. These components include pre-hospital care, transporta-tion, and hospital care. Each component is impor-tant, but all three must interact to produce a dura-ble effect on the health of the population. When the hospital transportation is weak or nonexistent, death is occurred, which could be avoidable using cheap methods (1, 2).

According to the World Health Organization (WHO), cardiovascular diseases are the most pre-valent causes of mortality in Iran. Unintentional accidents are the second cause of mortality. Iran

Analysis of the relationships between the determinants Influential in performance of Pre-hospital Emergency system of Iran using the dEmATEL ApproachAmir Ashkan Nasiripour1, Mohammadkarim Bahadori1,Shahram Tofighi2, Mahmoudreza Gohari3 1 Department of Health Care Management, Science and Research Branch, Islamic Azad University, Tehran, Iran2 Health Management Research Centre, Baqyattallah University of Medical Sciences, Tehran, Iran3 Department of Statistics, Iran University of Medical Sciences, Tehran, Iran

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with 28000 annual deaths is one of the countries with the highest mortality rate of car accidents in the world. The road accidents statistics in Iran is 20 times higher than the world average. According to the statistics issued in Iran, one person loses his/her life every 19 minutes due to car accidents. Poi-soning, falling, drowning and burning are the next causes of mortality due to unintentional accidents (3, 4, 5, 6, 7, and 8). On the other hand, 50% of the traffic accidents mortalities occur during the first hour after the collision, 25% during transpor-ting the injured to the hospital, and 25% relates to the infections due to the side effects occurring in the hospitals. It has been increasingly noticed that many of the deaths and long term disabilities can be avoided through empowering the trauma cen-ters and pre-hospital emergencies (9, 10, and 11).

The results of a study about the emergency pa-tients show that most instances of avoidable fai-lures included the time lapse in primary care, the lack of care during transportation of the patient to the medical care centers, and inappropriate com-munications (12). The paramedic personnel and the ambulance in some countries are only used to convey the patients to the medical care centers and no treatment is performed until arriving at hospi-tals (13, 14). According to the results of a study, the shortage of medical emergency transportation is an important obstacle in caring for the patients. The reasons for such a shortage include: shortage of the suitable facilities, inadequate roads, and ina-ppropriate transportation system (15).

Based on the accomplished studies, the most important variables of the pre-hospital emergen-cy include the “organization,” “training,” “man power,” “transportation,” and “communications.” Nevertheless, the comprehensive and integrated emergency system will be available through allo-cating the resources, making preparations for the future, cooperation, transparency, and sense of responsibility of all the health system (16).

Most of the studies made regarding the pre-hospital emergency have dealt with the present status of the pre-hospital emergency in the coun-tries, and no systematic relationships among the determinants of such systems have been identifi-ed. Today, the countries of the world try to use the European or American model of presenting medi-cal and emergency services. But due to population

and economical changes, and the pressing need for such services, most of the developing countri-es are not able to suitably use the European model, causing inevitable wasting of the vital resources (17, 18, and 19).

Considering the high statistics of the cardiovas-cular diseases and unintentional accidents in Iran, the pre-hospital emergency system in the country needs fundamental changes. The aim of this study is to identify the determinants influential in the performance of the pre-hospital emergency sy-stem of Iran and an analysis of the relationships among them.

Methodology

This research is of the comparative-descriptive studies type and has been accomplished by using cross –sectional design during the first half of the year 2009. The pre-hospital emergency systems of eight countries (United States of America, Uni-ted Kingdom, France, Germany, Italy, Norway, Netherlands, and Canada) have been selected in different regions of the world. The researcher co-mmunicated with the scholars who have conduc-ted research about the pre-hospital emergency ma-nagement, and based on the available experience principles, the available data and the similarity between Iran’s and the above countries pre-hos-pital emergency systems’ overall conditions has identified the successful countries in this regard. The results of the research show that the USA and some European countries have been relatively su-ccessful compared with other countries. Also the European and American system exist as two dis-tinct systems, and the usage and customization are different from each other.

The required data about the countries were ga-thered based on the predefined structure, and then different structural parts used for gathering the data for different countries were compared accor-ding to the comparative tables and the main de-terminants were exploited. The exploited determi-nants were finalized using the views of ten experts in the pre-hospital emergency system of Iran and using the Nominal Group Technique (NGT). Ul-timately, the final model was designed using the DEMATEL model and the MATLAB software.



The DEMATEL method was first introduced for studying the complicated and integrated pro-blems by the Science and Humanity Program of the Battle Memorial Institute, Geneva, during the years 1972 and 1976. The purpose behind the de-velopment of the DEMATEL method was that su-itable usage of the scientific research methods can help in better recognition of the structure of com-plicated problems and contribute in finding prac-tical solutions with hierarchal structure (20). The DEATEL method is based on directional graphs (diagraphs) capable of segregating the intervening factors in two groups of the causes and the effects. These diagraphs depict the dependency relation-ship between the elements of a system so that the numbers on each diagraph is indicative of the in-fluence intensity of an element on the other. The DEMATEL method therefore can convert the rela-tionship between the factors’ causes and effects to a comprehensible structural model of the system. The causal diagram is gained by depicting the

adjusted couples ( )kkkk RDRD -+ , in which the horizontal axis ( )RD + named “superiority” is

constructed by adding kR to kD and the vertical axis ( )RD - named “relationship” is constructed

by subtracting kR from kD .In causal diagram, the “superiority” horizontal

axis shows the significance of a criterion, while the vertical axis of “relationship” divides the criteria into two groups of causes and effects. When the

( )kk RD - value is positive, the criterion belongs

to the ‘cause’ group. If the ( )kk RD - value were negative, the criterion belonged to the ‘effects’ gro-up. Therefore, the causal diagrams can convert the compound causal relationships existing among the criteria into a visible structural model and create an accurate insight for solving the problem. In addition, with the help of causal diagrams and recognizing the difference between the cause and effect criteria, ma-king good decisions would be possible (21).

Findings

Based on the research findings, the most im-portant determinants influential in the pre-hospital

emergency performance have been specified in ta-ble 1 according to the comparative study and the final views of the experts (Table 1).Findings of the research showed that the ‘organization’ (P1), ‘re-gulations’ (P8), and ‘training’ (P5) determinants are penetrating determinants and are classified in the causes group. The ‘accessibility’ (P3), ‘manpower combination’ (P4), ‘transportation’ (P6), ‘commu-nications’ (P7), and the ‘caring model’ (P2) deter-minants are penetrated determinants and are cla-ssified in the effects groups (Table 2).

Also the relationships between the determi-nants influential in the pre-hospital emergency performance and their hierarchy indicates that the ‘organization’ (P1) determinant affects other deter-minants, but it is not affected by any determinant. On the other hand, the ‘caring model’ (P2) deter-minant is affected by the other determinants. The priority of the determinants is as follows: ‘organi-zation’ (P1), ‘regulations’ (P8), ‘training’ (P5), ‘co-mmunications’ (P7), ‘accessibility’ (P3), ‘transpor-tation’ (P6), ‘manpower combination’ (P4), and the ‘caring model’ (P2). (See fig. 1).

Discussion

Health care in developing countries is not tradi-tionally focused on emergency medical care. Pro-moting health and preventing harm must be the main values of any health system. Paying atten-tion to the emergency medical care in the health care systems may have a significant effect on the population welfare and reduce the economical and human expense of the diseases (22). Based on the results of the present research, the most significant determinants effective on the performance of pre-hospital emergency system of Iran are ‘organiza-tion’, ‘accessibility’, ‘training’, ‘transportation’, ‘communications’, ‘manpower combination’, ‘re-gulations’, and the ‘care model’. These finding are similar to the findings of the research titled “Esti-mating the pre-hospital systems of the developing countries” carried out by Michael Van Rooyen in 1999 (23). Meanwhile, surveying the pre-hospital emergency system of the developed countries in-dicates that, existing as an independent organizati-on, such systems together with the hospital emer-gency are practically under an integrated manage-

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ment with the online medical direction being one of their most important features. The pre-hospital emergency system of Iran however lacks such fe-atures, and this may be the cause for the “organi-zation’ determinant to be recognized as the most penetrating determinant in the interaction model (24, 25, 26, 27, 28, and 29).

The ‘regulations’ is the second penetrating ele-ment after the ‘organization’ determinant which is the most causal determinant in the performance of the pre-hospital emergency system of Iran. Based on the comprehensive investigations carried out in Iran, the standards and the regulations relating to the pre-hospital emergency system of Iran are still in preliminary stages. For example, in some coun-tries, the one who is not properly certified may not operate as the responder of the pre-hospital emer-gency. According to the study titled “The medical emergency systems in the developed and underde-veloped countries” carried out by the Carlos Risa et al. in 2007, the international attempts have been focused on developing the minimum standards for the traumatized patients. These standards can spread all over the world. In investigating the pre-hospital emergency system of Australia carried out by Patrick Weninger in 2005, the most impor-tant result of the estimation was establishing the required regulations for training the practitioners in 1998 which was a fundamental step towards in-tegrated care (30, 31).

One of the penetrating determinants influential in the performance of the pre-hospital emergen-cy system of Iran belonging to the ‘causes’ group is the ‘training’ determinant. The training in Iran includes three types of ‘common’, ‘specialized’, and ‘continual’ training. This training system is developing, but it is not yet classified in different levels, and it is not similar to the developed coun-tries in regard to quality and quantity. In the UK, training occupies the top position in the pre-hos-pital emergency management agenda. Based on the results of the research carried out in Portugal by Gomes in 2004, improving the management skills of the pre-hospital emergency management system requires investment for training and tech-nology. Development of the exceptional status of the intervening teams is an example in this regard. These teams include a number of the practitioners, nurses, psychologists, ambulance technicians, and

programming experts who receive special trai-ning. The continual quality improvement proce-dure includes optimization of the protocols, data bases, and education standards (32, 33).

Based on the findings of the present research, the penetrated determinants of ‘communications’, ‘transportation’, ‘manpower combination’, ‘acce-ssibility’, and the ‘care model’ are included in the “effects” group. In other words, these groups of determinants are penetrated by the ‘organization’, ‘regulations’ and ‘training’ determinants. In com-parison, based on the studies implemented in the countries like Mexico, the UK, the USA, Nether-lands, France and Germany whose present pre-hospital emergency status has been described, the ‘organization’ and ‘training’ determinants have been surveyed first, and then the other determi-nants have been dealt with (34, 35, 36, 37, 38, 39, 40, and 41).

Conclusion

Although the prep-hospital emergency system of Iran has grown quantitatively, to develop funda-mentally from both qualitative and quantitative po-ints of view, it needs structural modifications whi-ch establishes an independent emergency medical care organization, and integrates management of pre-hospital and hospital emergency systems. Also, compiling the regulations and standards of reviewing the specialized training courses and tra-ining the people is of considerable importance for implementing the proposed reforms.

Appendix:

Table 1. The eight essential pre-hospital determi-nants

1. Organization2. Access3. Model of Care4. Manpower Combination5. Communication6. Transportation7. Regulation,8. Education



Figure 1. The influence-relation-map the 8 deter-minants

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Table 2. The total –relation matrix of 8 determinantsD-R D+R R D P8 P7 P6 P5 P4 P3 P2 P1

1.3900 1.3900 0.00 1.3900 0.1382 0.1872 0.1872 0.1518 0.2157 0.2589 0.2510 0 P1-0.6233 1.3013 0.9623 0.3390 0.0010 0.0228 0.0228 0.0081 0.1712 0.1003 0.0128 0 P2-0.4646 1.6044 1.0345 0.5699 0.0101 0.1035 0.1035 0.0843 0.1127 0.0440 0.1118 0 P3-0.5852 1.2512 0.9182 0.3330 0.0010 0.0906 0.0906 0.0085 0.0134 0.1048 0.0241 0 P40.4762 1.2474 0.3856 0.8618 0.1219 0.1516 0.1516 0.0160 0.1703 0.0784 0.1720 0 P5-0.5172 0.8938 0.7055 0.1883 0.0012 0.0124 0.0124 0.0101 0.0135 0.1253 0.0134 0 P6-0.3028 1.1082 0.7055 0.4027 0.0014 0.0161 0.0161 0.0114 0.0409 0.1413 0.1755 0 P70.6269 1.1995 0.2863 0.9132 0.0115 0.1213 0.1213 0.0954 0.1805 0.1815 0.2017 0 P8

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Corresponding author Amir Ashkan Nasiripour, Department of Health Care Management, Islamic Azad University, Science and Research branch, Iran, E-mail: [email protected]



Abstract

A prospective study was conducted to observe the clinical features and complications of dengue infection. Admitted patients to the medical ward of dengue virus infection were studied in General Hos-pital Penang, Malaysia. A total of 756 patients were enrolled from January 2007 to December 2007. The clinical features were evaluated and compared in terms of age groups. The patients were distribu-ted in four different age groups. Children ≤18 ye-ars, young age groups 19 to 36 years, adult 37 to 54 years and the elderly age group ≥55 years old. The differences of clinical features between children and adults require further evaluation to find out and recognize the substantial factors involved, ascites, jaundice, pleural infusion are the complications of dengue which is required early identification in associ-ation with thoroughly monitoring and basic medi-cal support to save the life of dengue patients.

Key words, clinical features, dengue fever, den-gue hemorrhagic fever and dengue shock syndrome.

Introduction

Today dengue is a fast growing health problem in tropical and sub tropical countries. The definition of dengue fever is stated as “an acute illness caused by a virus belonging to the family flaviviridae, un-der the genus flavivirus”. Dengue is the most com-mon disease among all arthro-borne viral infection in the world today. There are four serotypes of den-

gue virus namely as Den-1, Den-2, Den-3, Den-4, which are existed in an urban transmission cycle in tropical and sub tropical areas by the aedes aegypti (WHO, 1997). Infection with one serotype deve-lops long life immunity against re-infection by that same serotype, but not against the other serotype.


A retrospective study was conducted on the ad-mitted patient’s hospital Penang during the period January 2007 December 2007. There were 756 patients studied from the data was collected from the charts and records of admitted patients Gene-ral Hospital Penang. Information’s of the patients was regarded on the standard form.

Ethical consideration

The study was approved by Clinical Research Committee (CRC) Penang GH, reference number was 2008/05.

Selection criteria of the Study population It consists of the following two criteria.

Inclusion Criteria1. Patients were considered for study with

confirmed diagnosis, complete records of dengue fever from Jan-2007 to December- 2007.

2. All in patients were included for study.

Evaluation of clinical features and complications of dengue infected patients in Penang general hospital, malaysia Khurshid Alam 1*, Syed Azhar Syed Sulaiman1, Asrul Akmal Shafie 1, Rozina Ghazali2

1 School of Pharmaceutical Sciences, Universiti Sains Malaysia, Malaysia2 Department of Pathalogy, Penang General Hospital, Malaysia

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Exclusion Criteria 1. Missing or incomplete data (record) was

excluded.2. Patients with complicated cases like surgical

cases, cancer, HIV were excluded

The clinical features of dengue fever in patients were mentioned in terms of age groups. and were divided into four groups. Children were conside-red less then 18 years or equal, 19-36 young age groups, from 37-54 years adult and ≥55 years old. The patients with dengue infection were diagno-sed by WHO, 1997 criteria.

Statistically the features were tested by appl-ying Fisher exact test to determine the clinical fe-atures in terms of age.

Results

Fever was defined as the fever prior to admissi-on and during hospitalization. Fever was present in all 756 admitted patients. Arthralgia and myal-gia were observed among children 43 (20.3%) and 53 (25.0%) respectively. While in the young age groups (19 –36yrs) arthralgia and myalgia were common and found 160 (52.5%) and 195 (63.9%) respectively. In the age 37 – 54 years old patients arthralgia and mayalgia were found prominently 104 (65.5%) and 110 (59.8%) respectively. While lowest level was seen in the age of 55 years and above 24 (43.6%) and 26 (47.3%) respectively, the (P <.001) found statistically significant.

The rashes were evaluated in different age gro-ups, island of white in the sea of red, macular po-pular rashes, were present to a mild level in the age group 18 years and below 4 (1.9%), 12(5.7%), respectively. On the other side in the age group of 19 – 36 years occurred 8 (2.6%), 28 (9.2%), 22 (12.0%), 43 (14.1%), 30 (9.8%) respectively. The-se features were also occurred in the age group of 37 – 54 years 11 (6.0%), 12 (6.5%), 9 (4.9%), 22 (12.0%), 15 (8.5%) respectively. While in the age of 55 years and above these clinical features were found as 5 (9.1%), 4 (7.3%), 4 (7.3%), 1 (1.8%), 5 (9.1%) respectively and were statistically non signi-ficant (P=0.096). Positive Hess test was found 26 (12.3%) in the children (≤18 years) ,69 (22.6%) had in the young age group (19 – 36 yrs), was positive

57 (31.0%) in the middle age group(37 – 54yrs), while in the older age group (≥55 yrs ) was noted 13 (23.6%) and was found non significant (P<.005). Gum bleeding, epistaxis, bleeding from GIT (mele-na and hemetemsis) were observed in all age groups with different proportions, 18 (8.5%), 14 (6.6%), 1 (.5%) were found respectively in children and the results were statistically non significant (P=0.200). In young age groups (19 – 36yrs) these features were noted as 40 (13.1%), 26 (8.5%), 1 (0.3%) res-pectively. In the middle age (36 – 54yrs) were seen 22 (12.0%), 11 (6.0%), 0 (.0%) respectively, while in the older age group (≥55 yrs) were stated as 3 (5.5%), 1 (1.8%), 0 (0.0%) respectively and were statistically non significant (P=0.200). Retro orbi-tal pain was found common in the young age group (19-37 yrs) and observed 93 (30.5%), 36 (17.0%) in children, 34 (18.5%) was in age group (37 – 54 yrs) and 11 (20.0%) in the elderly patients (≥55 yrs) and was found statistically significant (P=0.001). Headache was noted common in all ages group, in children was elicited 81(38.2%), 138 (45.1%) was present predominantly in young age patients, 83 (45.1%) was seen in middle age group, 22 (40.0%) was stated in the older patients (≥55 yrs) and found statistically non significant (P=0.370). Episgastric pain was common in children and demonstrated 98 (46.2%), 128 (42.0%) was found in young (19 – 36 yrs), 64 (34.8%) in elderly patients (≥55yrs) and noted statistically non significant (P=0.094). Chi-lls and rigors highly were observed in the children 78 (36.8%), in young age (19 – 36yrs) patients it was found 91 (29.8%), in the middle age patients (37 – 54 yrs) found 63 (34.2%), while in elderly patients it was found 16 (29.1%) and was noticed as non significant (P= 0.353). Loss of appetite was observed in children as 60 (28.3%), while in young age group(19 – 36yrs) were found 71(23.3%), 67 (36.4%) was observed in the middle age (37 – 54 yrs), while in the elderly (≥55yrs) patients loss of appetite was observed 23(41.8%) and was statisti-cally significant (P=0.003).

Cough was present in children 62 (29.2%), in young (19 – 36yrs) was observed 60 (19.7%), in the patients of middle age (37 – 54 yrs) was noted 33 (17.9%), in the elderly noticed 11(20.0%) and was statistically significant (P=0.025). Nausea was observed in children as 39 (18.4%), in young age it was found 58 (19.0%), in middle age (36 – 54yrs)



it was 45 (24.5%), in elderly patients it was 17 (30.9%) and was found non significant (P=0.104). Vomiting was found to have high in children and was present 121 (57.1%), in the young age group (19 - 36yrs) was mentioned 119 (39.0%), in the middle age(37 – 54yrs) 58 (31.5%), while in the el-derly (≥55yrs) it was found as 20 (36.4%) and was statistically significant (P=<0.001). Skin flushing was seen in children as 10 (4.7%), in young (19 - 36yrs) patients was noted as 26 (8.5%), in middle age (37 -54yrs) was 18 (9.8%) and in the elderly (≥55yrs) patients it was found 5 (9.1%) and was

statistically non significant with (P=0.245). The de-tails are demonstrated in the table 1.

The prevalence of the disease DF, DHF and DSS were evaluated in different age groups. Dengue fe-vers (DF) in this one year showed the highest pro-portion in ≥55 and less than 18 years of age and DHF were more prevalent in the young age groups (19 – 36 yrs). The details are demonstrated in the table 2.

Complications were studied generally, as myo-carditis was found (0.7%), encephalitis (0.9%), tachycardia (0.4%), bradicardia (0.4%), ascites (1.7%), Jaundice (1.2%) table 3.

Table 1. Clinical manifestations distribution of dengue fever in the age groups

Clinical manifestationsChildren≤ 18 yrs

n (%)

Young(19-36 yrs)

n (%)

Middle age37-54 yrs

n (%)

Elderly≥ 55 yrs

n (%)P-value

Fever 212 (100) 305 (100) 184 (100) 55 (100) ---Arthralgia 43 (20.3) 160 (52.5) 104 (65.5) 24 (43.6) <.001Myalgia 53 (25.0) 195 (63.9) 110 (59.8) 26 (47.3) <.001Island of white in the Sea of red 4 (1.9) 8 (2.6) 11 (6.0) 5 (9.1) 0.096Macular popular rashes 12 (5.7) 28 (9.2) 12 (6.5) 4 (7.3)Hess test 26 (12.3) 69 (22.6) 57 (31.0) 13 (23.6) <.001Gums bleeding 18 (8.5) 40 (13.1) 22 (12.0) 3 (5.5)

0.200Epistaxis 14 (6.6) 26 (8.5) 11 (6.0) 1 (1.8)GIT(haematemesis and melena) 1 (.5) 1 (.3) 0 (.0) 0 (.0)Retro orbital pain 36 (17.0) 93 (30.5) 34 (18.5) 11 (20.0) 0.001Headache 81 (38.2) 138 (45.1) 83 (45.1) 22 (40.0) 0.37Epigastric pain 98 (46.2) 128 (42.0) 64 (34.8) 19 (34.5) 0.094Chill/rigors 78 (36.8) 91 (29.8) 63 (34.2) 16 (29.1) 0.353Loss of appetite 60 (28.3) 71 (23.3) 67 (36.4) 23 (41.8) 0.003Cough 62 (29.2) 60 (19.7) 33 (17.9) 11 (20.0) 0.025Nausea 39 (18.4) 58 (19.0) 45 (24.5) 17(30.9) 0.104Vomiting 121 (57.1) 119 (39.0) 58 (31.5) 20 (36.4) <.001Skin flushing 10 (4.7) 26 (8.5) 18 (9.8) 5 (9.1) 0.245

*Fisher exact test

Table 2. Frequency of DF, DHF and DSS according to AgeAge DF DHF DSS≤18 145 (68.4%) 66 (31.1%) 1 (.5%)

19 – 36 197 (64.0%) 106 (34.8%) 2 (.7%)37 – 54 112 (60.9%) 70 (38.0%) 2 (1.1%)

≥55 39 (70.9%) 15 (27.3%) 1 (1.8%)Total 493 (65.2%) 257 (34.0%) 6 (.8%

Table 3. Complication of dengue fever found in hospitalized patientsTypes of complications in patients Percentage N (%)

MyocarditisEncephalitis TachycardiaBradycardia

Ascites Jaundice

Pleural infusion

5 (0.7%)7 (0.9%) 3 (0.4%)3 (0.4%)13 (1.7%)9 (1.2%)25 (3.3%)

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Discussion

Fever was present in all 756 admitted patients. age were distributes as ≤ 18 age were defined as children, (19 -36yrs) were called young; (37 – 54yrs) middle age and (≥55 yrs) were consi-dered as elderly. Arthralgia and myalgia were si-gnificantly present in all age groups in our study. However arthralgia was observed significantly hi-gher 65.5% in the middle age group, while mayal-gia was found significantly higher 63.9% in young group (P=<0.001). Agreeable results were found in another study in which adults had higher mayal-gia 83% and arthralgia 82% symptoms of disease (Harris et al., 2000).

In our results symptoms like island of white in the sea of red, macular popular rashes, were present none significantly in all age groups. These symp-toms were predominant in the young age groups with percentage 2.6%, 9.2%, respectively, except the condition of ‘island of white in the sea of red’ which was found higher in the elderly patients. In children dengue like symptoms may be not promi-nent in adults (Chadwick et al., 2006), in their study has stated that the presence of myalagia, flushing, macular popular rashes or scattered petechiae were the most predictive features of dengue fever as ot-her non dengue diseases. Similar symptoms were prevalent in the study evaluated in a hospitalized patient in children were noted 62% and in adults were 64 % (Lum et al., 2002). In a study conduc-ted by Harris slightly conflicting results were obta-ined (rash in children were observed 62%, while in adults were noted 56 % (Harris et al., 2000).

Hess test is used as a screening test for dengue infection. In our study Hess test was seen in all age groups and was statistically significant (P=<.001). However Hess tests significantly higher in the middle age of 37 – 54 years old patients. Our re-sults not agree with the findings found in the study performed by Harris et al., 2000. It was reported in a study that positive tourniquet test among children in 98.1% for DHF and 63.3% for DF and it was concluded that tourniquet test is helpful in diffe-rentiation from other illnesses disease such as chi-kungunya (Nimmannitya et al., 1987). According to Hammond and Whichmann, in their study they evaluated that the findings of positive tourniquet tests were in similar frequency between adult and

child groups admitted to the hospital, this may not support the identification of positive tourniquet tests as the only sign of severe adult infection. The-refore a tourniquet test might be used as an addi-tional simple diagnostic tool for both children and adults with dengue virus infection (Hammond et al., 2005; Whichmann et al., 2004).

In our study gums bleeding and epistaxis were common than gastrointestinal bleeding hemate-mesis 0.5% and melena 1.3% but these are the im-portant sign which are needed to be examined ca-refully. Gastrointestinal bleeding may be initially dormant and usually manifest as an abdominal pain or tenderness, a distended abdomen, pallor, tachycardia or a drop in haematocrit without cli-nical improvement found in young patients than children (Lum, 1997). Bleeding was studied from various sites and was found 72%, gum bleeding and epistaxis were the common bleeding manife-stations 40% (Singh et al., 2005). Lum and his colleague were reported in their study bleeding in children was present 62% and in adults was 64%(Lum et al., 2008). Retro-orbital pain was co-mmon symptoms of dengue in the present study and was significant statistically (P= <.001). Retro orbital pain in the present study was found high significantly in the young age 30.5% patients. In a study conducted by Lum et al., 2008 similar re-sults were reported in which the retro orbital pain was found 40% in children while in adults no-ted 51%. Headache was observed more in adults 45.1% than in children and elderly patients in the present study. It was reported from the Lum et al., 2008 study that 83% had headache in children and in adults was seen 96%.in our results headache, melena, retro-orbital pain, Hemetemesis were fo-und more in adults than children. Similar results were observed in a study of (Kittigul et al., 2007).

Epigastric pain is one of the important warning signs that DSS is impending (Gibbons and Vaughn, 2002). In our results the frequency of epigastric pain was found non significant in all age groups. However was found higher 46.2% in children than the other age groups. Epigastric pain may be alar-ming sign predict gastro-intestinal bleeding among the symptoms reported more frequently in different case series from 23 to 86 %( Diaz et al., 1988; Val-des et al., 1999). Similar results to our study were found in a study which was explored in children



87% and in adults 74%. Chills and rigors had repor-ted in many studies. In this study on the basis of age groups chills and rigors were assessed and found in all age groups statistically non significance. In children 36.8% was comparatively found high than the other age groups. Loss of appetite was found as a sign of dengue in our study. In all age groups was seen statistically significance (P=0.003). However in elderly patients 41.8% was significantly demon-strated high than the other age groups. Matching results were reported in a study in which the sign loss of appetite was present 78% in DF and 93% in DHF (Kalayanrooj., et al 1997). Cough was present significantly in all age group levels, however found significantly higher 29.2% in children (P=0.025). In adults sign and symptoms of dengue were evalu-ated in a study conducted in Puerto Rico 35% cou-gh was found (Cobra et al., 1995).

Halstead was observed cough in his study in chil-dren 21 % suffered of dengue Nausea and vomiting was assessed in our study and was found in all age groups with statistical significant different (Halste-ad et al., 1965). Nausea was noted non-significantly high in elderly patients 30.9%, while vomiting was found significantly (P=0.007) high in children 57.1% as compared to other age groups. On the ba-sis of present study findings, the clinicians should carefully evaluate and to observe closely vomiting, nausea, diarrhea in the febrile phase of disease to assess weather patient needs hydration or hospita-lization. Nausea and vomiting were analyzed in a study conducted by Kittigul et al., 2007 and found in children 50.2% and in adults 76.4%.

Skin flushing was also observed non-signifi-cantly in all age groups. In our study was found 9.8% non-significantly higher in the middle age groups. In one of the study skin flushing was re-ported 45% which is higher than the present study (Chadwick et al., 2006).

Jaundice was seen but to a lesser extent non-significantly in all age groups. However was fo-und comparatively higher in the elderly cases with 1.8%. Jaundice 5.13% was reported in a study carried out in Jeddah (Ayyub et al., 2006). In the present study tachycardia was observed non-signi-ficantly in children. There was only 1.9% of the tachycardia present in the children. It was reported that tachycardia may reduce the likelihood of the diagnosis of dengue fever (Chadwick et al., 2006).

Complications

In complications myocarditis was found (0.7%), encephalitis (0.9%), tachycardia (0.4%), bradicardia (0.4%), ascites (1.7%), Jaundice (1.2%), in these complications myocarditis was found in 5 (0.7%), which mostly lead to the hy-potension and circulatory failure. All the patients were recovered except one was died. This may be due to excess fluid might have been given in DSS status and producing fatal acute heart failure. This was discussed in a study conducted by Kularatna et al., 2005. In other studies similar results were reported in which circulatory complications occu-rred (Kamath and Suchitra, 2006). In our study 7 (0.9%) patients the encephalitis was found. In these patients were observed the change of men-tal status. This may be due to many factors such as shock, electrolyte disturbance and Acute Liver Failure (ALF) might contribute to encephalopathy (Malavige et al., 2007). Encephalitis in other study the reason for neurological manifestations contri-buting cerebral edema, direct neurotropic effect of dengue virus resulting in encephalitis or encepha-lopathy ,or secondary to hepatic dysfunction and metabolic derangements such as hypoglycemia and hyponatremia (WHO ,1999;Lum et al.,1996; Hendarto and Hadinegoro.,1992). Tachycardia 3 (0.4%) and bradycardia 3 (0.4%) was observed in our results. These cardiovascular complications were reported in the study conducted by Kama-th and Suchitra, 2006. Ascites the fluid collection was found in our results 13 (1.7%), pleural effusi-on 25 (3.3%), which is abnormal fluid collection. Jaundice was observed in 9 (1.2%) patients which is unusual characteristics of dengue. Hepatic in-volvement has been accepted in dengue and serum albumin level is considered as a prognostic marker (Nimmannitya et al., 1987). In another study he-patic dysfunction may be multifactorials, the most important causes are prolong shock associated metabolic acidosis and disseminated intravascular coagulation (DIC) with resultant ischemic hepati-tis (Nimmannitya et al., 1987; George et al, 1988, Mohan et al.,2000).

The total patients of dengue fever (DF) were 493 (65.2%), dengue hemorrhagic fever (DHF), 257 (34.0%) and DSS patients were noted 6 (0.8%). This could be due to rising of the popula-

578



tion in Penang. Here it shows that the highest pro-portion of dengue victims were children. Almost similar results were reported by the Social Statisti-cal Bulletin Peninsular Malaysia in 1973 and 1974 (Department of statistic 1973, 1974) that most of the children age was below 14 years. In 1973, over 50% of cases occurred in children below 14 years (Fang et al., 1984). In Malaysia the worst dengue and dengue hemorrhagic fever out break in 1982 that most cases occurred in the Chinese population over the age of 15 years.

At a conclusion the differences of clinical fea-tures between children and adults require further evaluation to find out and recognize the substan-tial factors involved. Dengue hemorrhagic fevers, dengue shock syndrome, ascites, Jaundice, Pleural infusion are complications; all these markers co-uld be helpful to make an early diagnosis of den-gue infection and on severity of infections, com-plications can be fatal for patients, which is required early identification in association with thoroughly monitoring and basic supportive care to save the life of the patients.

Limitations and recommendations

In retrospective study we cannot recognize the actual conditions of the patients and what were the physical and clinical observations of the medi-cal provider towards diagnosis, to depend on the written record which is not sure so the important data or observation may be not there. Various cli-nical features and complications were found in children and adults and complications especially in children, there is a need to conduct study on molecular genetic bases in context of these com-plications.

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Corresponding author Khurshid Alam School of Pharmaceutical Sciences, Universiti Sains Malaysia, Malaysia, E-mail: [email protected]

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Abstract

Background: Beta thalassaemia is highly pre-valent in Pakistan with a carrier rate of 5%. The thalassemic patients are given frequent blood tran-sfusions with irregular iron chelation treatment in our country.

Objectives: To determine frequency of endo-crine dysfunction in relation to iron overload in β-thalassaemic major children at Rawalpindi-Pa-kistan.

Subjects and Methods: A cross-sectional study was carried out on 131 β-thalassaemic ma-jor (BTM) children at Rawalpindi, Pakistan. Dia-gnosis of endocrine dysfunction was established by clinical and hormonal assays in the clinical laboratory of AM College Rawalpindi. Provocati-on tests for growth hormone (GH) secretion were applied. Plasma glucose, calcium and phosphate were measured on auto analyzer. Serum ferritin, GH, parathyroid (PTH) and thyroid profile were analyzed on Access 2 (Beckman, USA).

Results: One hundred and thirty one thalassae-mic children with median age of 10 (range 3-25) ye-ars (73 males; 58 females) participated in the study. The pre transfusion hemoglobin and serum ferritin were 8.7(3.2-14.8) g/dL and 2478(792-15334) µg/L respectively. Out of 131 patients, GH deficiency was found in 30%, hypoparathyroidism 17.5%, hypothyroidism 5%, subclinical hypothyroidism 3 %, diabetes mellitus 1.5% and impaired fasting glu-cose metabolism in 4% of patients. Patients were grouped according to recommended serum ferritin levels, group-I (<2500 µg/L) and group-II (>2500 µg/L). The group -II children had significantly low

levels of GH, PTH and FT4 hormones than group-I and endocrine complications were more prevalent among these patients (p=<0.01).

Conclusion: Multiple endocrinopathies espe-cially GH deficiency and hypoparathyroidism are the most frequent complications among our po-orly managed thalassaemic children. Endocrine dysfunction has positive correlation with serum ferritin which signifies the need for regular the-rapeutic interventions with the aim of preventing iron overload complications.

Key words: Beta Thalassemia, Endocrine Dysfunction, Iron overload, Ferritin, Growth re-tardation, Hypoparathyroidism, Hypothyroidism, Diabetes mellitus

Introduction

Thalassaemia is one of the most common ge-netic disorders worldwide and occurs more frequ-ently in the Mediterranean region, South Asia, and West Africa (1). The estimated frequency of mu-tated β-gene carriers in Pakistan is 5 % (2) and about 9000 β-thalassemic major children are born each year (3). Thalassemic patients require regu-lar blood transfusions and iron chelation therapy for their survival. Transfusion of erythrocytes has significantly increased the life expectancy of tha-lassaemic patients but at the same-time results in iron- induced complications (4). Even in carefu-lly managed patients, the endocrine dysfunction including hypogonadism, growth retardation, hy-poparathyroidism, hypothyroidism and diabetes mellitus have been reported (5-6)

Endocrine dysfunction in beta-Thalassaemic major Patients atrawalpindi, PakistanDilshad Ahmed Khan1, Asma Naseer Cheema1, Masood Anwar2, Farooq Ahmad Khan1

1 Department of Pathology Army Medical College, National University of Sciences and Technology, Pakistan,2 Islamic International Medial College, Riphah University, Pakistan.



The prevalence of endocrine complications among BTM children varies in different populati-ons depending on the literacy rate, socio-economi-cal status, availability of regular chelation therapy and transfusions. Hypoparathyroidism is found in 13.5% to 17% of thalassemic patients (7-8). The incidence of thyroid disorders is highly variable (1-18%) among different populations (9)(10). Lastly, the less common endocrine complication is diabetes mellitus (DM) which is limited to 10 % (11-12).

The guidelines regarding the schedule, dosage and optimal age to start chelation therapy in tran-sfusion-dependent thalassaemia patients are not properly followed in our country. The children are given frequent blood transfusions with irre-gular iron chelation treatment. That`s why Paki-stani thalassaemic patients are leading a misera-ble life as compared to advanced countries. The current status of endocrine complications among Pakistani β-thalassaemic major children is poor-ly characterized. So, the objectives of the present study were to determine the frequency of endocri-ne complications in relation to serum ferritin le-vels in irregularly chelated transfusion-dependent β-thalassaemic patients at Rawalpindi, Pakistan.

Subjects and Methods

Subjects:

We performed a cross sectional study on BTM patients at Army Medical College in collaboration with Thalassaemia center Rawalpindi. The study protocol was approved by institutional review and ethical committees of Army Medical College, Rawalpindi, NUST Pakistan.

Total 140 BTM children were randomly selec-ted from registered thalassaemia center Rawalpin-di patients. The subjects aged 3 to 25 years of eit-her sex were enrolled after informed consent. The medical history including age of diagnosis, tran-sfusion and chelation therapy were recorded. The children suffering from acute or chronic illness, auto immune disease and thalassaemia intermedia were excluded.

Clinical Evaluation

Clinical examination was carried out and ant-hropometric data was collected. Height and wei-ght percentiles were taken with the help of growth chart. Body mass index (BMI) was calculated. Diagnosis of BTM was re-confirmed with com-plete blood count and electrophoresis. Endocrine evaluation was carried out by qualified chemical pathologist. Two provocation tests including L dopa and exercise stimulation tests were applied for growth hormone assessment. Hypogonadism was evaluated as complete absence of secondary sexual characters after 12 years of age.

Biochemical Analysis

Blood sample (5ml) was drawn between 7:00-9:00am following an overnight fast. All the assays were performed at Clinical Pathology Laboratory, Army Medical College, Rawalpindi-Pakistan. Bi-ochemical analysis was carried out on Selectra E auto analyzer (Vita lab, Netherlands) following standard procedures of biochemistry by using pi-oneer diagnostic kits (USA). Plasma glucose was measured by glucose oxidase method (13). Serum phosphorous and total calcium were measured by using phosphomolybdic (14) and cresolphthalein complexone (15) colorimetric methods respec-tively. The Coefficient of variation (C.V) of the methods was found to be 3.7%.

Serum Ferritin and Hormonal assays

Serum Ferritin, GH, Parathyroid hormone (PTH), free thyroxin (FT4) and thyroid stimula-ting hormone (TSH) were estimated on Access II based on Chemiluminescent based immunoassay (Beckman, USA) by following the kit instruction. Two values of serum ferritin were obtained opti-mally one month apart, which were then averaged. Patients were divided according to recommended serum ferritin levels group-I (<2500 µg/L) and group II (>2500 µg/L). The diagnosis of hypo-parathyroidism was made in the presence of low serum PTH, low serum calcium, and high serum phosphate. CV of the assays was less than 5%.

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The data were analyzed by using standard SPSS software version-17 (SPSS Inc, Chicago). Des-criptive statistics including frequency and percen-tages were calculated. Chi-square test was applied for comparison of frequency of endocrine disor-ders. Mann-Whitney U test was applied for com-parison of analytes and hormones levels between group -I and II of BTM patients. The correlation between serum ferritin levels and hormones were determined by Spearman’s correlation. A p-value <0.05 was considered significant.

Results

Out of 140 BTM children selected by random draw, 131 participated in the study. Six children were unwilling to participate and three had acute illness. Baseline characteristics of BTM children are shown in the table-1. They comprised of 73 (56%) males and 58 (44%) females. The patients were receiving transfusions 3-4 times per month from thalassemia center but not taking regular chelation therapy at home.

Frequency distribution of different age group of the BTM patients is shown in figure -1. The majority of BTM children were younger than 10 years of age. Only thirty nine patients with age greater than 12 years were selected for evaluation of secondary sexual characteristics and hypogo-nadism was observed in 18(46%) patients. Out of 131 BTM patients, 48 (37%) had short stature (< 3rd centile). GH deficiency was diagnosed based

on peak median (range) GH hormone response va-lue 1.28(0.3-2.15 mIU/L) in 39 (30%) of children (Figure -2). Hypoparathyroidism was present in 23 (17.5%) of the study population. The patients had median (range) PTH levels 4.9(1.9-12ng/L) with serum calcium 1.8(1.38-1.90 mmol/L) and with high phosphorus concentration 2.4 (2.29-2.69mmol/L). Seven patients were diagno-sed hypothyroidism and 4 had subclinical hypot-hyroidism. Two patients had fasting blood glucose levels of 7.4 and 8.8mmol/L and diagnosed as dia-betics while 5 had impaired fasting blood glucose (5.6 to 6.9 mmol/L).

Figure 1. Frequency distribution of BTM patient’s age groups

GH deficiency was found in 30%, hypoparat-hyroidism 17.5%, hypothyroidism 5%, subclinical hypothyroidism 3 %, diabetes mellitus 1.5% and im-paired fasting glucose metabolism in 4% of patients

Table 1. Baseline characteristics of BTM children at Rawalpindi (n =131) Parameters Mean ± SD Median (Range)

Age(years) 10.13 ± 4.67 10 (3-25)Height(cm) 128±19.05 126(80-189)Weight(Kg) 25.49 ± 9.4 24 (11-60)BMI(kg/m2) 14.86 ± 3.05 14.79 (5.28-25.60)Ferritin(µg/L) 4498 ± 3479 2478 (792-15334)Hemoglobin(g/dL) 8.52 ± 2.17 8.7(3.2-14.8)HCT (%) 25.84 ± 6.6 26.8 (9.8-43.7)MCV(fL) 80.64 ± 5.01 81.2 (63.8-91.3)MCH(Pg) 26.77 ± 2.00 26.9 (19.9-31.7)MCHC(g/dL) 35.2 ± 23.5 33.2 (25-30)



Figure 2. Percentage distribution of endocrine complications in BTM patients

BTM patients were placed in two groups based on the recommended serum ferritin levels of 2500 µg/L levels. Seventy two (55%) BTM children had serum ferritin less than recommended levels with median of 2034 ug/L (group -I) while rest 45% (group-II) patients had greater than 2500ug/L. The group -II children had significantly low levels of GH, PTH and FT4 hormones than group-I (Ta-ble-2). There was significant negative correlation of serum ferritin levels with GH r=0.419(p<0.01) and PTH r=0.338 (P<0.01). Endocrine complicati-ons were dominantly observed in group II patients (Figure- 3). There were 48% BTM patients with one complication, 13% with two complications and 2% with three complications, while no endo-crine disorder was found in 37%.

Figure 3. Comparison of endocrine complicati-ons between group I and group II

Discussion

We have studied the current status of endocrine complications among BTM patients. This paper is the first attempt to compile data on endocrine com-plications in such a large cohort in Rawalpindi Pa-kistan. Growth retardation due to GH deficiency was the common problem among our thalassaemi-cs especially in the children who had high level of serum ferritin. Increased iron deposition gradually deteriorates the anterior pituitary function and is responsible for GH insufficiency. Besides GH de-ficiency, hypothyroidism was also contributing to short stature but to lesser extent (16). Our results of GH dearth were slightly lower than studies con-ducted in Tehran and Israel in which GH paucity was reported as 39.3 %(5) and 36 % (6) respecti-vely Scacchi et al, reported (17) GH deficiency in 22.3% of the patients. GH reserves were lacking in 53 % of the thalassemic patients of Taiwan (18). The last two study results were observed in only

Table 2. Comparison of hormones, calcium, phosphorus and blood glucose levels between two groups of BTM patients.

Parameters Group I (< 2500ug/L)n =72

Group II (>2500ug/L)n =59 P value

Ferritin (ug/L) 2034(792-2498) 7057(2650-15334) 0.001GH (µg/L) 7.6 (1.01-17.7) 2.04(0.3-13.2) 0.001Calcium (mmol/L) 2.11(1.99-2.66) 1.94(1.03-2.18) 0.001Phosphate (mmol/L) 1.84(1.30-2.56) 2.14(1.99-2.69) 0.001Parathyroid (ng/L) 16.6(3-53) 11.9(1.90-35.2) 0.001Blood Glucose (mmol/L) 4.9(3.8-5.6) 5.2(4.4-8.8) 0.001Free T4 (pmol/L) 11.7(8.13-16) 10.8(4.4-14.2) 0.001TSH (mIU/L) 1.4(0.4-4.4) 1.9(0.8-75) 0.20

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29 and 59 patients respectively. So, these variations from our results can be attributable to management of patients and low power of detection due to small sample size.

The second important endocrinopathy present in our patients was hypoparathyroidism and frequen-cy was in close concordance with 13.5 & 16.93% reported in a study (7-8). But this figure was higher as compared to studies reported by (5, 19) Chern and Shamshirsaz, 7.6% and 10.7% respectively. The significant numbers of patients with hypopara-thyroidism indicates that careful monitoring of pa-rathyroid function of BTM patients is also required in routine clinical practice.

The frequency of hypothyroidism in our study population was comparatively low. The prevalence of hypothyroidism is tremendously variable in dif-ferent studies (20-21). We also observed only four cases of subclinical hypothyroidism which is quite lower as compared to 20 % reported by study con-ducted in India (22). Majority of our patients are from 6 to 10 years. So, it can be explained as “hy-pothyroidism is a complication of advanced age”.

Diabetes was found infrequent in our study. This is considerably lower than the 4.3% and 5.4% of patients developing IDDM in studies conducted by other researchers (9-10) However our results were in close proximity to study done in 2008 (23). The severity and type of glucose disturbances depends on iron overload as well as genetic, and socioecono-mical factors. We observed that we had only 3.9% patients with impaired fasting in contrast to a recent report revealing 28.6% (24). The low incidence of DM and impaired fasting levels indicate that our thalassaemic patients have good insulin reserves.

Our study highlighted that Iron overload is di-rectly related to frequency of complications. High prevalence of endocrine complications were obser-ved in the BTM patients who had high serum ferri-tin levels than recommended levels. It indicates that iron overload due to multiple transfusions and with irregular chelation therapy is main culprit in this scenario. Ignorance about the treatment regimen reduces the significance of chelation therapy for affected community. They think that transfusion is sufficient to save the life of thalassaemia children. They are completely ignorant about the monster of iron overload resulting from irregular chelation. Therefore, they pay less attention towards this as-

pect of therapy. The second important factor is poor compliance of the patients for iron chelation. Oral chelation therapy has its own side effects like nau-sea and vomiting while use of subcutaneous injec-tions (5 days/week) is limited due to pain and bot-heration. The third one cause is stumpy purchase power of our people thalassaemia major is frequent problem of people belonging to low economical status due to high ratio of cousin marriages in this group. Chelation is a costly regimen. People some-how manage blood transfusion but they are unable to afford the chelation therapy. We could not evalu-ate gonadal dysfunction with GnRh stimulation test because of unwillingness of majority of patient for the test.

Conclusion

Multiple endocrinopathies especially GH defi-ciency and hypoparathyroidism are most frequent complications among our poorly managed thalas-saemic children. Endocrine dysfunction has positi-ve correlation with serum ferritin. These important facts regarding high frequency of endocrine com-plications suggest and necessitate regular therape-utic interventions to improve the quality of life and enhance life expectancy of Pakistani thalassemic children.

Acknowledgement

This study was a part of the Project No. 20-837/R&D/07/319 entitled, “Quantitative Evaluation of Iron Overload among Pakistan B-Thalassaemic Major Children” supported by Higher Education Commission (HEC) Pakistan

References

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2. Zafar AM, Zuberi L, Khan AH and Ahsan H. Uti-lity of MRI in assessment of pituitary iron overload. J Pak Med Assoc 2007; 57(9): 475-477



3. Rehman M and Lodhi Y. Prospects and future of conservative management of beta thalassemia major in a developing country Pak J Med Sci 2004 ; 20(2): 105-112

4. Malik S, Syed S and Ahmed N. Complications in tran-sfusion–dependent patients of ß-thalassemia major: A review. Pak J Med Sci 2009; 25(4): 678-682.

5. Shamshirsaz AA1, Bekheirnia MR, Kamgar M, Pour-zahedgilani N, Bouzari N, Habibzadeh M, Hashemi R, Shamshirsaz AA2, Aghakhani S, Homayoun H and Larijani B. Metabolic and endocrinologic com-plications in beta thalassemia major: a multicenter study in Tehran. BMC Endocr Disord 2003; 3(1)4.

6. Shalitin S, Carmi D, Weintrob N, Phillip M, Mi-skin H, Kornreich L, Zilber R, Yaniv I and Tamary H. Serum ferritin level as a predictor of impaired growth and puberty in thalassemia major patients. Eur J Haematol 2005 ;74(2):93-100.

7. Angelopoulos NG, Goula A, Rombopoulos G, Kal-tzidou V, Katounda E, Kaltsas D and Tolis G. Hy-poparathyroidism In Transfusion-Dependent Pa-tients With Β-Thalassemia. J Bone Miner Metab 2006;24(2): 138-145

8. Moaddab MH, Hashemipour M & Mahmoud Nade-ri. The prevalence of endocrine complications in pa-tients with thalassemia major. Endocrine Abstracts 2008; 16: 578.

9. Mehrvar A, Azarkeivan A, Faranoush M, Mehrvar N, Saberinedjad J, Ghorbani R and Vossough P. Endocrinopathies in patients with transfusion-de-pendent beta-thalassemia. Pediatr Hematol Oncol 2008; 25(3): 187-94.

10. Jaruratanasirikul S, Wongcharnchailert M, La-osombat V, Sangsupavanich P and Leetanaporn K. Thyroid function in beta-thalassemic children receiving hypertransfusions with suboptimal iron-chelating therapy. J Med Assoc Thai 2007; 90(9): 1798-802

11. Cunningham MJ, Macklin EA, Neufeld EJ and Cohen AR. Complications of β-thalassemia major in North America. Blood 2004; 104(1): 34-39

12. Khalifa AS, SalemM, Mounir E, El-Tawil MM, El-Sawy M and AbdAl-Aziz MM. Abnormal glucose tolerance in Egyptian beta-thalassemic patients: possible association with genotyping.Pediatric Di-abetes 2004; 5(3): 126—132.

13. Barham D and Trinder P. An improved colour rea-gent for the determination of blood glucose by the oxidase system. Analyst 1972; 97(151): 142-145

14. Endres DB and Rude RK. Disorders of bone. In: Brutis BA, Ashwood ER, Bruns BE editors. Tietz Fundamentals of Clinical Chemistry, 6th ed. Else-vier; 2008, pp. 711-731.

15. Lorentz K. Improved determination of serum calci-um with 2-cresolphthalein complexone. Clin Chem Acta 1982; 126(3): 327-334.

16. Fuchs GJ, Tienboon P, Linpisarn S, Nimsakul S, Leelapat P, Tovanabutra S, Tubtong V, DeWier M and Suskind RM. Nutritional factors and thalassa-emia major. Arch Dis Child 1996; 74(3):224–227

17. Scacchi M, Danesi L, Cattaneo A, Valassi E, Pecori Giraldi F, Argento C, D’Angelo E, Mirra N, Car-nelli V, Zanaboni L, Cappellini MD and Cavagnini F. Growth hormone deficiency (GHD) in adult tha-lassaemic patients. Clin Endocrinol (Oxf) 2007; 67(5): 790-795.

18. Wu KH, Tasi FJ and Peng CT. Growth hormone de-ficiency in patients with Beta thalassemia major and the efficacy of recombinant GH treatment. Ann Hematol 2003; 82(10): 637-640

19. Chern JP & Lin KH .Hypoparathyroidism in tran-sfusion dependent patients with beta-thalassemia. J Pediatr Hematol Oncol 2002; 24(4): 291–293

20. Phenekos C, Karamerou A, Pipis P, Constantoula-kis M, Lasaridis J, Detsi S and Politou K: Thyroid function in patients with homozygous beta-thalasse-mia. Clin Endocrinol (Oxf) 1984; 20(4): 445-450.

21. Farmaki K, Tzoumari I, Pappq C, Chouliaras G, Berdoukas V. Normalization of total body iron with very intensive combined chelation reverses cardiac and endocrine complications of thalassaemia ma-jor. Br J Haematol 2009; Nov 12 [Epub ahead of print].

22. Jain M, Sinha RS, Ghellani H and Anand NK. Assessment of thyroid functions and its role in body growth in thalassaemia major. Indian pediatrics 1995; 32(2): 213-219

23. Siklar Z , Citak FE, Uysal Z, Al G, Ertem M, En-giz Z, Adıyaman P, leri T , Gzdaolu S and Berbe-rolu M .Evaluation of glucose homeostasis in tran-sfusion-dependent thalassemic patients. Pediatric Hematology and Oncology 2008; 25(7): 630 – 637

24. Najafipour F, Aliasgarzadeh A, Aghamohamad-zadeh N, Bahrami A, Mobasri M, Niafar M and Khoshbaten M. A cross-sectional study of metabo-lic and endocrine complications in beta-thalassae-mia major.Ann Saudi Med 2008; 28(5): 361-366.

Corresponding author Dilshad Ahmed Khan Dept of Pathology, Army Medical College, Pakistan, E-mails: [email protected]; [email protected]

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Abstract

Background and aims: Chronic periapical di-sease with pulp origin is an inflammatory disorder caused by bacterial infection. Cytokines such as interleukin-6 have important role in pathogenesis of inflammatory diseases. Chronic infection could affect general health by increasing production of cytokines such as interleukin-6 that probably play some roles in pathogenesis of pulpal and periapi-cal diseases. The aim of present study was a com-parative evaluation of serum interleukin-6 in pati-ents with periapical lesions and healthy controls.

Materials and Methods: This analytical case-control study included 40 patients suffering from chronic periapical lesions and 40 healthy persons without any oral diseases. After informed consent was obtained, clinical and radiographical exami-nation was carried out and blood samples were collected. Serum interleukin-6 was measured by ELISA method. Data were subjected to SPSS sof-tware and t-test was used for statistical analysis.

Results: Serum interleukin-6 concentration was significantly higher in patients group in com-parison with healthy controls (P<0.001).

Conclusion: The results of present study indi-cate that interleukin-6 produced in periapical lesi-ons may serve as a marker of pathologic inflam-matory activities in chronic periapical lesions.

Key words: Cytokine, Interleukin-6, Periapi-cal lesions

Introduction

Periapical lesions are inflammatory diseases that develop as the result of root canal bacterial infections1, and may result in periapical bone de-struction because of host defensive-microbial di-sturbances.2 The host defensive reactions against root canal pathogens may elicit cellular and ho-moral immune responses as well as inflammatory processes.3 Cytokines are low-weight messenger molecules between the host cells and secreted by different immune cells that are believed to have important role in treatment and pathogenesis of many inflammatory diseases, such as periradicu-lar lesions.4 The term interleukin (IL) is used to describe a cytokine that communicate leukocytes as an intracellular way.5 Interleukin-6 (IL-6) has traditionally been considered a pro-inflammatory cytokine that may have a role in inflammatory process of periapical lesions.6 However, emerging data suggests that IL-6 is a multifunctional cyto-kine which is mostly produced by several types of immune cells such as monocytes, macrophages, Th-2 lymphocytes, activated B cells and PMN ce-lls.7 Furthermore, this type of interleukin may re-lease locally in inflamed pulpal and periradicular lesions, especially in chronic forms.8

Chronic infections could affect general health by increasing production of cytokines, therefo-re levels of these biologic markers could be em-ployed to enhance the early detection of inflamma-tory pulpal diseases. In fact, quantitation of these molecules in healthy and diseased pulpal tissues is very important.5 Elevated levels of IL-6 and IL-8

serum Interleukin-6 as a serologic marker of Chronic Periapical Lesions; A Case-control studyBehnoush Bakhtiari1, Hamed Mortazavi2, Mehrdad Hajilooi3, Shahrzad Nazari4

1 Dental School, Hamedan University of Medical Sciences, Iran,2 Dept of Oral Medicine, Dental School, Hamedan University of Medical Sciences, Iran,3 Research Center of Dental School, Hamedan University of Medical Sciences, Iran,4 Dept of Endodontics, Dental School, Hamedan University of Medical Sciences, Iran,



in fibroblast subpopulation have been demonstra-ted in adult periodontitis.9 Moreover, serum IL-6 amounts were reported significantly higher in mo-re-extensive periodontal lesions than the normal periodontal tissues.10

On the other hand, an increased locally produc-tion of IL-6 in periapical lesions was shown in se-veral studies8,7,11 with a clear association between levels of IL-6 and the extent of tissue damages.11 To our best knowledge, the alteration of IL-6 in serum in patients suffering from such lesions is poorly understood. Therefore, the purpose of this study was to determine the serum concentrations of IL-6 in patients suffering from chronic periapi-cal lesions compared to healthy controls.


During one and half year (2006-2007), all the patients with periapical lesions (total of 40 pati-ents) were reported to a referral dentistry center (department of Oral Medicine) in the city of Ha-medan, Iran and 40 healthy individuals whom their gender and age matched, were included the study as control group.

Inclusion criteria consisted of lack of any hi-story of diabetes, hepatitis, HIV infection, immu-nosuppressive chemotherapy, bleeding disorder and severely compromised immune function.12 Exclusion criteria included:

1) known systemic diseases such as inflammatory or autoimmune diseases (BehÇet’s syndrome, arthritis, Reiter syndrome, AIDS, etc…),

2) history and/or presence of other infections, 3) specific physiological condition (pregnancy

or menstruation),4) periodontal diseases,5) any type of oral ulcers,6) current smokers or a history of smoking,7) treatment with any medication known to

affect the serum level of inflammatory markers in recent 3 months,

8) any clinical signs of spontaneous pain, swelling, and tenderness around the periapical overlying mucosa, pain during percussion, presence of pus or sinus tract as well as positive dental vitality signs/symptoms.

All of the patients were healthy volunteers with clinically diagnosed periapical lesions. Prior to the study, each patient was informed of the nature and risks of the study and signed a consent form approved by the Ethics Committee of Hamedan University of Medical Sciences, Hamedan, Iran.

A periapical radiographic was taken after de-tection of periapical lesion in panoramic radio-graphic view of involved tooth. The samples were observed by two examiners (an Oral Medicine specialist and an Endodontist) to determine the size of radiolucent area around the periapex by di-gital caliper (Mitutoyo, USA). On the basis of the-ir radiographical presentations, the subjects were divided into two groups: 40 patients with 2-5 mm radiolucent asymptomatic periapical lesion (case group), and 40 healthy individuals without any ra-diolucent periapical lesions, as well as matching gender, age and place of residence, as the control group.

Fasting venous blood samples were taken all within the time frame between 8 AM to 10 AM. Serum was extracted from the blood samples by centrifuge and then stored at -700C for subsequent analysis. The concentrations of IL-6 were analy-zed using a commercial enzyme-linked immu-nosorbent assay kit (Bender Med System, serial number BMS213/2, Austria). The assays were performed according to the manufacture’s instruc-tion. The concentration of IL-6 present in serum of participants was calculated with the reference to the standard curve that was constructed using 450 nm wave length spectro-photometer (ELISA reader, Averneas, USA) as pictogram per millili-ter (pg/ml).

The statistical analysis was performed using the SPSS software (SPSS Inc., Chicago, IL,

USA) and the differences between case and control group’s distribution for serum IL-6 were determined by student t-test.

Results

This study quantified the levels of IL-6 in se-rum of 80 participants, with the age range between 17 to 46 years (Table 1). No significant differences were found among different groups according to the age and sex (P>0.05).

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Table 2 illustrates the average levels of IL-6 in each group.

Serum concentrations of IL-6 were signifi-cantly correlated with presence of periapical lesi-ons (P<0.001).

Discussion

The present study was undertaken to establish the association between periapical lesions and in-creased IL-6 in serum of affected patients. A we-alth of information has shown that cytokines have become one of the most important aspects of cli-nical research because they are new markers for diagnosis or treatment and have been implicated in the pathogenesis of several diseases including peritoneal sepsis, inflammatory bowel diseases, systemic sclerosis , cardiovascular diseases and periapical lesions.13-16 It has been clarified that various cytokines and prostaglandins are being produced locally in periapical lesions and they are important communication factors for cross-regu-lation of immune responses.16 Furthermore, these substances may consider the diagnostic markers of periapical periodontitis.16 T-cell derived cyto-kines in turn categorized to Th-1 such as IFN-γ, IL-2, IL-1 and cytokines produced by Th-2 lymp-

hocytes including IL-4, IL-10 and IL-13.11 Eleva-ted levels of prostaglandins and cytokines such as IL-8 16-18 , IL-1RA4 IL-6 4,8,12,19 , TGFβ-1 4 , IL-2 5 , IFN-γ 20 , IL-4 20 , IL-1β 12,19 , TNF-α 19,21 , IL-10 12 and IL-1α 21 were found to be highly associated with periapical lesions in human and animal stu-dies. IL-6 was also detected in neutrophils from peripheral blood and inflammatory periradicular tissues.22 It was shown that IL-6 plays a complex and central role in regulation of the immune res-ponse.2 An animal study has demonstrated that IL-6 is a proinflammatory mediator because it is produced locally and early, following stimulation by IL-1 and TNF-α in the inflammatory cascade.6 Finally, IL-6 increases number of osteoclasts from colony-forming granulocyte macrophage 6 and has been demonstrated to have the capacity of bone resorption.11 On the other hand, it has been shown that IL-6 interferes with apoptosis in neutrophils.22 Kawashima et al. observed the production of IL-6 in normal murine periapical tissues, especially it is further induced fourteen days after pulp expo-sure. However, they mostly failed to detect IL-6 mRNA.11 Redics et al. suggested that higher con-centration of the local IL-6 present in symptoma-tic lesions containing epithelial cells, represent an active stage of apical periodontitis rather than asymptomatic, poor PMN cell, non-epithelialized

Table 1. Demographic data of the study participantsStudy groups Sex Age

(Number) Number (%) (Mean + SD)

Case (40)Female 14 (35)

27.8 +3.1Male 26 (65)

Control (40)Female18 (45)

23.8 + 8.2Male 22 (55)

Table 2. Serum IL-6 concentration in case and control groupsStudy group Serum IL-6 level MD* SE ** CI*** t P.value

(Number) (Mean + SD)Case (40) 1.92+1.55

1.31 0.27 0.59 » 2.03 4.81 0.001Control (40) 0.61+0.73

* Mean Difference** Standard Error*** Confidence Interval



chronic and healing lesions.7 In a recent study in terms of periradicular lesions, the effect of a new antibiotic (linezolide) was evaluated on the level of some cytokines such as IL-6. Concentrations of this cytokine was reported between 0.1 to 2.9 pg/ml in periapical tissue extracts.4 This discrepancy could be attribute to the difference in the type of samples, methods and units used for measuring IL-6. To our knowledge, cytokines were produced and affected locally in inflammation areas, as we verified in the present study. Because of the close location of capillaries to the endodontic microbial flora, there would be the likelihood of increasing cytokines in blood.

Some in vitro studies have shown that signi-ficant blood levels of IL-6 were produced by neutrophils stimulated with microbial lipopoli-saccharides when compared with unstimulated cells. Their findings indicated that there was a dose-dependent increase in IL-6 level when cells were stimulated with A. actinomycetecomitans extract and to a less degree after stimulation with P.gingivalis.22 These results are relatively in agree-ment with present study. Since IL-6 can be produ-ced by a variety of cells such as neutrophils, the levels of IL-6 in mentioned study were higher than our findings. In addition, these differences may be the result of direct stimulation of neutrophils.

In conclusion, the results of the present study show that serum level of IL-6 in patients with pe-riapical lesions was significantly higher (by 3.14 fold) than healthy controls. Moreover, it highli-ghts the potential for improving our understanding about IL-6 as a sensitive indicator of inflammation and may be useful for evaluation of periapical pe-riodontitis. These data might provide the first step toward association between elevated levels of se-rum IL-6 and chronic periapical lesions.

Acknowledgments

This study was a general graduate thesis carri-ed out in Dental School, Hamedan University of Medical Sciences. The authors would like to thank Dr. M. Safari; the dental student, Dr. H. abdolsa-madi, and Dr. M. Farshchian for their kind assi-stance, Mr. Kh. Mani Kashani for statistical ad-vice, and all the patients who participated in the

study. Also authors would like to thank Deputy of Research, Hamedan University of Medical Scien-ces for supporting towards the publication of this paper through grant No: 48933

References

1. Colić M, Vasilijić S, Gazivoda D, Vucević D, Mar-janović M, Lukić A. Interleukin-17 plays a role in exacerbation of inflammation within chronic peria-pical lesions. Eur J Oral Sci 2007;115:315-20.

2. Prso IB, Kocjan W, Simić H, Brumini G, Pezelj-Ri-barić S, Borcić J, et al. Tumor necrosis factor-alpha and interleukin 6 in human periapical lesions. Me-diators Inflamm 2007;2007:38210.

3. Takahashi K. Microbiological, pathological, infla-mmatory, immunological and molecular biologi-cal aspects of periradicular disease. Int Endod J 1998;31:311-25.

4. Danin J, Linder L, Lundqvist G, Wretlind B. Cyto-kines in periradicular lesions: the effect of linezolid treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96:492-8.

5. Rauschenberger CR, Bailey JC, Cootauco CJ. De-tection of human IL-2 in normal and inflamed den-tal pulps. J Endod 1997;23:366-70.

6. Balto K, Sasaki H, Stashenko P. Interleukin-6 de-ficiency increases inflammatory bone destruction. Infect Immun 2001;69:744-50.

7. Radics T, Kiss C, Tar I, Márton IJ. Interleukin-6 and granulocyte-macrophage colony-stimulating factor in apical periodontitis: correlation with cli-nical and histologic findings of the involved teeth. Oral Microbiol Immunol 2003;18:9-13.

8. Barkhordar RA, Hayashi C, Hussain MZ. Detection of interleukin-6 in human dental pulp and periapi-cal lesions. Endod Dent Traumatol 1999;15:26-7.

9. Dongari-Bagtzoglou AI, Ebersole JL. Increased presence of interleukin-6 (IL-6) and IL-8 secreting fibroblast subpopulations in adult periodontitis. J Periodontol 1998;69:899-910.

10. Bretz WA, Weyant RJ, Corby PM, Ren D, Weissfeld L, Kritchevsky SB, et al. Systemic inflammatory markers, periodontal diseases, and periodontal infections in an elderly population. J Am Geriatr Soc 2005;53:1532-7.

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11. Kawashima N, Stashenko P. Expression of bo-ne-resorptive and regulatory cytokines in mu-rine periapical inflammation. Arch Oral Biol 1999;44:55-66.

12. de Sá AR, Moreira PR, Xavier GM, Sampaio I, Kalapothakis E, Dutra WO. Association of CD14, IL1B, IL6, IL10 and TNFA functional gene pol-ymorphisms with symptomatic dental abscesses. Int Endod J 2007;40:563-72.

13. Yamamoto T, Umegae S, Kitagawa T, Matsumoto K. Intraperitoneal cytokine productions and their relationship to peritoneal sepsis and systemic in-flammatory markers in patients with inflammatory bowel disease. Dis Colon Rectum 2005;48:1005-15.

14. Meloni F, Caporali R, Marone Bianco A, Pas-chetto E, Morosini M, Fietta AM, et al. Cytokine profile of bronchoalveolar lavage in systemic sc-lerosis with interstitial lung disease: comparison with usual interstitial pneumonia. Ann Rheum Dis 2004;63:892-4.

15. Cesari M, Penninx BW, Newman AB, Kritchevsky SB, Nicklas BJ, Sutton-Tyrrell K, et al. Inflamma-tory markers and cardiovascular disease (The He-alth, Aging and Body Composition [Health ABC] Study). Am J Cardiol 2003;92:522-8.

16. Shimauchi H, Takayama S, Narikawa-Kiji M, Shimabukuro Y, Okada H. Production of interleu-kin-8 and nitric oxide in human periapical lesions. J Endod 2001;27:749-52.

17. Huang GT, Potente AP, Kim JW, Chugal N, Zhang X. Increased interleukin-8 expression in inflamed human dental pulps. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;88:214-20.

18. Marton IJ, Rot A, Schwarzinger E, Szakáll S, Radi-cs T, Vályi-Nagy I. Differential in situ distribution of interleukin-8, monocyte chemoattractant prote-in-1 and Rantes in human chronic periapical gra-nuloma. Oral Microbiol Immunol 2000;15:63-5.

19. Ataoğlu T, Ungör M, Serpek B, Haliloğlu S, Ataoğlu H, Ari H. Interleukin-1beta and tumour necrosis factor-alpha levels in periapical exuda-tes. Int Endod J 2002;35(2):181-5.

20. Kabashima H, Nagata K, Maeda K, Iijima T. Pre-sence of IFN-gamma and IL-4 in human periapi-cal granulation tissues and regeneration tissues. Cytokine 2001 7;14:289-93.

21. Muglali M, Komerik N, Bulut E, Yarim GF, Cele-bi N, Sumer M. Cytokine and chemokine levels in radicular and residual cyst fluids. J Oral Pathol Med 2008;37(3):185-9.

22. Euler GJ, Miller GA, Hutter JW, D’Alesandro MM. Interleukin-6 in neutrophils from peripheral blood and inflammatory periradicular tissues. J Endod 1998;24:480-4.

Corresponding author Behnoush Bakhtiari Dental School, Hamedan University of Medical Sciences, Iran, E-mail : [email protected]



Abstract

Background: İn order to improve the quali-ty of medical education, medical faculties of the universities are searching for better pedagogical approaches. To determine the frequency of the ar-ticles about new approaches in medical education is our main objective in this study.

Method: An exclusive search was conduc-ted in PubMed and other life science journals for biomedical articles. Total of 120 articles were comprised the term constructivism. 6 were con-ducted between 1980-1989, 41 were conducted between 1990-1999 73 were conducted between 2000-2007. Total of 44 articles were found to in-clude the term IBL. Most of the research (n=32) were conducted in the last decade whereas. Total of 3080 articles were cited that included the term PBL. PBL was the most commonly encountered teaching strategy on the 21st.century (n=2133). Total of 1030 articles were found to comprise the term CL. CL was the most commonly encountered learning strategy on the 1980’s (n=161).

Conclusion: It is evident that there has been a considerable increase in research about construc-tivist teaching and learning in the medical field from 1980’s to the 21st. Century. More research about constructivism need to be published in the medical journals.

Key words: constructivism, inquiry-based le-arning, problem-based learning, cooperative lear-ning.

Introduction

In order to improve standarts and the quality of medical education, medical faculties of the uni-versities are searching for new and better pedago-gical approaches. The purpose of this research is to explain the theoretical base for the constructi-vist teaching strategies through the review of lite-rature in medical education.

In medical education, assessment of medical competence and performance, important changes have taken place in the last 5 decades (1). Especi-ally ın the last 10 years, these changes have affec-ted the basic concepts and constructivism became increasingly popular as referent for professional actions in education, as a conflicting paradigm to positivism (2). John Dewey, asserts that people construct their own understanding and knowled-ge of the world, through experiencing things and reflecting on those experiences (3). In assessment the change from trait-orientated to competency or role-orientated thinking has given rise to a whole range of new approaches (1).

Prior knowledge, reflection and discussion are the key steps of constructing new knowledge. Espe-cially; class group discussions, where students can test their previous knowledge and their alternative conceptions, lead students to assimilate or accom-modate new knowledge. Constructivist view of le-arning can be transferred to active teaching strategi-es like inquiry, and real-world problem solving (4). At the same time, these kind of teaching techniques

use Of Constructivist Approach for medical Education: review of LiteratureKurtulus Ongel¹, Sevinc O.Erdal², Selnur Erdal³, Haluk Mergen4, Nazan Karaoglu5

¹ Department of Family Medicine, Faculty of Medicine, University of Suleyman Demırel, Turkey.² Science Educator, Columbus, OH, USA³ The Ohio State University Medical Center, OH, USA4 Uludag University of Bursa, Turkey.5 Department of Medical Education and Informatics, Meram Faculty of Medicine, Selcuk University, Turkey.

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can improve medical care by boosting doctors’ per-formance and enhancing patient safety.

A worldwide association for medical educa-tors, The Association for Medical Education in Europe (AMEE), again emphasized the importan-ce of integration classical teaching styles with new pedagogical approaches (5).

Material and Methods

An exclusive search was conducted in PubMed; a medical database of the U.S. National Library of Medicine that includes over 16 million citations from MEDLINE and other life science journals for biomedical articles. Keywords such as constructi-vism, inquiry-based learning (IBL), problem-based learning (PBL) and cooperative learning (CL) were searched. Various medical journal types (clinical, dental, and nursing) were analyzed.

Results

İt was evident from our research that in the last decade constructivism has gained considera-ble attention in the medical field. It is found that between 1980-2007 there has been a considerable increase in research about constructivist teaching and learning in the medical field. (See in table-1)

Figure 1. Analysis of research articles based on time periods

Constructivism

Constructivism theory takes human being as the center of teaching. According to this theory,

everything is created by the individual. Construc-tivism mentions that personal experience cannot be separated from knowledge. Especially cogni-tive and social experiences are very important in constructivist hypothesizes (6). Shortly, we can call constructivism as a philosophical view on how we come to understand or know . Constructi-vism has some basic principles different from the other education techniques.

Total of 120 articles were comprised the term constructivism. 6 were conducted between 1980-1989, 41 were conducted between 1990-1999 73 were conducted between 2000-2007. (See in ta-ble-2)

Figure 2. Analysis of research articles based on constructivism

Inquiry–based learning (IBL)

One of the most important teaching techniques used today, is the inquıry-based learning. İnquıry-based learning can provide valuable opportuniti-es for students to improve their understanding of both science content and scientific practices (7). At the same time, we can call inquiry-based learning as a flexible holistic adaptation of problem-based learning (8). By this method, students determine what they need to learn and asses their progress by themselves.

Total of 44 articles were found to include the term IBL. Most of the research (n=32) were con-ducted in the last decade whereas, no articles were found on the 80’s.



Figure 3. Analysis of research articles based on inquiry-based learning

Problem-based learning (PBL)

Problem-based learning (PBL) is a learner-cen-tered educational method and it is widely used in higher education. İn this method, learning is based on real world problems. These teaching style gives more responsibility to students for their own edu-cation. There is evidence avaible that students and faculty are higher satisfied with PBL (9).

Total of 3080 articles were cited that included the term PBL. PBL was the most commonly enco-untered teaching and learning strategy on the 21st.century (n=2133).

Figure 4. Analysis of research articles based on problem-based learning

Cooperative learning (CL)

Working situations and conditions of the me-dical doctors are changing day by day. Especially information-based, expensive high technologies are becoming more important. Neverthless, we

can not deny the role of basic medical skills in pa-tient examination. Because of this; medical facul-ties must produce doctors capable of professional medical skills, thinking skills, communication and social skills. And that can be proper by cooperati-ve learning.

Total of 1030 articles were found to comprise the term CL.CL was the most commonly encoun-tered teaching and learning strategy on the 1980’s (n=161).

Figure 5. Analysis of research articles based on cooperative learning

Analysis of research articles based on journal group

Three groups of journals were analyzed: 1) Clinical Trial Journals (CJT),2) Dental Journals (DJ), and 3) Nursing Journals (NJ).

Out of 120 articles that include the term Con-structivism, 109 were cited in Nursing Journals, 7 were cited in Dental Journals, and 4 were cited in Clinical Trial Journals.

Inquiry-based Learning:Out of the 44 articles that included IBL, 27

were found in Nursing Journals, 11 were in Clini-cal Trial Journals, and 6 were in Dental Journals.

Problem-based Learning:Out of the 3080 articles that included IBL,

1509 were found in Nursing Journals, 986 were

594



in Clinical Trial Journals, and 585 were in Dental Journals.

Cooperative Learning:Out of the 1030 articles that included IBL, 783

were found in Nursing Journals, 144 were in Clini-cal Trial Journals, and 103 were in Dental Journals.

Discussion

It is evident that there has been a considerable increase in research about constructivist teaching and learning in the medical field from 1980’s to the 21st. Century. Most of this researches howe-ver; were cited in Nursing Journals. More research about constructivism need to be published in the medical journals.

In order to become life-long learners and pro-blem solvers medical students need to be exposed to constructivist teaching strategies as well as un-derstand the theoretical bases of constructivism in their medical education programs (10,11).

We can say that, there is a need to reconceptu-alise the role of medical education and to renego-tiate relationships between teachers and students in medical faculties by the help of new education techniques.

Acknowledgement

This study was presented in the 18. WONCA World Conference – Singapore - 2007

References

1. Schuwirth LW, Van Der Vleuten CP. Changing edu-cation, changing assessment, changing research?, Med. Educ. 2004, 38(8): 803-804.

2. Tobin, K; and Tippins, D. (1993). Constructivism as a referent for Teaching and Learning. In K. To-bin (ed), controvert in classroom research, Sec. Ed) Buckingham. Open University Press.

3. Dewey, J., (1933). How We Think. Chicago: Henry Regnery.

4. Öngel K, Mergen H, Katırcı E, Ak C, Sarıkan I, Özkan S, Cesur G, Erdal S. “Investigating the sci-entific process skills of medical students in relati-on to medical decision making: Research on basic medical science competence”, Zdravniski Vestnik 2009 November, 78:626-632.

5. Segouin C, Hodges B, Byrne PN. World conference on medical education: a window on the globalizing world of medical education ? Med. Teach. 2007, 29(2-3): 63-66.

6. Whitman N. A review of constructivism: understan-ding and using a relatively new theory. Fam Med. 1994, 26(3): 141-142.

7. Daniel CE, Douglas NG, Roy DP. Addressing the Challenges of Inquiry-Based Learning Through Technology and Curriculum Design, Journal of the Learning Sciences 1999, 8(3-4): 391-450.

8. Magnussen L, Ihsıda D, Itano J. The impact of the use of ınquıry-based learning as a teaching metho-dology on the development of critical thinking. Eric database; documentatıon number: EJ615973.

9. Dolmans DH, De Grave W, Wolfhagen IH, Van Der Vleuten CP. Problem-based learning:future chall-enges for educational practice and research. Med Educ. 2005, 39(7): 732-741.

10. Erdal SO, Ongel K. Promoting Learner-Centered Instruction through the Use of Cooperative and Inquiry Learning Strategies, Educational Resour-ces Information Center; Documentation number ED479308, 2003.

11. Öngel K, Mergen H, Kayacan H, Yıldızhan A. “Medical Students’ İmpressions and Satisfactions from Medical Professional Skill Education Le-ssons” Education Resources İnformatıon Center, Documentation number ED 500512, 2008.

Corresponding author Kurtulus Ongel, Suleyman Demirel University Faculty of Medicine, Department Head of Family Medicine, Turkey, E-mail: [email protected]



Abstract

Background: The objectives of the study in-clude: 1) Therapeutic adherence of patients to the MMT program, and 2) National protocol compa-rison with that of active practices by practitioners and its impact on patient’s compliance to therapy.

Methodology: Medical records of all the out-patients from Jan. 2007 until May 2008 were re-viewed. A year’s retrospective and six months’ prospective study was done. Exclusion was made for the defaulted out-patients (drop-outs, relapses etc.). Medical records were reviewed from all the three Government registered methadone clinics of Penang State, Malaysia.

Results: A total of 283 patients were registe-red, 97.5% of which were males and 2.5% fema-les. Only 215 patients’ records were reviewed in the study. Majority of defaulted cases were untra-ceable (70.1%) and these drop-outs were found among the Chinese. Only 1 case was successfully treated. 54.0% were well adhered to the therapy and stayed over a year of treatment. 99 patients re-started the methadone treatment after an interval of 1-7 months. Withdrawal symptoms were observed among 57.3% of patients, while 89.8% of out-pa-tients had medical complications during the met-

hadone therapy with: musculo-skeletal problems (48.2%), neurological disorders (41.5%). Dose management showed that 69.4% of patients were on ineffective therapeutic comfort dose. 60.0% had positive urine analysis (>14 times) during 6 months of treatment, while a negative correlation (r = - 0.492) was found between counselling and positive urine analysis with 24.2% shared varian-ce. Regression modelling identified (R-square = 0.793) 79.3% functional change in the adherence to therapy with p < 0.001 and CI 95%.

Conclusions: High rate of defaulted cases were observed as compared to the therapeutic adheren-ce to MMT program. Inconsistent practices were found. Although the practices were inconsistent to National Protocol, it was found that there is a strong need to review the National Guidelines on present evidence-based therapeutic approach. To control the positive urine analysis further research is needed to identify the social stigma among the patients during the treatment paradigm.

Key words: Methadone, MMT, therapeutic adherence, methadone addiction, addiction trea-tment, full agonist addiction treatment.

Therapeutic Adherence with methadone Treatment: Evaluation of therapeutic adherence and withdrawal management in methadone maintenance treatment program in Penang, malaysiaWasif. S. Gillani1, Azhar. S. Sulaiman1, Forouzan Bayat Nejad2, Tahir Mehmood Khan1, Amir Hayat Khan1,1 School of Pharmaceutical Sciences, Universiti Sains Malaysia Malaysia.2 Tehran University of Medical Sciences, Iran

596



Introduction and background

The Global epidemic of opiate use continue to spread and causes an increasing burden to both de-veloped and under developing countries (Ali, R., et al., 2005). Inevitably, Malaysia is just another country that has to deal with this Global Burden. A lot of studies have been carried out in Malaysia re-lated to drug abuse and addiction. Simultaneously, Malaysian anti narcotics taskforce is progressive-ly working on the preventive and control measures of narcotics abuse (Deva, M.P, 1977).

Malaysia has the fastest growing economies in South-East Asia with a population of approxima-tely 26 million people; experiencing extreme pro-blems associated with the use of illicit drugs, there were 235,495 registered drug users and offenders are registered into 2002. Similarly, heroin acco-unts for 63% of drug abuse treatment admissions and 69% of drug related criminal offenses in Ma-laysia (National Drug Information, 2005).

According to the National Anti-Drug Agency, Ministry of Internal Security 2007, declared the increase in drug addicts from 5,976 to 6,403 du-ring 2004-2005. After the official declaration of ‘drug abuse as a country enemy’ from the Prime Minister of Malaysia on 13th Feb. 1983, Malaysian ministry of Health and Anti-drug task force wor-ked efficiently for the harm reduction.

In that context, the government of Malaysia first introduce the Buprenorphine for the treatment of narcotic addiction but due to the non-evidence ba-sed dose practices Buprenorphine became the drug of abuse in 2-3 years (Vicknasingam et al., 2007 & Mazlan M., 2006). Methadone maintenance was first developed as a treatment for heroin addiction in the mid-1960s (Humeniuk et al., 2000) and has been proven to be an effective and sate mode of tre-atment (Jarvis et al., 1995). Methadone maintenan-ce has been proven beyond a doubt to be important in the reduction of spread of HIV via intravenous drug use (Latowsky 1996; NSW health Department 1999) and an important factor in reduction of cri-me rates. Despite the overwhelming evidence on the benefits of methadone and intense lobbying by certain quarters locally, methadone was registered locally in 2005 for use in the treatment of opiate dependence. The objectives of this program were to determine the best possible program that suited the

local setting and determination of best average dose for the local population. From the middle of 2006, Malaysian government initiated the large scale use of methadone for the treatment of narcotic addiction as methadone maintenance treatment (MMT). By the start of our study there are three-3 methadone clinics in whole Penang state so our aim is based on the evaluation of therapeutic outcomes of this pro-gram. Under the National Policy of Drug substitu-tion therapy (DST) direct observed therapy (DOT) is employed as a role of treatment and no take way doses allowed for the first 4-6 weeks except during weekends under strict family/guardian supervision. Henceforth take away doses will depend on the pro-gress of the patient, presence of family support and the discretion of prescribing doctor in accordance with guidelines (MOH Malaysia 2005). A dose of less than or equal to 20mg or a 70kg patient can be presumed to be safe, even in opioid-naïve users (MOH Malaysia 2006).

The objectives cover; 1) therapeutic adherence of patients to the MMT program, 2) National pro-tocol comparison with that of active practices by practitioners and impact on patient’s compliance to therapy and 3) dose management plan and ef-fective treatment of medical complication invol-ved in the treatment plan. Our specific secondary objectives were aimed in accordance to evaluate the quality of care in patients under MMT pro-gram, patients outcomes in term of adherence or withdrawal or by medical complications under methadone therapy, physician practices against the national guidelines and comparison against the evidence-base practice guidelines to determine the possible causes of relapse or program withdrawal or non-compliance with methadone.

Methodology

Penang state has three health centers registered to methadone maintenance treatment program: Hospital Bukit Mertajam, Health clinic Butterwor-th, and General hospital Pulau Pinang. Data was collected from all the three registered methadone clinics of Penang state. Penang was selected beca-use of the increasing trend of narcotic addiction.

Medical records of all the out-patients of met-hadone maintenance treatment (MMT) program



from Jan. 2007 until June 2008 were reviewed. Methadone maintenance treatment program was initiated during the middle of 2006, but only in one of the health clinic of Penang state. The re-maining two other health clinics started the MMT program in the beginning and middle of 2007, so the study was started from the Jan. 2007. It was found that until the mid of 2007 only one Health center started MMT program, General Hospital Pulau Pinang.

A year’s retrospective (Jan–Dec. 2007) and six months’ prospective (Jan–June. 2008) study was done. Exclusion was made for the defaulted out-patients (drop-outs, relapses etc). Data was collec-ted in self-development data-collection form, and data analysis was done with Statistical Package for the Social Science (SPSS 13®). Analysis met-hods were; Chi-square, Spearman correlation, and regression modeling.

Results and findings

A total of 283 respondents from three different methadone maintenance clinics of Penang state participated in the study. Total of 215 (76.0%) were accepted for the study because of active on MMT program, while remaining of 68 (24.0%) were exc-luded after being defaulted from MMT program.

Complete descriptive data is available in table 1 & table 2. Forty-seven about (70.1%) relapse cases were detected in Pinang hospital. There was only 1 (1.9%) patient who had successfully followed to MMT program and was free from drugs. Figure 1 represents the information regarding the relapse data during the last one and half year of MMT pro-gram, it was found that Pulau Pinang Hospital has highest percentage with 31.1% (47), although the MMT program was started in the mid-2007.

Table 1. Methadone maintenance treatment (MMT) program

Methadone clinics of Penang state Gender

Total Male N (%)

Female N (%)

a. Centre Pulau Pinang General Hospital (P.P)a

Bukit Mertajam Hospital (BM)b

Butterworth Health Clinic (B.W)b

145(96.0) 83 (98.6) 48 (100.0)

6 (3.9)1 (1.4)

-

1518448

Total 276 (97.5) 7 (2.5) 283b. MMT treatment active Pulau Pinang General Hospital Bukit Mertajam Hospital Butterworth Health Clinic

99 (95.2) 68 (97.1) 41 (100.0)

6 (4.8)1 (2.9)

-

1056941

Total 208 (96.7) 7 (3.3) 215a patient consensus from march 2007 to May 2008.b patient consensus from Jan 2007 to May 2008.

Table 2. Frequency of related outcomes of the MMT program

Characteristics N (%)P.P BM B.W

Defaulted casesa. Prisonb. Untraceablec. Rehabilitation centreSuccessful treatment casesDeaths: a. Abnormal liver function b. Aspirated Pneumonia c. overdose of methadone

9 (15.7) 9 (13.4) 3 (4.5) 39 (76.5) - 2 (2.9)

- 2 (2.9) - - 1(1.9) -

- - 1 (1.9)

- 1(1.9) - - - 1 (1.9)

Total N = 68 (24.2) 47 (70.1) 13 (19.4) 7 (10.4)

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Figure 1. Percentage relapse from last 1½ year (Jan 2007- May 2008) among three methadone clinics of Pinang state.

Table 3 contains the information regarding the therapy module of MMT program, 116 (54.0%) taking methadone from 7 -12 months only 26 (12.1%) >12 months of treatment. High rate of relapse was significantly found among Chinese (table 4).

Significant data was found between dose setting & management, methadone centre of treatment, gender and restarting dose after interval (table 5). Baseline data on withdrawal sign & symptoms in-dicated that 57.3% of patients who undergone the treatment experienced some adverse sign & symp-toms during the treatment with methadone.

Table 3. Duration of treatment among the out-patients of MMT programDuration of Treatment

MMT clinics N % Total P.P BM B.W≤ 6 moths

7 – 12 months> 12 moths

24 (11.1)71 (33.0) 9 (11.2)

28 (13.0)33 (15.3)9 (4.2)

21 (9.8)12 (5.6)8 (3.7)

73 (33.9)116 (54.0)26 (12.1)

Total 104 (48.4) 70 (32.6) 41 (19.1) 215

Table 4. Percentage of relapse among races in MMT Program

Race

Methadone clinic P.P B.M B.W N (%) N (%) N (%)Enroll active sig enroll active sig enroll active sig

Malay ChineseIndian OtherTotal

52 (34.5) 40 (26.5) 37 (44.0) 35 (41.6) 29 (60.4) 26 (54.2) 70 (46.3) 43 (28.5) 0.001 31 (36.9) 31 (36.9) 0.000 10 (20.8) 8 (16.7) 0.02428 (18.5) 21 (13.9) 16 (19.1) 4 (4.8) 9 (18.8) 7 (14.6)1 (0.7) 0 (0.0) - - - - 47 (31.1) 14 (16.7) 7 (14.5)

Sig: chi-square

Table 5. Methadone dose setting consistent with responsive behavior of out-patientsCharacteristics

N (%)Induction dose

≤ 20mgMaintenance dose 30–80mg

Withdrawal dose(Varies) Sig.

Centre: (N = 215)P. Pinang. 104 (48.4)B. Mertajam. 70 (32.6)B. Worth. 41 (19.0)Active patients of MMT program:Male 208 (96.7)Female 7 (3.3)a Restarting MMT after interval: (N = 99)< 1 month 28 (28.3)1 – 3 months 37 (37.4)4 – 6 months 22 (22.2)7 > months 12 (12.1)

27 (26.0)12 (17.1)8 (19.5)

46 (22.1)1 (14.3)

20 (71.4)7 (18.9)

--

70 (67.3)53 (75.7)30 (73.2)

151 (72.6)2 (28.6)

5 (17.9)29 (78.4)17 (77.3)10 (83.3)

7 (6.7)5 (7.1)3 (7.3)

11 (5.3)4 (57.1)

3 (10.7)1 (2.7)5 (22.7)2 (16.7)

0.000*

0.000*

0.001*

a sorted reasons for interval: prison/ rehabilitation centre, unknown etc* Chi-square test



Chart 1 represents the diagrammatic presentati-on of the basic issues of therapeutic comfort dose (TCD), while data showed that 149 (69.4%) have ineffective TCD plan and pertaining to risk of re-lapse on MMT program. Medical complications before treatment (MCBT) 193 (89.9%) and me-dical complication during treatment (MCDT) 185 (95.9%) had a significant effect on MMT outcome of the therapy while only 8 (4.1%) were shown im-proved quality of health in MMT program (table

6). While information presented in table 7 showed that Chinese had significantly higher rate of medi-cal complication before and during the Methadone maintenance treatment.

A negative correlation (r = -0.492, CI= 95%) was found with 24.3% shared variance between positive urine analysis and counseling offered by practitioners by using spearman correlation. Urine analysis and positive responses were found highest during the week 3rd to 8th (table 8 and table 9).

Table 6. Clinical features associated with drug abuse and reliance on MMT treatmentCharacteristic N (%)

Medical complications before treatment with methadone a

General complications Injection related problems Cardio/ respiratory disorder Genitor-urinary disorder Musculo-skeletal problem Neurological disorder Gastro-intestinal disorder

Medical complications during methadone treatment b

Improved complications during treatmentNo MC c before treatment with methadone

193 (89.8) 130 (67.4)28 (14.5)57 (29.5)7 (3.6)93 (48.1)80 (41.4)73 (37.8)

185 (95.9) 8 (4.1)22 (10.2)

a MCBT b MCDT c Medical complicationsTable 7. Cross-tabulation between race and clinical outcomes of MMT

Race Clinical outcomes

MCBT MCDT Improve no MC N (%) N (%) N (%) N (%) Sig.

Malay ChineseIndians Total

95 (91.3) 88 (84.6) 7 (3.6) 9 (8.7) 60 (85.7) 59 (84.3) 1 (0.5) 10 (14.3) 0.001* 38 (92.7) 38 (92.7) - 3 (7.3) 193 (89.8) 185 (86.0) 8 (3.7) 22 (10.2)

* chi-square Table 8. Descriptive data for the urine analysis of MMT out-patients

Urine testsNo.

Positive urine analysis for drug of abuse 0-3 4-7 8-11 12-15 16-19 20≥ N (%)

N = 215 First 2 weeks 3rd – 8th week9th–32nd week33rd week ≥

2(100.0) - - - - - 2 (0.9) 1(2.1) 30(63.8) 16(34.0) - - - 47(21.9) 4(3.1) 39(30.2) 45(34.9) 38(29.5) 3(2.3) - 129(60.0) 1(2.7) 16(43.2) 13(35.1) 4(10.8) 2(5.4) 1(2.7) 37(17.2)

Table 9. Correlation of counseling with the positive urine analysis

No Number of Urine positive for drugs during treatment 0-3 4-7 8-11 12-15 16-19 20≥ N (%)

N = 215Sessions: 1 – 4 5 – 8 9 – 12 No session

1(0.8) 36(28.8) 47(37.6) 36(28.8) 4(3.2) 1(0.8) 125(58.1) 2(6.1) 22(66.7) 8(24.2) 1(3.0) - - 33(15.3) 4(10.3) 19(48.7) 11(28.2) 4(10.3) 1(2.6) - 39(18.2) 1(5.6) 8(44.4) 8(44.4) 1(5.6) - - 18(8.4)

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Table 10. Issues among the MMT protocol

Characteristics Prescription type Direction

Induction methadone dose titration Maintenance dose levelsWithdrawal dose settingChanging dose levels Concurrent use of illicit opioids Individual variation in methadone metabolism Use of other medications Poly-drug use Pregnancy Compliance Monitoring drug useAdjunct treatment Social services Counseling Takeaway home dosesMissed doses and ReintroductionChronic diseases (HIV, hep B&C, Impaired liver function)

Inconsistent Inconsistent Consistent

Consistent Inconsistent Consistent

Inconsistent Consistent

Inconsistent Consistent

Consistent Consistent

InconsistentConsistent

Inconsistent

Major *Major *

Major *

Minor **

Major **

Major **

Major *** major inconsistent identified more then 50% cases deviating the MMT protocol** minor inconsistent identified less then 50% cases deviating the MMT protocol

Table 11. Dose schedule for MMT program in out-patientsCharacteristic N (%) Dose level

Maintenance dose:Std dose (30–60 mg per day)Practice dose : 40 mg 45 mg 50 mg 55 mg 60 mg 65 mg 70 mg 75 mg 80 mg 85 mg 90 mg 95 ≥Withdrawal dose:↓ 10 mg / week↓ 5 mg / week↓ 10 mg / month

Retention to 40 mgRetention on > 40 mg

42 (27.5) Consistent1 (4.2)3 (1.4)5 (2.3)13 (7.0)20 (9.3)17 (12.6) 111 (72.5)11 (5.1) Inconsistent 4 (1.9)19 (18.1)22 (11.2)6 (7.4)32 (19.5)

10 (66.7)3 (20.0)2 (13.3) Consistent 13 (86.7)14 (93.3)1 (6.7)



Chart 1. Evaluation of MMT outcomes related to therapeutic comfort dose (TCD)

Majority of major inconsistencies were found among the practices in methadone clinics and stan-dard Malaysian protocol (table 10). Dose schedule among respondents showed 111 (72.5%) inconsi-stencies to the standard protocols (table 11).

Regression model

Model summary of logistic regression repre-sents R-square (0.793) with the CI of 95% showed significance (p < 0.000) among the given varia-bles; therapeutic comfort dose (TCD), medical complications both MCBT & MCDT, chronic di-seases, duration of therapy and psychotherapy.

Discussion

The treatment of opioid addiction was a contro-versial issue since late 70’s. A lot of clinical studi-es were carried out in this span of time to analyze the therapeutic effectiveness of the methadone tre-atment and majority of them have modest results (Einat Pele, 2005; Kreek M.J, 1992; Leavitt S.B et al., 2000; Ward, J., 1995; Sarz Maxwell, 1999; Jeff ward, 1999; Lubmir Okruhlica, 2002; Icro Maremmani, 2007; Yasukazu Ogai et al., 2007; Jason Luty, 2003).

The effectiveness of Methadone maintenance treatment in this study was observed by:

1. Checking the rate of relapse among the MMT patients

2. Medical complication found during the treatment

3. Life quality (medical complication before the treatment and medical complication found during the treatment).

Chronic disease and subsequent supportive pharmacotherapy and therapeutic comfort dose setting correlation with urine analysis and psyc-hotherapy session during the treatment.

Findings of this study showed high controver-sial practices in the implementation of MMT pro-gram that were found inconsistent to the National guidelines of methadone maintenance treatment protocol of Ministry of Health Malaysia (2006).

Relapse and Defaulted percentage

A total of 283 drug addicts were registered in the three registered methadone clinics of Penang state during Jan. 2007 to May 2008. The mean S.D age was 41.0 (9.32), majority of them were males (97.5%) and they also formed the majority (53.4%) of admission were found in Hospital Pu-lau Pinang. The total of 54.6% relapsed cases were identified from all three registered methadone cli-nics among them 70.1% relapse cases were found in Hospital Pulau Pinang, 19.4% in Butterworth methadone clinic and 10.4% in Bukit Meratajam Hospital. Majority of relapses were identified in the first six month of MMT program, few relapse cases were found after the completion of 1 year treatment. Majority of relapse cases were reported in Chinese least in Indian.

Currently only 12.1% patients were successfu-lly completed the 1 year of treatment, while majo-rity of them only followed the treatment program for a period of 7 – 12 months of treatment. Few studies identified that craving for drugs, emotio-nal pressure and boredom were the main reasons found among relapsed cases (Research report No. 31, 1997; Yasukazu Ogai et al., 2007). As Ame-rican National consensus Development panel (1998) reported 50% common drop-out from the MMT studies.

Yasukazu Ogai and his collegues worked in Japan for the development and validation of re-lapse prevention technique through risk scale assessment for drug abusers in Japan, while his basis emphasize were focused on craving, quality of life experience by drug addicts and time frame spent in the treatment program. Modest findings and outcomes were associated with his study but the limitation was applied for Opioid users as the

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validation was done on cocaine users alone. Such model may help the Ministry of Health Malaysia to control the relapse cases of drug addiction. As certain reliability was found in the study to esta-blish such a model as a preventive measures to re-duce relapse cases.

Medical complication and withdrawal sign & symptoms

Baseline data show evidences that 57.3% pati-ents were experienced withdrawal sign & symp-toms, while 15.1% suffered with intoxication symptoms and only 27.6% MMT patients showed no evidence of any sign & symptoms during the treatment of methadone. Majority of withdrawal symptoms were yawning and anxiety. The rea-son behind these findings represent the low dose setting of methadone which would revealed the withdrawal symptoms and cravings that will also associated with the risk of relapse.

The current study showed that 89.8% of drug addicts were pre-suffered with different types of medical complications before starting the methado-ne treatment, while after the completion of 3 mont-hs therapy 95.9% of drug addicts among them still carrying the medical complication only 4.1% drug addicts improved, predominantly in Malays.

Majority of patients were experiencing General complications (like; fatigue, trouble sleep, loss of appetite, teeth problem and eye/vision problem), musculo-skeletal problem (joint pain and stiffne-ss), neurological disorder (headache, numbness, dizziness, forgetting things), gastro-intestinal pro-blems (constipation, stomach pain, diarrhea) and cardio-respiratory disorders (persistent cough, wheezing, chest pain). These findings could be the possible cause of psycho-disturbance which may lead to depression. It could be the predictive factor for relapse; similar findings were reported by Icro Maremmani (2007) in his study in Italy.

Therapeutic comfort dose (TCD)

The therapeutic comfort dose (TCD) is ter-med as “the effective dose plan that will suppress the withdrawal sign & symptoms while will not

show any sign of intoxication”. The objective wi-thdrawal sign & symptoms can be suppressed by increasing the potency dose of methadone while subjective intoxicated symptoms can be overco-me by decreasing the potency dose. So TCD is the middle dose setting between objective and subjective sign & symptoms. The findings of the study reported only 30.6% of all the patients were on TCD setting while remaining 69.4% seemed to be on ineffective dose setting. These findings were directly correlated with the evidences of wi-thdrawal and intoxications symptoms found in the study previously discussed in section 5.3.2.

Urine analysis for the drug of abuse is one of the reasonable marker to determine the therapeu-tic effectiveness of the dose. Several studies sug-gested that with an increase in the average dose of methadone could possibly lower down the number of drop-out cases and also a lower proportion of urine tested positive for opioid (Maxwell S, Sc-hinderman, 1999; Strain E. C., Stitzer M. L., Lie-bson I. A., Bigelow G. E, 1998; Strain E.C et al., 1999; Ceplehorn J.RM., Bell J., 1991).

Findings of our study showed that 93.5% of total 215 patients induce positive urine analysis for opioid. Majority reports were positive from 8 to 16 month of therapy. Surprisingly 98.8% were on maintenance dose setting of 30 – 80 mg / day. Similar findings were explained in several studies (Einat Peles et al., 2005; Maxwell, Shin-derman, 1999; levit S. B., et al., 2000; Ward. J., 1995; Lubomir. Okruhlica. et al., 2002 and Jason Lutty., 2003). They reported the direct proporti-on of methadone dose and urine positive results. Their findings were preliminary based on Serum methadone level (SML), majority of them repor-ted that to achieve an effective therapeutic dose it is necessary to adopt a methadone serum level of 600ng/ml or higher for the completely suppressi-on of opioid withdrawal symptoms. Overall all the above researchers suggested that effective thera-peutic treatment was achieved with 100mg dose of methadone and above.

Cepelhorn & Bell (1991) did a research on drug respondents in Italy, the results of this study con-cluded that patients who were on methadone dose less than 60 mg have five times more risk of dro-pping out as those receiving doses of 80 – 100mg. Similarly a double-blind trials done by Strain et



al. (1999) revealed that 53% positive urine results were found after 30 weeks in patients receiving methadone dose 80 – 100 mg as compared to 62% of those on 40 – 50 mg of methadone dose setting.

In view the results of this study and the stan-dard MOH (Ministry Of Health, Malaysia) guide-lines of MMT and other dose optimizing studies conducted all over the World, following were the important issues found in the protocol;

1. No therapeutic monitoring of methadone as SML value.

2. Maintenance dose range was between 30 – 80 mg.

3. Highly Urine positive results were found even after the 14 months of treatment.

4. Increase relapse rate was found during the first six months of treatment.

5. Increased evidences of withdrawal and intoxicated sign & symptoms.

Low percentage of improve quality of life in term of medical conditions in the treatment with methadone. In those who follow the therapy.

Protocol comparison

The findings of our study showed the inconsi-stent practices in MMT program in the following sub-sections of National MMT guideline, 2006;

1. Induction methadone dose titration2. Individual variation in methadone

metabolism3. Poly-drug users.4. Compliance 5. Take away home doses6. Chronic diseases and managements.

It is highly suggested in our study that above mention sub-sections were in major level of in-consistency to protocol; as more than 50% cases deviating from standard National MMT protocol practices.

Regression modeling

Logistic regression modeling was performed in our study to identity the risk parameters of our

clinical management of methadone and MMT ef-fectiveness. We find high significance (CI= 95%, P < 0.001) with R-square value (0.793) in the fo-llowing variables;

1. Therapeutic comfort dose (TCD)2. Medical complications both (MCBT and

MCDT)3. Chronic infectious diseases (hepatitis C, B.

HIV/AIS etc..)4. Duration of therapy 5. Psychotherapy offered by institute.

While TCD, chronic infectious diseases and duration of therapy alone will produce functional change in R-square value up to (0.613) & if eli-minated from the model the significance reduce tremendously (P < 0.025).

Conclusion

High rate of defaulted cases were observed as compared to the therapeutic adherence to MMT program. Inconsistent practices were found altho-ugh the practices were inconsistent to National Protocol but it was found that is a strong need to review the National Guidelines on present eviden-ce-based therapeutic approach. To control the po-sitive urine analysis, further research is needed to identify the social stigma among the patients du-ring the treatment paradigm.

References

1. Ali, R., et al., (2005) the WHO collaborative study on substitution therapy of Opioid Dependence and HIV/AIDS, WORLD HEALTH ORGANIZATION (W.H.O). http://www.who.int/substance_abuse/ac-tivities/substituion_therapy_opioid_dependence_general_protocol%20_v2.pdf (Last Aceess: March 03, 2009)

2. Deva, M.P., 1977, A survey of 47 Opiate dependant persons who sought Treatment. Med. J. Malaysia 31, 183-187.

3. National drug information (NADI), National Drug agency, Malaysia, 2005, Annual report, 5-21

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4. Mahmud. Mazlan., Schottenfeld. R. S., Chawarski. M.C., 2006, “New Challenges and Opportunities in Managing Substance Abusers in Malaysia”., Drug and Alcohol Review, 25, 473-478.

5. Viknasingam. B., et al., 2007, “Injecting Buprenor-phine in Malaysia: Demographic and drug use cha-racteristics of Buprenorphine Injectors”., Abstract submitted to NIDA International Conference, Que-bec City, Canada, June 16-21

6. Einat peles, Shaul Schreiber, Miriam Adelson., 2006, “ Factors predicting retension in treatment: 10-year experience of a methadone maintenance treatment (MMT) clinic in Israel”. Drug and Alcohol depen-dence, 2006, volume 82, issue 3, pp: 211-217.

7. Kreek, M. J., 1992, “Rationale for maintenance phar-macotherapy of opiate dependence”, In: O’Brein, C.P., Jaffe, J.H. eds. Addictive states. Research pu-blications: Association for research in Nervous and mental disease. Ravan Press, New York: 210.

8. Levitt S.B., et al., 2000, “When Enough is not Eno-ugh: New perspectives on optima methadone main-tenance dose”, Mt Sinai J Med. 67(5-6): 404-411.

9. Ward. J., 1995, “Factor influencing the effecti-veness of methadone maintenance treatment: An evaluation of change and innovation in the metha-done program in New South Wales, Australia 1985-1995”, PhD thesis. National Drug and Alcohol Re-search Centre.

10. Saraz Maxwell, Marz Shinderman., 1999, “Op-timizing Response to methadone maintenance treatment: Higher Dose Methadone”, Journal of Psychoactive Drugs: Vol-31(2)

11. Jeff ward., Wayne Hall., Richard. P. Mattick., 1999, “Role of maintenance treatment in opioid dependence”, The Lancet, vol. 353. issue: 9148. pp. 221-226.

12. Lubomir Okruhlica., et al.,2002. “Does therape-utic threshold of methadone concentration in pla-sma exist?”, Heroin Addiction & Rehabilitation Clinical Problems, 4(1): 29-36.

13. Icro. Maremmani., 2007, “Substance use and qu-ality of Life over 12 months among buprenorphine maintenance-treatment and methadone mainte-nance-treatment heroin-addicted patients”., Jour-nal of Substance Abuse Treatment 33. 91-98.

14. Yasukazu Ogai., et al., 2007, “Development and validation of the stimulant relapse risk scale for drug abusers in Japan”, Drug and Alcohol De-pendence, 88: 174-181.

15. Jansson. L. M,, Velez. M., Harrow. C., 2004, Met-hadone maintenance and lactation: a review of the literature and current management guideli-nes. J Hum Lact; 20:62–71

16. National Methadone Maintenance Therapy Gui-deline., 2006, second edition February, NCD pre-vention and control, Ministry Of Health Malaysia.

17. Research Report No.31, 1997, “A Follow-up study on Drug addicts after treatment and Reha-bilitation”, Centre of Drug research, University Sains Malaysia (USM).

18. Strain. E. C., et al.,1998, “Useful predictors of outcome in methadone-treated patients: Results from a controlled clinical trial with three doses of methadone”, Journal of Maintenance in the ad-diction, 1(3): 15-28.

19. Strain. E. C., et al., 1999, Moderate- vs high-dose methadone in the treatment of opioid dependence: a randomized trial. JAMA; 281:1000–1005

20. Ceplehorn J.R.M., Bell J. (1991), “Methadone dosage and retention of patients in maintenance treatment”, Med J Aust, 154(1): 195-199

Corresponding author Syed Wasif Gillani School of Pharmaceutical Sciences, Universiti Sains Malaysia, Malaysia, E-mail: [email protected]



Abstract

Basal cell carcinoma is the most common cancer of epidermis. Histologically, the tumor cells have a remarkably uniform morphology and resemble normal basal keratinocytes. Known karyotypic abnormalities in this tumor are nonrandom, rarely including evidence of clonal evolution. The aim of this research was to investigate possibilities of establishing primary cultures of basal cell carci-noma and observing chromosome aberrations in analyzed tumor samples. The study was con-ducted over 24 patients with diagnosed basal cell carcinoma. Chromosome analysis was conducted over 7 samples, successfully harvested and with the sufficient number of metaphases. Observed chromosome aberrations were scored according to the International System for Human Cytogenetic Nomenclature. In 63.27% of analyzed metaphases chromosome aberrations were registered. Both numerical and structural chromosome aberrati-ons were registered, as well as chromosome and chromatide type aberrations. The most frequent structural aberrations were acentric fragments, while aneuploidy was the most frequent numerical aberration. It was concluded that must be realized karyotyping for determining specific chromosome abnormalities in basal cell carcinoma and, as the fibroblasts present the main problem in obtaining

tumor keratinocytes, fibroblast growth should be carefully observed and cultures harvested before fibroblasts overgrow tumor keratinocytes.

Key words: Basal cell carcinoma, cell culture, chromosome aberrations

Sažetak

Bazocelularni karcinom je najčešći karcinom epidermisa. Histološki, tumorske ćelije imaju izrazito uniformnu morfologiju i liče normalnim bazalnim keratinocitima. Poznate kariotipske ab-normalnosti u ovom tumoru su neslučajne i rijetko uključuju tragove klonalne evolucije. Cilj ovog istraživanja je bio istražiti mogućnosti uspostav-ljanja primarne kulture bazocelularnog karcinoma i opservacija hromosomskih aberacija u analizira-nim tumorskim uzorcima. Studija je provedena na 24 pacijenta sa dijagnosticiranim bazocelularnim karcinomom. Analiza hromosoma je provedena na 7 uzoraka, uspješno kultiviranim i sa dovolj-nima brojem metafaza. Uočene hromosomske aberacije su evidentirane prema Međunarodnom sistemu citogenetičke nomenklature. U 63.27% analiziranih metafaza registrirane su hromosom-ske aberacije. Registrirane su numeričke i struk-turne, kao i aberacije hromosomskog i hromatid-nog tipa. Najfrekventnije strukturne aberacije bile

basal Cell Carcinoma: Cultivation Potential and results of Chromosome Aberrations AnalysisBazocelularnI karcInom: mogućnosTI kuLTIVACIjE I rEzuLTATI AnALIzE HrOmOsOmskIH AbErACIjAIbrulj S. 1, Haveric A. 2, Haveric S. 2, Rahmanovic A. 2, Alendar F. 3 1 Medical Faculty, University of Sarajevo, Bosnia and Herzegovina2 Institute for Genetic Engineering and Biotechnology of Sarajevo, Bosnia and Herzegovina3 Dermatovenerology Clinic, Clinical Center University of Sarajevo, Bosnia and Herzegovina

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su acentrični fragmenti, dok su aneuploidije bile najfrekventnije numeričke aberacije. Zaključeno je da je u cilju determinacije hromosomskih abe-racija specifičnih za bazocelularni karcinom neop-hodno realizirati kariotipizaciju, te da s obzirom na to da fibroblasti predstavljaju glavni problem u dobijanju tumorskih keratinocita, rast fibroblasta treba biti pažljivo praćen i kulture izvedene prije njihovog nadrastanja tumorskih keratinocita.

Ključne riječi: bazocelularni karcinom, kultu-ra ćelija, hromosomske aberacije

Introduction

Clinical cytogenetics has had the great impact on many aspects of the medical and basic sciences, perhaps the most notably, hematology and oncolo-gy. Progress in understanding the clinical signifi-cance of cytogenetics in hematologic malignanci-es was made more quickly than in solid tumors. Less number of published solid tumor karyotypes can be associated with technical problems to obta-in sufficient quantities of quality metaphases from solid tumor samples (1).

Most mutagens and genotoxic carcinogens are efficient inducers of chromosomal alterations in ex-posed cells. These aberrations are associated with congenital abnormalities and neoplasia in humans (2). The most cancers result from the multiple gene-tic changes, including the activation of oncogenes and inactivation tumor-suppressor genes. In many cases, these changes result from decades of exposu-re to the mutagenic effects of carcinogens (3). The most common cancer in humans is basal cell carci-noma. Histologically, the tumor cells have a remar-kably uniform morphology and resemble normal basal keratinocytes (4). The connective tissue adja-cent to basal cell carcinomas is frequently abnormal and contains increased numbers of fibroblasts and increased extractable collagenase (5). The karyoty-pic abnormalities in this tumor are nonrandom, ra-rely including evidence of clonal evolution. Clonal chromosome abnormalities are present in 22 of 23 short-term cultured basal cell carcinomas (6).

The aim of this research was to investigate possibilities of establishing primary cultures of basal cell carcinoma and observing chromosome aberrations in analyzed tumor samples.

Material and methods

The study was conducted over 24 patients with diagnosed basal cell carcinoma. After excision, with patient consent, samples were put in transport me-dium containing RPMI 1640 (GIBCO-Invitrogen, Carlsbad, CA, USA), penicillin/streptomycin and amphotericin B, placed on room temperature for two hours. After bathing in transport medium tumor samples were incubated overnight in 0.25% trypsin (1:250) at 4°C to disperse. Dissociated cells were seeded in 7.5 ml of medium in flasks (Easyflasks - NUNC, Rochester, NY, USA). Two different media were used for cultivation. One was prepared with RPMI 1640, while the other with Dulbecco’s mini-mal essential medium – DMEM (Sigma-Aldrich, St. Louis, MO, USA). Both media were supplemented with 10% fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin and amphotericin B. Culti-vation at 37°C, lasted 5-15 days - depending on cells division rate, with medium replacing every third day.

Cell division in cultivated cells was blocked by addition of colcemid (GIBCO BRL) at final con-centration of 0.2 µg/ml, 1.5 hours prior the end of the cultivation.

After the cultivation period, cultures were cen-trifuged for 10 min. at 1000 rpm and subjected to hypotonic treatment with 0.56% KCl. Hypotonic treatment lasted for 20 min. at 37°C. Hypotonic treatment was followed by centrifuge for 10 min. at 1000 rpm and fixation in ice-cold glacial acetic acid:ethanol (1:3) fresh fixative. Fixation was repe-ated three times. Fixed cells solution was dropped on ice-cold microscope slides. Air-dried slides were stained with 5% Giemsa stain for 7 minutes.

Metaphase slides were analyzed under oil-im-mersion objective at 1000× magnification, with a BX51 Olympus microscope (Japan). Aberrant metaphases were documented using digital came-ra DP50 (Japan) attached to the microscope.

Observed chromosome aberrations were scored according to the International System for Human Cytogenetic Nomenclature (7). Verified aberrati-ons were subclassified as chromosome-type: chro-mosome breaks (chrb), acentric fragments (acea), double minute fragments (dmin); chromatid-type aberrations: chromatid breaks (chrb), minute fra-gments (min); as well as pulverizations (pvz). Gaps were not scored as aberrations (8,9).



Results and discussion

Establishing primary culture of basal cell car-cinoma has been extremely difficult as it was con-firmed earlier (4). We found that both used media are sufficient for cultivating basal cell carcinomas. The main problem was contamination that over-took 17 samples, which were not harvested. The second problem was fibroblasts overgrowth that usually appeared during second week of cultiva-tion. Tumor keratinocytes obtained from one of the processed samples are presented on figure 1. Tumor keratinocytes with sporadic fibroblasts are presented on figure 2, while pure fibroblasts cultu-re is presented on figure 3.

Figure 1. Tumor keratinocytes in culture

Figure 2. Tumor keratinocytes with sporadic fi-broblasts in culture

Figure 3. Fibroblasts in culture

Chromosome analysis was conducted over 7 samples, successfully harvested and with the suf-ficient number of metaphases. In 63.27% of analy-zed metaphases chromosome aberrations were re-gistered. It is generally accepted that chromosomal mutations are causal events in the development of neoplasia, and it has earlier been postulated, but not proven, that increased chromosomal damage may reflect an enhanced cancer risk (10). Both numerical and structural chromosome aberrations were registered, as well as chromosome and chro-matide type aberrations. Percentages of observed chromosome aberrations are presented in figure 4.

Figure 4. Percentages of registered chromosome aberrations

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Within registered structural aberrations acen-tric fragments dominated, while aneuploidy was the most frequent numerical aberration. Some of the most frequent observed chromosome aberrati-ons are presented on figures 5, 6 and 7.

Figure 5. Metaphase with acentric fragment

Figure 6. Poliploidy

Figure 7. Metaphase with numerous double mi-nute fragments

Aneuploid cells are found as it was expected. Aneuploidy is probably the only mutation capa-ble to clarify all aspects of carcinogenesis (11) as gene mutation hypothesis is not sufficient to iden-tify cancer-specific mutations and to explain why cancer develops certain time after the mutation or why cells of solid tumors are aneuploid (12).

In one processed tumor sample we have registe-red three metaphases with numerous double minute fragments (figure 6), indicating gene amplifications (amp). Double minutes are tiny spherical chromatin bodies of a few mega-base pairs of size, which are found occasionally in hematopoietic neoplasia and more or less often in human solid tumors. They have been associated with worse prognosis and poor out-come of the malignancies where present (13).

Conclusion As the fibroblasts present the main problem in

obtaining tumor keratinocytes, fibroblast growth should be carefully observed and cultures harvested before fibroblasts overgrow tumor keratinocytes.

Majority of analyzed metaphases were aberrant. The most frequent structural aberrations were acen-tric fragments, while aneuploidy was the most frequent numerical aberration.

In order to determine specific chromosome ab-normalities in basal cell carcinoma, karyotyping must be realized which is further planned.



Aknowledgements

The research was approved and financially su-pported by the Ministry of education and science, Canton Sarajevo (number: 11-14-21632.1/07).

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10. Hagmar L, Stromberg U, Tinnerberg H, Mikoczy Z (2001) The usefulness of cytogenetic biomarkers as intermediate endpoints in carcinogenesis. Int J Hyg Environ Health. 204, 43-47.

11. Duesberg,P., Rasnick,D. (2000) Aneuploidy, the somatic mutation that makes cancer a species of its own. Cell Motil Cytoskeleton. 47(2), 81-107.

12. Li,R., Sonik,A., Stindl,R., Rasnick,D., Duesberg,P. (2000) Aneuploidy vs. gene mutation hypotesis of cancer: Recent study claims mutation but is found to support aneuploidy. Proc Nat Acad Sci U S A. 97(7), 3236-3241.

13. Gebhart E. (2005) Double minutes, cytogenetic equivalents of gene amplification, in human neo-plasia – a review. Clin Transl Oncol. 7(11), 477-485.

Corresponding author Ibrulj S., Medical Faculty, University of Sarajevo, Bosnia and Herzegovina, E-mail: [email protected]

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Therapy effect on ejection fraction in patients with hypo- and hyperthyroidismIsmana Surkovic1, Ismet Suljevic2, Azra Kudumovic3

1 Clinic of Endocrinology, Diabetes and Metabolic Deseases, University Clinical Centre Sarajevo, Bosnia and Herzegovina,2 Clinic of Anestesiology, University Clinical Centre Sarajevo, Bosnia and Herzegovina,3 General Hospital, Sarajevo, Bosnia and Herzegovina

Abstract

Introduction: Hypothyroidism and hyper-thyroidism are followed by cardiovascular struc-tural and functional changes which entirely with-draw after timely treatment with l - thyroxin and thyreostatics.

Aim of research: To demonstrate cardiovas-cular changes caused by hyperthyroidism and hy-pothiroidism by analyzing ejection fraction (E.F.)gained by doppler echocardiography; to check cardiovascular changes caused by hypothyroidism and hyperthyroidism by comparative methodolo-gy upon achivement of euthyroidism by medica-ment therapy.

Patients and methods: One hundred patients took part in the study: 50% with hypothiroidism, being of either sex, aged over 14, hospitalised at the Clinic of Endocrinology, Diabetes and Me-tabolic Deseases, Clinic for Heart Deseases and Reumathism, or observed at the their ambulan-ces over the period 1997-2003. Patients suffering from sub-clinical hypo- and hyperthyroidism took part in the study as well. All patients have under-gone thyroid hormonal status, echocardiography before being treated by l-thyroxin and thyreostati-cs, and after achivement of euthyroidism. Patients suffering from central and secondary hyperthyroi-dism, those who have record of rheumathoid fever in their personal anamnesis were excluded from the study.

Results: Hypothyroidism was associated with significant increase ejection fraction after at le-ast six months therapy with levothyroxin. Hyper-

thyroidism was associated with nonsignificant increase ejection fraction after at least six months therapy with antithyroid drugs.

Conclusion: Our study showed particularly statisticaly significant improvement in disfunction of thyroid gland and consequential cardiovascular changes after treatment of medications, but not sa-tisfactory by clinical aspect.

Key words: hypothyroidism, hyperthyroidism, ejection fraction, l-thyroxin, antithyroid drugs.

Introduction

Myocardium is the main target of effect of thyroid hormones. Of all endocrine glands in or-ganism, thyroid gland has the greatest hormone effect on heart.

Multiple effects of thyroid hormones on cardio-vascular system are reflected directly, on myocar-dium, and indirectly, through the effect on sym-pathetic nervous system and basal metabolism. In hypothyroidism, organism preserves heat, which leads to peripheral vasoconstriction and slower circulation. As a result of these and other physi-ological effects, patients with a deficit of thyroid hormone usually suffer from bradycardia, reduced systolic contractility, delayed ventricular diastolic relaxation and increased systemic vascular resi-stance (1,2,3,4,5,6).

Hyperthyroidism is characterized by the incre-ased heat release. Organism defends itself against overheating by increasing the circulation, so that excessive heat is emitted to the body surface and



then removed by radiation and perspiration. Peripheral vasodilatation occurs due to the incre-ased heat release. Contractility, automatism and irritability of myocardium are increased, as well as coronary bloodstream and oxygen consumpti-on. However, the work effect is decreased.

Systolic and diastolic function of the left ven-tricle (LV) at rest is not always reduced in hyper-thyroidism. At load, ejection fraction of the left ventricle can significantly drop, but despite this clearly pathological response, the expressed con-gestive heart failure is not usual in hyperthyroidi-sm, unless the patient already has a heart disease or atrial fibrillation occurs (7,8,9,10,11,12,13).

Problem definition

Slight reversible functional and morphological changes on heart were detected in conditions of subclinical hypo- and hyperthyreosis as well. The-refore, it is recommended to treat these conditions by an adequate medicament therapy, in order to prevent the expressed hypo- and hyperthyroidi-sm and their possible complications on time. The issue of occurrence of cardiovascular changes in subclinical hypo- and hyperthyroidism, as well as the therapies for these conditions, is still contro-versial, due to the absence of symptoms in patients and the possibility of overdosed therapy (1,9,10).

The basic hypothesis: Cardiovascular changes in hypo- and hyperthyroid patients significantly su-bside after applying the therapy with l-thyroxine and thyreostatics, i.e. achieving the euthyroid con-dition.

Objective of the study:To examine reversibility of disordered ejection fraction of heart in hypo- and hyperthyroidism after medicament therapy, in the achieved euthyroidism, by comparative method.

The patients and study methods: The study involved 100 randomly selected patients (random sample method): 50 with hypothyroidism and 50 with hyperthyroidism, aged over 14, both genders, hospitalized at the Clinic for Endocrinology, Diabe-tes and Metabolism Diseases, Clinic for Heart Di-seases and Rheumatism, or observed through their counseling. The study also involved the patients with subclinical hypo- and hyperthyroidism. The patients were observed in the period from the year

of 1997 to 2003. Hormone status of thyroid gland and echocardiography were determined for all pa-tients: before the ordinance of medicament therapy (l-thyroxine for hypothyroidism and methimazole or propylthiouracil for hyperthyroidism), regardless of duration of the previous dysfunction, and after at least six months of medicament therapy.

Secondary and central hypo- and hyperthyroi-dism and the patients whose anamnesis reveals the history of rheumatic fever and congenital heart de-fect were excluded from the study.

Analytical method was used in the study and the work techniques were:

Collecting the anamnesis data on existence and dating of symptomatology related to dysfunction of thyroid gland and cardiovascular system.

Determination of hormone status of thyroid gland (FT4, T4, FT3, T3, TSH) was carried out using the radioimmunoassay and immunometric method. Reference ranges (Ref. r.) TSH 0.3-4.2, FT4 11.4-21.3, FT3 3.2-7.4, T4 70-180, T3 1.3-3.3.

Analysis of the echocardiographic finding. Ec-hocardiographic tests were carried out by cardiolo-gist, using the echocardiograph Toshiba Power Vi-sion 7000, equipped with a transducer of 2.5 MHz. Exact measures of hearth cavities and wall thickne-ss were obtained by the M-method of measuring. Dimensions obtained were inserted into formulas and ejection fraction (E.F.) was calculated.

Ejection fraction is the most important para-meter for determination of systolic function. It re-presents the ratio (percentage) of systolic volume (difference of the end-diastolic and end-systolic volume) to end-diastolic volume. Reference ran-ges are 53-66%.

The study is clinical, comparative, retrospective.

Statistical evaluation

The data will be evaluated by statistical anal-ysis using the computer program SPSS, by adequ-ate statistical analyses of the data collected (t-test, correlation…). All tests and analyses are conside-red statistically significant, on the level of signifi-cance of p<0.05. The results obtained will be pre-sented on charts.

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Results

The main value of TSH in hyperthyroid pati-ents 0.05+/- 0.05

The main value of TSH in hypothyroid pati-ents 15.67+/- 10.31

In the test of independent samples, the valu-es of ejection fraction (E.F.) do not significantly differ in hyper- and hypo- conditions, in the test P=0.16 and retest P(r)=0.06.

Arithmetic means E.F. – test in hyperthyroid patients is 49.96, with standard deviation of 10.58.

Arithmetic means E.F. – test in hypothyroid pa-tients is 52.48, with standard deviation of 7.121.

Arithmetic means E.F. – retest in hyperthyroid patients is 50.10, with standard deviation of 10.23.

Arithmetic means E.F. – retest in hypothyroid patients is 53.36, with standard deviation of 6.84.

Chart 1. Change of the value of E.F. before and after the therapy, by groups

Therapy effect on E.F. is evident and it is stati-stically significant in hypo- P=0.000, and insigni-ficant in hyperthyroidism P=0.43

The values of E.F. are higher in hypo-, with tendency towards an increase, and lower in hyper-thyroidism, with tendency towards a slight decre-ase.

Resultant of the value range of E.F. in hyper- and hypo- is an increasing line.

Chart 2. Change of the value of E.F. before and after the therapy

Discussion

The difference in the value of E.F. was not sta-tistically significant in hypo- and hyperthyroid patients, neither at basal point nor after the thera-py. Average value in relation to the reference ran-ge was reduced in both hyper- (49.96+/-10.6) and hypo- (52.48+/-7.1). It is more reduced in hyper-, and tendency towards an increase is lower than in hypo-. Therapy effect is statistically significant only in hypo-, with the value (53.36+/-6.8) at the lowest limit of the reference range. The value in hyper- is still reduced after the therapy (50.10+/-10).

Goldman L. E. et al. demonstrated that the re-duced ejection fraction of the left ventricle, with congestive heart failure in a female patient with thyrotoxicosis, was possibly reversible after three weeks of treatment with thyreostatics. They asse-ssed that possible cause was the effect of thyroid hormone on the change of gene expression in car-diac cells, which confirms reversibility after the application of thyreostatics. Possible etiology of this myocardiopathy is chronic tachycardia asso-ciated with thyrotoxicosis.(14)

Biondi et al. detected the reduced E.F.: 53+/-8% at load and 62+/-7% at rest in subclinical hyperthyroidism.

Foldes et al. detected lower ejection fraction of the left ventricle in subclinical hypothyroidism than in euthyroid controls, both at rest and physi-cal load.

Bell et al. found reduction of ejection fraction of the left ventricle only at maximum load. The



se differences might be due to different selecti-on (age, gender, inclusion of patients with previo-us hyperthyroidism or unstable subclinical hypot-hyreosis), different diagnostic criteria (excessive-ly wide range of TSH levels) and impossibility to determine the occurrence and duration of thyroid disease in most of the examined populations.(15)

Ridgway E. C.et al 1982, Cooper D. S. et al. 1984, Forfar J. C. et al. 1985, Foldes J. et al. 1987 detected a damage of systolic function of the left ventricle in subclinical hypothyroidism, both at rest and at load, with clear improvement after l-T4 substitution therapy.

Shapiro et al. were observing a group of 17 asymptomatic patients on TSH-suppressive L-T4 therapy. They did not detect a difference between these and control patients in terms of the avera-ge heart frequency, prevalence of premature atrial contractions or average ejection fraction.

Vitale et al. emphasized the advantage of pulse tissue Doppler in evaluation of myocardium functi-on: using the standard Doppler echocardiography in subclinical hypothyroidism, they did not detect changes in systolic function of the left ventricle (fraction reduction, stroke volume or heart output), nor significant difference in global diastolic indices of the left ventricle between subclinical hypothyre-oses and euthyroid controls; using the pulse tissue Doppler, they demonstrated significantly slight da-mage to Em/Am (m – myocardium) ratio on the level of posterior septum in subclinical hypothyreoses. In their study, they determined that stable subclinical hypothyreosis was associated with cardiac functio-nal disorders of systolic and diastolic phases.

Fabio Monzani et al. demonstrated significant damage to both diastolic and systolic function of the left ventricle in subclinical hypothyroidism and that these changes were completely reversi-ble after l-T4 substitution therapy. This is the first study reporting on the presence of both functional and tissue damages to myocardium in subclinical hypothyroidism, which are reversible after speci-fic hormone compensation (16,17).

Said Gazibegovic et al. concluded that cardi-ovascular disorders associated with subclinical hyperthyroidism were significantly reduced after the treatment with methimazole and the achieved euthyroidism.

Konstantinos et al. presented a patient with su-

bclinical hyperthyroidism, expressed congestive heart failure and dilatation of the left ventricle and reduced ejection fraction, which is not usual in hyperthyroidism. After six months of therapy with thyreostatics, the changes were reversible (18).

Tuncel D. et al. detected the ejection fraction of 42% in Hoffman syndrome with hypothyroidism; after 6 months of therapy, E.F. was 55%, with re-versible dilating cardiomyopathy (19).

S. J. Lee et al. registered euthyroid sick syndro-me and reduced ejection fraction of the left ven-tricle in stress myocardiopathy (20).

Conclusion

Systolic function of the left ventricle is reduced both in hyper- and hypothyreosis in our patients.

Therapy effect on reduced systolic function of the left ventricle in patients with dysfunction of thyroid gland was statistically significant and in positive sense, but clinically still unsatisfactory.

References

1. Fabio Monzani at all.: Effect of Levothyroxine on Cardiac Function and Structure in Subclinical Hy-pothyroidism. A Double Blind, Placebo – Contro-lled Study. The Journal of Clinical Endocrinology and Metabolism. Vol.86.No3 Printed in USA Co-pyright 2001 by The Endocrine Society, 1110.

2. S.B. Ligget Shah SD, Cryer PE.: Increased fat and skeletal muscle beta adrenergic receptors but unal-tered metabolic and hemodynamicsensitivity to epi-nephrine in vivo in experimental human thyrotoxi-cosis. J Clin Invest 1989; 83: 803.

3. T. Kovač, L.Lepšanović. Endokrinologija Beograd 1996; 59.

4. Imaizumi et al. Subclinical Hypothyroidism and Heart Disease. J Clin Endocrinol Metab July 2004; 89(7): 3369.

5. Fabio Monzani et al. Myocardial Dysfunction in Hypothyreoidism. JCE&M 2001; Vol.86 No.3: 1113.

6. Obuobi et al. Hypothyroidism and Central Arterial Stiffnes. J Clin Endocrinol Metab 2002; October 87(10): 4662-4666.

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7. G. Mintz, R. Pizzarelo, I. Klein.: Enhanced left ven-tricular diastolic function in hyperthyroidism: non-invasive assement and response to treatment. J Clin Endocrinol Metab 1991;73:146.

8. J.C.Forfar et al. : Abnormal left ventricular function in hyperthyroidism: evidence for a possible reversi-ble cardiomyopathy.N Engl J Med 1982;307:1165.

9. Wildemberg LE, Sousa LL, Fonseca LP, Souza MV. Servico de Endocrinologia, Instituto Estdual de Di-abetes e Endocrinologia Luis Capriglione, Rio de Janeiro: Reversible dilated cardiomyopathy related to hyperthyroidism. Arq Bras Endocrinol Matabol. 2007 Dec; 51(9): 1533-8.

10. Tariq Nasser, Abdulhalim Kjnsarah, Abdulah Ka-rawgh, King Abdulaziz National Guard Medical City, Jeddah/Western Region, Saudi Arabia: Re-versible thyrotoxic cardiomyopathy, European Congress of Endocrinology 2010, Prague, Czech Republic, 24 April 2010-28 April 2010, European Society of Endocrinology

11. Nicolas Rodondi, MD, MAS, Douglas C. Bauer, MD, Anne R. Capopola, MD, ScM, Jacques Cor-nuz, MD, MPH, John Robbins, MD, MHS, Linda P. Fried , MD, MPH, Paul W. Ladenson, MD, Eric Vittinghoff, PhD, John S.Gottdiener, MD, FACC and Anne B. Newman, MD, MPH: Subclinical Thyroid Dysfunction, Cardiac Function, and the Risk of Heart Failure, The Cardiovascular Health Study; J Am Coll Cardiol, 2008; 52: 1152-1159, doi: 10.1016/j.jacc.2008.07.009 2008 by the Ame-rican College of Cardiology Foundation.

12. S.J.Lee, J.G.Kang, O.H.Ryu, C.S. Kim, S.-H.Ihm, M.G.Choi, H.J.Yoo, and K.S. Hong.: The relati-onship of thyroid hormone status with myocardial function in stress cardiomyopathy. Eur. J. Endo-crinol., May 1, 2009; 160(5): 799-806.

13. Tsarouhas K, Kafantaris I, Antonakopoulos A, Vavetsi S, Pavliddis P, Constantinou I.L. Rever-sible Dilated Cardiomyopathy Due to Subclinical Hyperthyreoidism, 2 nd Department of Cardiolo-gy, „Amalia Fleming „ General Hospital, Athena, Greece. Hellenic J Cardiol 2010; 51: 67-70.

14. Goldman L.E, et al. Royal Victoria Hospital, McGill University, Montreal, Canada. : A case of thyrotoxicosis and reversible systolic cardiac dysfunction. Canadian Journal of Cardiology. Jul. 1999;15(7): 811.4.

15. Vitale G, Galderisi M, Lupoli G.A et al. Myocar-dial Evaluation by TD in Subclinical Hypothyroi-dism . J.Clin.Endocrinol. Metab. September 2002; 87(9): 4353.

16. L.E. Shapiro et al.: Minimal cardiac Effects in Asymptomatic Athyreotic Patients Chronically Treated with Thyrotropin-Supressive Doses of L-Thyroxin. Journal of Clinical Endocrinology and Metabolism1997; 82: 2592.

17. Monzani F, Di Bello V, Caraccio N, et all. Myo-cardial Dysfunction in Hypothyreoidism JCE&M. 2001; Vol. 86.No.3: 1110-1114.

18. Gazibegović S, Jakubović – Čičkušić A, Kušljugić Z, Baraković F: Treatment of subclinical hyper-thyroidism significantly reduces the rate and symtomps of associated cardiovascular abnorma-lities, Acta Med.Sal 2009; 38(1): 30-35.

19. Tuncel D, Cetikaya A, Kaya B , et al. Hoffmann syndrome: A case report. Medical Principles And Practice 2008; 17(4): 346-348.

20. Seong Jin Lee, Jun Goo Kang, Ok Hyun Ryu, Chul Sik Kim, Sung.Hee Ihm, Moon Gi Choi, Hyung Joon Yoo, Kyung Soon Hong: The relationship of thyroid hormone status with myocardial function in stress cardiomyopathy.Devision of Endocri-nology and Metabolism, Division of Cardiology, Department of Internal Medicine, College of Me-dicine, Hallym University, ChunCheon, Republic of Korea.Copyright 2009 European Society of En-docrinology.

Corresponding author Ismana Surkovic, Clinic of Endocrinology, Diabetes and Metabolic Deseases, University Clinical Centre Sarajevo, Bosnia and Herzegovina, e-mail: [email protected]



Abstract

Problem: Blood glucose variations damage tissue by activation of the oxidative stress. Chil-dren with diabetes mellitus TYPE 1 have great variations of blood glucose (BG) because of fast metabolism and variations in appetite and activity. Variations of BG can’t be realistic present by cap-illary blood glucose measuring ( SMBG). Contin-uous glucose monitoring (CGMS) performed by subcutaneously placed sensor for at least 3 days is new method for evidence of glucose variation.

Aim of the study: to compare results of glu-cose level obtained by CGMS with results ob-tained by SMBG from the same period.

Patients and methods: Glycaemia of paedi-atric T1DM patients treated for diabetes mellitus TYPE 1 (T1DM) at Paediatric Clinic in Sarajevo was monitored by CGMS and simultaneously and by SMBG. Number of glucose results, mean glycaemia, span of glycaemia, standard deviation of all glucose results are presented by tables and graphics.

Results: CGMS monitoring was performed in 44 patients: 19 boys and 25 girls mean age of 12,1 ± 4,5 years. 28 / 44 patients had HbA1c > 10,0%;

4/44 HbA1c < 7 %, and 12/44 patients had HbA1c between 7,0-8,0%. Indications for CGMS moni-toring were: poor metabolic control of T1DM - 68%; hypoglycaemia - 27%; changing thera-py - 27%; false results of SMBG in record book - 22%. Mean number of glycaemia results from CGMS monitoring was 801 ± 58, and for SMBG 7 ± 3. ( p< 0,0001). Mean glycaemia for CGMS was 11.5 mmol/l and for SMBG was 14,8 mmol/l (p<0,01). Percent of glycaemia values higher than 10,0 mmol/l, for CGMS was 52% and for SMBG was 36% (p<0,05). Percent of glycaemia results in target range was 36% for CGMS and 58% for SMBG (p<0,05). Hypoglycaemic values were obtained in 12% measurements by CGMS and in 4% measurements from SMBG (p<0,001). Standard deviation of all results for CGMS was 6,6 mmol/l, and for SMBG 5,0 mmol/l ( p<0,05).

Conclusion: CGMS monitoring of glycaemia in paediatric T1DM patients is very important because of precise data about events like hypo- and hyperglycaemia monitored in real time. This method is more superior than classic SMBG from capillary blood for reproducing moment and range of glucose variation. These results are useful for correction of the therapy and habits of the child

Comparisson of the results from continuous glucose monitoring and results from Capillary Glycaemia in children with type 1 diabetes kOmPArACIjA rEzuLTATA kOnTInuIrAnOG mOnITOrInGA GLukOzE sA rEzuLTATImA kAPILArnE GLIkEmIjE kOd djECE sA TIP 1 dIAbETEs mELLITusOmSnjezana Hasanbegovic

Paediatric Clinic, University Clinical Centre Sarajevo, Bosnia and Herzegovina

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to improve metabolic control of t1DM and escape chronic micro vascular complications of T1DM.

Key words: Diabetes mellitus TYPE 1, glu-cose variation, continuous glucose monitoring, SMBG.

Sažetak

Problem: Varijacije glikemije oštećuju tkiva putem aktivacije oksidativnog stresa. Djeca sa TIP 1 diabetes mellitusom (T1DM) imaju velike varijacije glikemije (ŠUK) zbog brzog metaboliz-ma te promjenjivog apetita i aktivnosti. Varijacije ŠUK-a se ne mogu realistično prikazati putem sa-mokontrole glukoze iz kapilarne krvi. Kontinuira-ni monitoring glukoze (CGMS) putem supkutano plasiranog senzora u toku najmanje 3 dana je nova metoda za prikazivanje varijacija glikemije.

Cilj studije: komparirati rezultate glikemije dobivene putem CGMS-a i rezultate glikemije do-bivene u istom vremenskom periodu iz kapilarne krvi (ŠUK).

Pacijenti i metode: Glikemija pedijatrijskih pacijenata sa T1DM koji se liječe na Pedijatrijskoj Klinici u Sarajevu je monitorirana putem CGMS metode i putem kapilarnog ŠUK-a istovremeno. Broj rezultata glikemije, srednja glikemija, raspon glikemije i standardna devijacija svih rezultata su prikazani tabelarno i grafički.

Rezultati: CGMS monitoring je urađen kod 44 pacijenta: 19 dječaka i 25 djevojčica srednje dobi 12,1 ± 4,5 godina. 28 pacijenata je imalo HbA1c > 10,0%; 4 pacijenta HbA1c < 7 %, a 12 pacijena-ta su imali HbA1c između 7,0-8,0%. Indikacije za CGMS monitoring su bile: loša metabolička kon-trola T1DM - 68%; hipoglikemije - 27%; promje-na terapije - 27%; lažni rezutati ŠUK-a u dnevniku - 22%. Prosječan broj registrovanih glikemija za CGMS monitoring je bio 801 ± 58, a za kapilar-ni ŠUK 7 ± 3. (p<0,0001). Srednja glikemija za CGMS je bila 11.5 mmol/l, a za kapilarni ŠUK 14,8 mmol/l (p<0,01). Procenat glikemija viših od 10,0 mmol/l za CGMS je bio 52% a za kapilarni ŠUK 36% (p<0,05). Procenat rezultata glikemije u ciljnom rasponu je bio 36% za CGMS a 58% za kapilani ŠUK (p<0,05). Hipoglikemijske vrijed-nosti su se javile u 12% mjerenja za CGMS i u 4% mjerenja kapilarnog ŠUK-a (p<0,001). Standard-

na devijacija za sve rezultate CGMS-a je bila 6,6 mmol/l, a za sve rezultate kaplarnog ŠUK--a 5,0 mmol/l ( p<0,05).

Zaključak: CGMS monitoring glikemije kod pedijatrijskih pacijenata je veoma značajan zbog preciznih podataka o događajima kao što su hipo- i hiperglikemije monitorirane u realnom vremenu. Ova metoda je superiornija nego klasčni kapilarni ŠUK za reprodukciju trenutnih glikemija i raspo-na glikemijskih varijacija. Rezultati CGMS-a su korisni za korekciju terapije i navika djeteta, te po-stizanje bolje metaboličke kontrole T1DM u cilju izbjegavanja hroničnih mikrovaskularnih kompli-kacija T1DM.

Ključne riječi: Diabetes mellitus TIP 1, va-rijacije glukoze, kontinuirani monitoring glukoze, ŠUK

Introduction

Glycaemia is continuously changing in non-diabetic persons but in the range of normal gly-caemic results. But, in diabetic persons, variations of glycaemia are higher as comparing every result with the previous one obtained by seven- point capillary glucose profile.(1)

Paediatric TYPE 1 Diabetes mellitus patients (T1DM) achieve normal glycaemic results with much difficulties because of growth, physical de-velopment, fluctuation of appetite and unpredict-able physical activity. Puberty and psychological burden of chronic, life-long lasting disease and traumatic therapy provoke incompliance of the child and get him to poor metabolic control of T1DM ( glycosylated haemoglobin - HbA1c ≥ 7,0 %).(2, 3)

In spite of numerous, self-controls of glucose (SMBG) performed from capillary blood by glu-cose meter, even 7-10 times daily, it is impossible to obtain realistic information about all variations of blood glucose during 24 hours and especially from longer period of time. In the periods between SMBG points hyper- and hypo-glycaemias are frequent, and the variability of blood glucose is increased. Hyperglycaemia has glucotoxic ac-tivity and hypoglycaemia, especially in children younger than 7 years, can make irreversible con-sequences on psychomotor development.(4, 5)



Numerous studies indicate to very harmful in-fluence of glucose fluctuations to incidence of acute and chronic diabetic complications. Mean glycaemia (MPG-mean plasma glucose) can be calculated from the result of HbA1c, and does not point to glucose variability. HbA1c value is also bad predictor of glucose variability, especially for hypoglycaemic episodes.(6, 7)

Glycaemic variability could be simplest ex-pressed by result of standard deviation (SD) of all glycaemia measured by SMBG.

Blood glucose variations produce their bad ef-fect by activation of the oxidative stress. Great fluctuations of glycaemia activate oxidative mech-anisms producing super oxide by electron transfer in mitochondria. This activation starts cascade of harmful events which favour “classic” patterns of arising of diabetic complications like: polyol pathway, increased protein glycation, synthesis of AGE (advanced glycation end) products and activation of protein-kinase C and nuclear B fac-tor. Flux of glucose to hexosamine pathway is also increased.(8, 9)

Development of sensors for 72-hour-long mon-itoring of glycemia (measuring of glucose

level subcutaneously every 5 minutes) on the basis of classic enzymatic glucose-oxidase meth-od enabled better insight in fluctuation of glucose than achieved with SMBG.

Medtronic Minimed was the first company who manufactured CGMS - continuous glucose monitoring system – which was approved for use in 1999 by FDA ( Food and Drug Administration. CGMS gold system and Guardian RT ( real time) are advanced versions of this system. Guardian RT enables actual visualisation of glucose level in real time.(10, 11)

Nowadays, systems for continuous glucose monitoring are the best means for presentations of glucose variability in the continuous period of at least 3 days. After downloading results to PC we get tables and graphics with precise glucose readings from every 5 minutes. These data enable correction of glucose results by more adequate insulin therapy and by improvement of quality and quantity for diet and for physical activity.(12)

Aim of the study

The aim of this study was to compare results of glycaemia obtained by continuous glucose moni-toring system with results obtained by SMBG in the same period from T1DM patients treated at Paediatric Clinic in Sarajevo.

Patients and methods

All T1DM patients age between 0-18, in whom CGMS monitoring was performed at Paediatric Clinic of University Clinical Centre in Sarajevo were enrolled in the study. This study is retrospec-tive analysis of CGMS records that were per-formed in the period from the 1st of May 2007 to the 1st of May 2009.

We analyzed: - patients according sex and age - therapy and the level of metabolic control

of T1DM - indications for CGMS monitoring - number of glucose results, mean glycaemia

and span of glycaemia for CGMS results and for SMBG results

- quantity of results between normal glycaemic range, and out of normal range for CGMS monitoring results and for SMBG results

- SD ( standard deviation) for CGMS results - SD (standard deviation) for SMBG results

CGMS was performed in all patients by CGMS gold system manufactured by Medtronic Min-imed.

Indications for CGMS start were set up by paediatric diabetologist.

At first, patient and parent s were educated for entering following data to the device in the same moment when that event was occurred:

- result of capillary blood glucose performed by glucose meter (SMBG) in mmol/l ( mili mol per litre) ,

- injection of insulin, - meal, - exercise, - hypoglycaemia

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Older patient or his parent must fill form with: SMBG results with the time of performing; time, type and dose of injected insulin; time of meal with quantity and quality of eaten food; time and sort of exercise; time of hypoglycaemia and pos-sible reason.

During 24 hours of the day there must be at least 4 SMBG measuring, and results must be en-tered in the device immediately after obtaining, because of calibration of the sensor.

Sensor can measure all glucose results between 2,2-22,2 mmol/l. Every result out of this range must be corrected (by food or insulin) and SMBG must be performed again and result appropriate to the range of sensor should be entered in the de-vice. Before each monitoring, device should be adjusted to actual time and identification number of patient must be entered.

After education, sensor is implanted subcutane-ously – mostly in lateral part of stomach, and one hour later, cable of the device is connected with sensor. Than, after the time for sensor initialization (1 hour) first SMBG result is entered in the device.

Next three days patient is doing hid everyday activities without limits becuse of the device, while monitoring of glucose is continuously per-formed. After 72 hours of monitoring sensor is re-moved and device is placed at Com-station (other device connected with personal computer - PC) by which, all data are transferred to PC. PC has al-ready downloaded system file for these activities.

All data are presented on tables and graphics with the symbols for all entered events that are synchronic with glycaemia plot.(13)

Level of metabolic regulation of T1DM was defined by HbA1c result and micro colon method was used for analysing HbA1c from vein blood.(14)

Data for hypo- and hyperglycaemia are read from tables, and they are expressed in percent (%) of the time of their lasting in the comparison with whole time of CGMS registration.

SD (standard deviation) for CGMS results and for SMBG results that were entered in the device has already been calculated and presented on the table.

All data were statistically analyzed by appro-priate statistical tests, and results were presented numerically on the tables. Difference between pa-rameters were statistically significance for p < 0,05.

Results

In this paper we compared results of glycaemia obtained by CGMS monitoring and the results of glycaemia obtained by SMBG simultaneously. All analysed patients were monitored in the period from the 1st of May 2007 to the 1st of May .

SD ( standard deviation) for CGMS results and for SMBG results were calculated, and according SD result we establish the level of variability of glycaemia.

Table 1 presents that CGMS monitoring was performed in 44 patients : 19 boys and 25 girls. Mean age of all patients was 12,1 ± 4,5 years.

From table 2 we can see that 28 of 44 CGMS monitored patients had HbA1c level over 10,0%, and 4 of the patients less than 7 %. Rest 12 pati-ents had HbA1c level between 7,0-8,0%.

Table 1. CGMS monitored patients according sex and age.Sex No (%) age (years)boys 19 (43 %) 12,4 ± 4,4girls 25 (57 %) 11,8 ± 4,6total 44 (100%) 12,1 ± 4,5

Table 2. Therapy of the patients in the moment of CGMS monitoring therapy

HbA1cclassic insulin

(%)insulin analogues

(%)insulin pump

(%)total(%)

≤ 7,0 % 1 (9 %) 1 (4 %) 2 (20 %) 4 (9 %)7,0 – 8,0 % 4 (36 %) 4 (17 %) 4 (40 %) 12 ( 27 %)

> 8,0 % 6 (55 %) 18 (79 %) 4 (40 %) 28 (64 %)total 11 (100 %) 23 (100%) 10 (100 %) 44 (100 %)



Indications for CGMS monitoring are presen-ted at table 3. Even 63% of all, had poor metabolic control of T1DM what was indication for CGMS, hypoglycaemic episode were indication in 27% of the patients and changing therapy in 27% of the patients. False results in record book were indica-tion for CGMS monitoring in 22% of all patients.

Table 4 presents number of glycaemia results performed by CGMS monitoring, and it was 801 ± 58 while number of glucose results performed by glucose meter which were entered in CGMS device was 7 ± 3. There was high statistical significance between number of results obtained by two diffe-rent methods. Mean glycaemia for CGMS moni-toring was 11.5 and for SMBG by glucose meter was 14,8. Differences between mean glycaemia, and between range of glycaemia for two methods were statistically high significant: p< 0,01 for mean glycaemia and p < 0,05 for range of glycaemia.

Table 5 represents percent of glycaemia valu-es higher than 10,0 mmol/l, and for CGMS met-hod it was 52%, but for SMBG method was 36%, with statistically significant difference between results (p<0,05). Percent of glycaemia results in target range was 36% for CGMS and 58% for SMBG method, with statistically significant dif-ference 0,05.

Hypoglycaemic values were obtained from 12% measurements by CGMS and from 4% mea-surements from SMBG (p<0,001)

Standard deviation of all measurements of gly-caemia for CGMS was 6,6 mmol/l, and for SMBG 5,0 mmol/l, with significant statistical difference p<0,05, what was presented at table 6.

Table 3. Indications for CGMS monitoringIndications for CGMS Yes (%) No (%) Totalpoor regulated T1DM 28 (63 %) 16 (37 %) 44 ( 100%)

hypoglycaemia 12 (27 %) 32 (73 %) 44 ( 100%)change of therapy 22 (50 %) 22 (50 %) 44 ( 100%)

false records 10 (22 %) 34 (78 %) 44 ( 100%)

Table 4. Number of glycaemia results, mean glycaemia and range of glycaemic results for CGMS and SMBG

Method No of glycaemia results

Mean glycaemia result (mmol/l)

Glycaemia rangeMin-Max (mmo/l)

CGMS 801 ± 58 11,5 2,6 – 22,2CGMS glucose meter 7 ± 3 14,8 4,1 – 21,1

p p < 0,0001 p < 0,01 p<0,05

Table 5. Percent of glycaemia values in and out of target results for CGMS and SMBG % glucose results method

glycaemia> 10 mmol/l

glycaemia3,9 – 10,0 mmol/l

glycaemia< 3,9 mmol/l

CGMS 52 % 36 % 12 %Glycaemia-glucose meter 38 % 58 % 4 %

p p < 0,05 p < 0,05 p < 0,001

Table 6. Mean glycaemia and SD (standard deviation) for all results from CGMS and SMBG method.method mean glycaemia (mmol/l) SD ( mmol/l)CGMS 11,5 ± 6,6 6,6

ŠUK-glucose meter 14,8 ± 5,0 5,0p p < 0,01 p < 0,05

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Discussion

Continuous glucose monitoring with parallel monitoring of glycaemia (SMBG) by glucose me-ter was performed in 44 patients.

There were 25 girls (57%). Numerous studies (15) pointed to higher incidence T1DM in boys, but Verroti and co. documented that girls attain good metabolic control more difficult than boys, what is the reason for more monitored girls. Mean age of patients in study was 12,1 ± 4,5, what was pubertal time.

Previous studies of the patients suffering from T1DM who were treated at Paediatric Clinic in Sarajevo pointed out that even 2/3 -3/4 of poor regulated patients were in pubertal age. (17) Pu-bertal patients also often wrote false results of SMBG in their record book, so results were not correspondent with poor level of metabolic regu-lation of T1DM. Suspicion to this situation was indication for CGMS monitoring in 22% of all pa-tients in the study.(18)

Poor metabolic regulation of diabetes (HbA1c > 8%) was indication for CGMS monitoring in 64% of all patients. Contemporary therapeutic regimens for patients in pubertal age recommend intensification of insulin therapy with insulin ana-logues or with insulin pump.(19)

Almost 79% of patients treated with insulin analogues were monitored by CGMS because of poor regulated T1DM or because of their in-tention/suggesting of medical professionals for changing therapy.

Very few patients using insulin pump was mon-itored by CGMS. Reason for this probably was ob-ligation of these patients for very frequent SMBG because of very intensified therapy. Record books of these patients were full of SMBG data what was one of the very important reasons for better metabolic regulation of their T1DM.

According previous studies of T1DM patients from Paediatric Clinic in Sarajevo, good meta-bolic regulated T1DM patients using pen thera-py, performed SMBG 2,5-3,5 times daily, while mean number of SMBG results of patients with HbA1c>10,0% was 1,25 SMBG daily.(18, 20)

Mean number of CGMS glucose results was 806 per one monitoring, what was significantly different from the number of results from SMBG

method. We must mention that number of SMBG results entered to CGMS device was higher than number of SMBG performed in the time out of CGMS monitoring.

In study of Boland and co. results of 8-point capillary glucose measurements and results of CGMS monitoring were compared. This study confirmed great benefit of CGMS monitoring ,be-cause there was not everyday compliance of pa-tients for frequent SMBG with glucose meter.(21)

Direct Net study and other similar studies from last five years pointed to benefit of periodi-cal CGMS monitoring, because of numerous data about glucose variability. CGMS records were analyzed by doctor and patient together and all mistakes in therapy and diet were discussed and patent got advice to improve his diabetes manage-ment. This process had long-lasting benefit for better metabolic control of T1DM.(22)

Significantly more measurements of glycaemia obtained by CGMS resulted in significantly dif-ferent mean glycaemia (11,5 mmol/l) than mean glycaemia from SMBG (14,8 mmol/l)

Owing to numerous measurements of glycae-mia by CGMS, range of glycaemia ( Min-Max) was more realistic (2,6-22,2 mmol/l) than by CGMS results (4,1-21,1 mmol/l)

More results of glucose measurements obtained by CGMS presented percent of results out of up-per limit (10,0 mmol/l) more close to the level of metabolic control , and also indicated to more fre-quent hypoglycaemias (12%) than SMBG (4%).

These data are correspondent to great number of patients who had CGMS monitoring because of poor metabolic control of T1DM (63%) and because of hypoglycaemia (27%)

Study of Brazilian authors pointed to the im-portance of CGMS for discovering of unrecog-nised hypoglycaemia and postprandial hyperg-lycaemias. Both of them are the base of glucose variability and in the same time both problems could be solved by adequate insulin therapy and adequate food.(23)

Standard deviation of mean glycaemia as mean marker of the degree of glucose variability was also significantly different for CGMS results (6,6 mmol/l) and for SMBG results ( 5,0 mmol/l).(7, 9)

Desirable SD result should up to half of mean glycaemia, although it is difficult to obtain it. In



spite of very broad range of measured glycaemia (min-max range) obtained from both methods, SD is not over recommended maximal values- half of mean SD for CGMS and for pen therapy. (7, 9).

Conclusion

CGMS method of glycaemia monitoring is very important for paediatric T1DM patients. It provides a lot of precise data about events like hypo- and hyperglycaemia monitored in real time. This method is more superior than classic SMBG from capillary blood. CGMS offered possibil-ity for more precise reproducing range of varia-tions of the glucose and the moment when they occurred. Graphics’ and tables’ presentation of the results for CGMS monitoring and SMBG monitoring done simultaneously offers to patient possibility for mistake visualization and advice from doctor for their correction. It will be help for attaining long-time better metabolic control of T1DM in paediatric patients, and for escape of chronic micro vascular complications..

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9. Kilpatrick E.S., Rigby A.S., Atkin S.L. The effect of glucose variability on the risk of microvascular complications in type 1 diabetes. Diabetes Care 2006;29:1486–1490.

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Corresponding author Snjezana Hasanbegovic., Paediatric Clinic, University Clinical Centre Sarajevo, Bosnia and Herzegovina, E-mail: [email protected]



Abstract

Introduction: Glucose intolerance (GI) is a major risk factor for coronary heart disease (CHD). The aim of this study was to determine the role of GI as risk factor in emergence of CHD. Subjects and methods. Prospective study included examination of 186 patients with established CHD. The diagnosis of CHD was determined according to the criteria of the European Cardiac Society (ESC). Based on the OGTT insulinemia and blood glucose levels by ADA/WHO criteria, patients were divi-ded into groups with glucose intolerance (GI) and group with normal glucose tolerance (NGT), then the group with GI was divided into prediabetes (PD) and type 2 diabetes subgroups.

Results: Patients group with GI had a 4.02 ti-mes (OR = 4.02; 95% CI: 2.66-6 .08, p<0.0001) had higher risk of CHD than patients with NGT. Prediabetes subgroup (IFG + IGT) had a 2.10 ti-mes (OR = 2.10; 95% CI: 1.35-3.27, p <0,001) hi-gher risk than patients with NGT. The subgroup of patients with type 2 diabetes had 1.94 times (OR = 1.94; 95% CI: 1.24-3.03, p <0.04) higher risk than those with NGT.

Results of meta analysis showed that visce-ral obesity (OR = 0.35; 95% CI :0.19-0 .65, p = 0.0009), hyperinsulinemia (OR = 0.47; 95% CI: 0.26-0.86, p = 0.02), fasting plasma glucose ( OR = 0.39; 95% CI :0.21-0.74, p = 0,004), 2hrs-po-stprandial glucose (OR = 0:40, 95% CI: 0.21-0 .79, p = 0,011), hemoglobin A1C (OR = 0.40; 95% CI: 0.22-0.72, p = 0.0031), hypertension (OR = 0.52; 95% CI :0.30-0.92, p = 0,034), hypertrigliceri-

demia (OR = 0.51; 95% CI :0.30-0.90, p = 0.02), decreased HDL - cholesterol (OR = 0.33; 95% CI :0.17-0.64, p = 0,001), apo B / apoA> 0.7 (OR = 0.31; 95% CI :0.16-0.90, p = 0.0005), small, dense LDL (OR = 4.22; 95% CI :1.71-10 .36, p = 0,001), hyperuricemia (OR = 0.46, 95% CI :0.23-0.93, p = 0,042) were independent risk factors for CHD in the GI . Good markers of coronary heart disease in the GI were microalbumin (OR = 0.50; 95% CI, 0.27-0.93, p = 0,038) and C-reactive protein (OR = 0.36; 95% CI :0.18-0 .70, p = 0,003).

Conclusion: The results of this study showed that multiple GI is a risk factor for CHD, while vis-ceral obesity, atherogenic dislipidemia, hyperinsu-linemia, hypertension and hyperuricemia are inde-pendent risk factors which when associated together generate multiple toxicity. Microalbuminuria and elevated C-reactive protein are markers of coronary atherosclerosis and cardiovascular vulnerability.

Key words: glucose intolerance, risk factor, coronary heart disease

Sažetak

Uvod: Glukozne intolerancije (GI) su veliki ri-zikofaktor za koronarnu srčanu bolesst (CHD). Cilj istraživanja je bio da se utvrditi ulogu GI kao rizi-kofaktora u nastanku CHD.

Ispitanici i metode: Prospektivnom studijom je obuhvaćeno ispitivanjem 186 bolesnika sa utvr-đenom CHD. Dijagnoza CHD je utvrđena prema kriterijima Europskog kardiološkog društva (ESC). Na osnovu OGTT inzulinemije i glikemije prema

Glucose intolerance is the major risk factors for coronary artery disease GLukOznA InTOLErAnCIjA jE VELIkI rIzIkO fAkTOr zA kOrOnArnu bOLEsTHajder M1., Samardzic R2., Kudumovic A.3

1 University Clinical Center Tuzla, Internal clinic, Department of Endocrinology, Bosnia and Herzegovina,2 Cazin Health Center with Outpatient clinic, Bosnia and Herzegovina,3 General Hospital, Sarajevo, Bosnia and Herzegovina

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ADA/WHO kriterija bolesnici su podjeljeni na grupu sa glukoznim intolerancijama (GI) i grupu sa normalnom glukoznom tolerancijom (NGT), a potom je grupa sa GI je podjeljena na podgrupe sa predijabetesom (PD) i tip 2 dijabetesom.

Rezultati: Grupa bolesnika sa GI imala je 4.02 puta (OR=4.02; 95%CI:2.66-6.08, p<0.0001) veći rizik CHD nego bolesnici sa NGT. Predijabetes podgrupa (IFG+IGT) imala je 2.10 puta (OR=2.10; 95%CI: 1.35-3.27, p<0.001) veći rizik nego boles-nici sa NGT. I podgrupa bolesnika sa tip 2 dijabete-som imala je 1.94 puta (OR=1.94; 95%CI: 1.24-3.03, p<0.04) veći rizik nego osobe sa NGT.

Rezultati meta analize su pokazali da je visceralna gojaznost (OR=0.35; 95%CI:0.19-0.65, p=0.0009), hiperinzulinemija (OR=0.47; 95%CI: 0.26-0.86, p=0.02), plazma glukoza natašte (OR=0.39; 95%CI:0.21-0.74, p=0.004), 2h-postprandijalna glukoza (OR=0.40; 95%CI:0.21-0.79, p=0.011), hemoglobin A1C (OR=0.40; 95%CI:0.22-0.72, p=0.0031), hipertenzija (OR=0.52; 95%CI:0.30-0.92, p=0.034), hipertrigliceridemija (OR=0.51; 95%CI:0.30-0.90, p=0.02), snižen HDL-holesterol (OR=0.33; 95%CI:0.17-0.64, p=0.001), apo B /apoA >0.7 (OR=0.31; 95%CI:0.16-0.90, p=0.0005), small, dense LDL (OR=4.22 ; 95%CI :1.71-10.36, p=0.001), hiperurikemija (OR=0.46; 95%CI:0.23-0.93, p=0.042) bili su samostalni riziko faktori u CHD u GI Dobri markeri koronarne srčane bolesti u GI bili su mikroalbumin (OR=0.50; 95%CI;0.27-0.93, p=0.038) i C-reaktivni protein (OR=0.36; 95%CI:0.18-0.70, p=0.003).

Zaključak : Rezultati ove studije su pokazali da je GI multipli riziko faktor GHD, dok su vis-ceralna gojaznost, aterogena dislipidemija, hipe-rinzulinemija, hipertenzija i hiperurikemija sa-mostalni riziko faktori, koji udruženi zajedno čine multiplu toksičnost. Mikroalbuminurija i povišen C-reaktivni protein su markeri koronarne ateros-kleroze i kardiovaskularne ugroženosti.

Ključne riječi: glukozne intolerancije, riziko faktor, koronarna srčana bolest

Introduction

Hyperglycaemia is a landmark of glucose into-lerance (GI), an individual independent risk factor for cardiovascular disease (1). Postprandial hyper-

glycemia, and not the fasting glucose, was an in-dependent risk factor for the occurrence of acute myocardial infarction (AIM) and total mortality in patients with type 2 diabetes recently diagnosed (2). Patients who had glucose above 7.4 mmol / L after AMI had a high risk of reinfarction, he-art failure and increased mortality (3). Results in large cohort studies have shown that people with prediabetes (PD) have increased risk of coronary heart disease and higher mortality which would mean that continuous hyperglycemia is a risk fac-tor for CHD (4). A recent Austrian study, which included patients scheduled for elective coronary angiography because of suspected CHD, has fo-und 85% of subjects with GI, and these data sug-gest that GI is significantly more often present in patients with CHD than in the general population (5). Approximately 28% of patients with coronary disease have type 2 diabetes, and 70% of patients with ACS have abnormal glucose metabolism (6). Prognosis after coronary events in the diabetic is worse than in the nondiabetic population. Af-ter AIM, 44.2% of men with type 2 diabetes and 36.9% of women with diabetes die within 1 year (7). It is estimated that the risk of AIM in patients with type 2 diabetes is the same as in patients with CHD present, and about four times more com-mon in women and two times in men compared to nondiabetic population (8). Mortality was higher in both sexes compared to patients without diabe-tes, and mortality was similar in both sexes of the same age (9). The aim of this study was to deter-mine the representation of GI in coronary disease.

Subjects and Methods

Prospective study included the examination of 186 patients with established CHD hospitalized in The Internal clinic-Department of intensive care of UKC Tuzla. Diagnosis of acute coronary syn-drome (ACS) patients was established by ESC criteria (10). Diagnosis of chronic coronary artery disease patients was determined on the basis of positive history of coronary artery disease, ECG changes, Echocardiography, stress test and coro-nography. Patients with established CHD were divided on the basis of OGTT insulinemia into two groups: group with normal glucose tolerance



(NGT) and the group with GI. According to the ADA / WHO (11) criteria group with the GI was divided into subgroup with PD (IFG + IGT) and subgroups with Type 2 diabetes. Excluded from the study were the patients who were not proven CHD and were older than 65 years.

Diagnostic methods

Classification of obesity was determined on the basis of BMI and the waist size according to WHO criteria (12). Blood pressure was measured with blood pressure monitors, in a sitting position, a value determined on the basis of the criteria of ESH (13). Electrocardiogram was recorded after the clinical examination carried out in 12 leads on the unit CILLER Cardidiovit AT-1 Switzerland. Left side heart catheterization and bilateral selecti-ve coronography, left side ventriculography, were done by right transfemoral approach under local anesthesia, standard Judkins technique, nejonski Optiray 350, catheter JR4 pigtial, 6F. Following equipment was used Siemens’ Axiom “GE” Adve-mit “USA. OGTT was performed with 75 grams of glucose dissolved in water taken on an empty stomach after 12 hours of fasting. Blood was taken for determination of insulin and fasting glucose, during 30, 60 and 120 min. For determination of insulin in plasma kit INSI-CTK Irma, was used, DiaSorin firms in gamma counter “RIASTAR - PACKARD.” Normal values of insulin in the blo-od on an empty stomach were in the range of 2 -15 MU / ml. HbA1c was used the original Flex reagent catridge, companies DADE BEHRING, the apparatus DIMENSION chemeitry clinical system, in the reference values: from 4.8-6.0%. Microalbumin was analysed in urine using the ori-ginal DADE BEHRING reagens, on the apparatus BNII system NEFELOMETAR and gave Bering, USA. Reference Value: up to 30 mg / L.

Lp (a) is determined on the basis of immuno-chemical reaction nephelometric method, was used for the analysis of original kit reagent Com-panies DADE BEHRING, the apparatus BNII system NEFELOMETAR and gave BEHRING, USA. Reference values: from 0.30 to 0.0 g / L. Biochemistry parameters were determined by cla-ssical methods in a reference laboratory of UKC.

Statistical analysis

Statistical analasys was done using SPSS sof-tware version 12.

Numerical data are presented by mean ± SD, as median (range), the number% and 95% CI. For hypothesis testing between two independent gro-ups t-test (Mann-Whitney test and Fisher exact test)was used, and more independent groups was done one way ANOVA (Kruskal-Wallis test) - nonparametrical tests with significance level of p <0.05. For testing hypotheses difference frequen-cy (distribution) of observed parameters and the possible risk, meta analysis odds ratio was used, Chi square test - 2 by 2 and are shown obtained p-value significance difference (p <0.05).

Results

From a total of 186 patients with CHD present, NGT had 37 (19.89%), IFG had 36 (19.35%), IGT had 42 (22.06%) that is PD had 78 (41.41%) and DM type 2 had 72 (38.70%) (Fig. 1)

Figure 1. Distribution of Glucose intolerances in Coronary heart disease

The study results show that the presence of coronary artery disease in the GI was 80.11% in the NGT is 19.89%. Relative risk in the NGT was 0.24 while the relative risk reduction was 76% (1-0.24 = 0.76 x 100 = 76%). Absolute risk reduction was 60.22% (80.11% -19.89% = 60.22%). The re-lative risk of coronary heart disease in the GI was 4.02 (OR = 4.02, 95% CI :2.66-6 .08, p = 0.0001) times higher than in normal glucose tolerance (Fi-gure 2).

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Figure 2. Relative risk (95%CI) of Coronary he-art disease in Glucose intolerance compared with Normal glucose tolerance.

Characteristics of coronary artery disease in glucose tolerance

Results of this study showed that fasting pla-sma glucose >5.6 mmol/L ( OR=2.23; 95% CI: 1.47-3.72, p <0.03), 2h-plasma glucose> 7mmol

/ L (OR = 12.91; 95% CI:3.04-54 .7, p <0.0001), HbA1c> 6% (OR = 11:25, 95% CI: 3.39-13.24, p <0.0001), fasting plasma insulin> 15 mu / ml (OR = 7.20; 95% CI: 2.49-20 .78, p <0.0001), 2h-pla-sma insulin> 60 mu / ml (OR = 16.1; 95 % CI: 2.16-22.70, p <0,007), body mass index> 25 kg/m2 (OR = 2.82; 95% CI: 1.38-5.76, p <0,005), the ratio of waist m> 94 cm and f> 80 cm ( OR = 2.97, 95% CI: .1.42-6.23 p = 0,004), hiperurichemia: men> 363 women and> 238 (OR = 3:13; 95% CI: 1.40-7.0, p <0,006), triglycerides> 1.69 mmol / L (OR = 3.1; 95% CI: 1.48-6.49, p = 0,002), HDL-cholesterol <0.9 mmol / L (OR = 2:50; 95% CI: 1.22-5.13, p = 0,015), VLDL> 0.77 mmol / L (OR = 3.72; 95% CI: 1.73-8.0, p = 0.0003), apoB / apoA> 1 (OR = 2:42, 95% CI; 1.23-4.76, p = 0,008), small, dense LDL> 3 (OR = 4:22; 95% CI: 1.71-10.36, p = 0,001) and hypertension> 130/85 mmHg (OR = 3.69; 95% CI: 1.65-8 .13, p = 0,001) had significantly higher risks of coro-nary heart disease in glucose intolerance in relati-on to the subgroup with normal glucose tolerance (Table 1). Others, already established risk factors are age, cigarette smoking (OR = 1.01, p = 1.0),

Table 1. Relative risk (95%CI) of risk factors for Coronary heart disease in glucose intolerance in com-paration with normal glucose tolerance

Parameters NGTn = 37 GIn = 149 Odds RatioCI95% P – value

PGN > 5.6 (mmol/L) 12(32,4%) 108 (72,5%) 2,23 (1,47-3,72) P= 0.032h-PG >7.4 (mmol/L) 2 (5,4 %) 104 (69,8%) 12.91(3.04 - 54.7) P < 0.0001HbA1C > 6.0 (%) 3 (8,1%) 136 (91,3%) 11,25(3,39 – 3.24) P < 0.0001PIN >12 (mU/ml.2h- PI > 60 (mU/ml.)

4 (10 %) 1 (2 %)

116 (77,9%) 65 (43,6%)

7.20(2.49-20.78) 16.1(2.16 - 22.7)

P < 0.0001 P = 0.007

TGL >1.69 (mmol/L) 10(27 %) 125(83,9%) 3.1(1,48 – 6.49) P= 0.002HDL-C < 0.9 (mmol/L) 11 (29 %) 111 (74%) 2,50(1.22 – 5.13) P = 0.015LDL-C >3.0 (mmol/L) 35(94,6%) 133(89,3%) 0.943 (0.56 - 1.58) P = 0.93VLDL > 0.77 (mmol/L) 9 (24 %) 135 (90,6%) 3.72 (1.73 - 8.0) P = 0.0003Apo(B) /Apo(A) > 0.7 13(35,1%) 127 (85,2%) 2.42 (1.23 - 4.76) P = 0.008Lp(a) > 0.4 (g/L) 10(27,3%) 79 (53%) 1.96 (0.92 - 4.15) P = 0.10BMI (kg/m2): m > 25; ž> 24 11 (29%) 125 (83 %) 2.82 (1.38 - 5.76) P = 0.005OS (cm): m > 94 ž > 80 10(27,3%) 120 (80,6%) 2.97 (1.42 - 6.23) P = 0.004BP > 130/85 (mmHg.) 8 (21 %) 119(79 %) 3.69 (1.65- 8.13) P = 0.001Microalbumin > 20 (mg/L) 4 (10,8%) 102(68,5%) 6.33 (2.18 - 18.3) P = 0.0003Acidum uricum m > 363; w > 238 (µmol/L) 8 (21,6%) 101 (67,7%) 3.13 (1.40- 7.0) P = 0.006Homocystein ≥ 10 mmol/L) 34(91,9%) 120 (80,5%) 0.87 (0.51 - 1.48) P = 0.71Familly CHD (%) 19 (51%) 76 (51,3 %) 0.99 (0.53 - 1.84) P = 1Clamidia Pneumon. (%) 26 (73%) 89 (59,7%) 0.85 (0.48 - 1.49) P = 0.67Smoking history (%) 12(32,4%) 49 (32%) 1.01 (0.49 - 2.09) P = 1



antibody Chlamidia pneumoniae (OR = 0.85, p = 0.67), homocysteine (OR = 0.87, p = 0.71), positi-ve history of coronary artery disease (OR = 0.99, p = 1.0) were proved to be independent risk factors of glucose abnormalities (Table 1).

Characteristics of coronary artery disease in type 2 diabetes

In this study, morbidity from coronary heart disease in type 2 DM subgroup was 38.70% and 19.89% NGT. The relative risk of coronary he-art disease was 1.94 times (OR = 1.94; 95% CI: 1.24-3 .03, p = 0.04) higher in type 2 DM than in NGT subgroup. Absolute risk reduction of coro-nary heart disease was 18.81% (38.70% -19.89% = 18.81%) (Figure 3).

Figure 3. Relative risk (95%CI) of Coronary heart disease in Diabetes type 2 compared with Normal glucose tolerance.

Characteristics of coronary artery disease in prediabetes (P = IFG + IGT)

In this study morbidity from coronary heart disease in prediabetes was 41.41% and morbidity in normal glucose tolerance was 19.89%. Abso-lute risk reduction of coronary heart disease was 21:52% (41.41% -19.89% = 21:52%). The rela-tive risk of coronary heart disease was 2:10 times higher (OR = 2.08; 95% CI: 1.36-3 .27, p = 0,001) in prediabetes than in NTG subgroup (Figure 4).

Figure 4. Relative risk (95%CI) of Coronary heart disease in Pre diabetes compared with Nor-mal glucose tolerance.

Discussion

Glucose intolerance is associated with vice-ral obesity, insulin resistence/hyperinsulinemia, essential hypertension, atherogenic dyslipidemia, protrobotic and proinflamotory state in metabolic syndrome (MetS) and together comprise multiple toxicity and cause atherotrombosis (14). Results of UKPDS study showed that the MetS has 10 ye-ars IR toxic effect, but when there is collapsing of 50% of beta cells diabetes appears, and each year further lose by 4%. With the emergence of type 2 diabetes, hyperglycemia is an additional risk factor for CHD (15). Only 20% of the me-tabolic syndrome develop type 2 diabetes, while 80% will delay type 2 diabetes manifestation by hypertrophy of pancreatic beta-cells (16). Com-pensatory hyperinsulinemia does not go without consequences, leading to formation of macrovas-cular complications (CHD), which are consequen-ces of insulin, hypertensive and lipid toxicity (17). These facts may explain the earlier occurrence of the microvascular complications. This is suppor-ted by results of UKPDS-35 study (18), reduction of HbA1c by 1% significantly reduces heart attack by 14%, stroke by 12 % cardiac death by 16% and reduce microvascular complications by 56%.

The results of this study showed that the re-presentation of GI was 81% in coronary disease. Of the total CHD 41.41% belonged to prediabe-

628



tes, and 38.70% type 2 diabetes disease. (Figure 1). Hyperglycemia has its effect on endothelial cells through toxicity. Acute hyperglycemia in the blood can damage the endothelial homeostasis by producing free radicals (19,20, 21). Norwegi-an study has shown that elevated fasting glucose values above 6.1 mmol / L has to do with cardio-vascular morbidity and with other risk factors can be considered a risk factor (22). Results in cardi-ovascular Quebec prospective studies (22) indi-cate that fasting hyperinsulinemia, normal fasting glucose with hyper apoB, the increased value of small, dense LDL particles, is associated with twenty times higher risk of CHD, which indicates atherogenic nature of this group of disorders. Ho-orn study results indicate that increasing fasting blood glucose> 6.1 mmol / L, 2 hrs postprandial blood glucose and HbA1c, were risk factors for the occurrence of total cardiovascular morbidity in the population who did not know that they su-ffer from diabetes (23). IGT is a major risk factor for formation of type 2 diabetes and CHD in the middle age population (24). Asymptomatic hyper-glycemia increases the risk for future cardiovas-cular morbidity and mortality (25). Norhammar et al. (3) reported that the level of blood glucose above 7.4 mmol / L was an independent predictor of occurrence of reinfarction, heart failure and ma-jor cardiovascular events. A recent Austrian study, which included patients scheduled for elective coronary angiography because of suspected CHD was found 85% of those with GI, and these data suggest that GI significantly more often present in patients with CHD than in the general populati-on (5). Approximately 28% of patients with CHD have type 2 diabetes, and 70% had abnormal glu-cose metabolism (26).

The results of other studies have also demon-strated that poor glycemic control (27) is major risk factor for future coronary artery disease. Meta analysis showed for all the prospective studies published 1998 years, including nondiabetic pa-tients with 2hrs-posprandial glucose greater than 7.8 mmol / L, an increased risk of cardiovascu-lar events by 58% in comparison with the level of glucose of 4.2 mmol / L (28). The past 7 ye-ars several large epidemiological studies DCCT (29) UKPDS (30,31,32) tried to find a connection between control of diabetes and occurrence of nu-

merous complications. The results match the the-ory that chronic hyperglycemia plays an important role in patogenesis of micro and macrovascular complications. One of the main conclusions.

These results show that visceral obesity, hype-rinsulinemia, hyperglycemia, atherogenic lipo-protein phenotype, essential hypertension and hyperuricemia are independent risk factors of glu-cose intolerance, and together they make multiple toxicity and cause atherosclerosis. Glucose into-lerance microalbumin> 20mg / L (OR = 6:33, 95 % CI: 2.18-18.3, p <0.0003) is a good predictor of coronary atherosclerosis (Table 1). Results of other studies are consistent with these results. Fra-mingham Heart Study (33) indicates that obesity and physical activity is positively associated with risk of CHD, NCEF (34) and AHA (35) showed that obesity is an important risk factor for CHD and put it in the major risk factors. Insulin toxicity (hyperinsulinemia, hiperproinzulinemia, hipera-milinemia) is present in the MetS and coronary di-sease. It is known that insulin regulates a number of AT-1 receptor identified in the intima of islets of blood vessels, and activate systemic and local RAAS, and is able to cause cross-reactions with AT-1 receptors (36,37,38). Therefore, the toxicity of Insulin (I) can be associated with toxicity of an-giotensin II (A), resulting in increased redox stress that is increased in the circulation system, the local endothelium and islets (39). Endogen hyperinsuli-nemia is also associated with an increased level of free fatty acid, PAI-1, increased sympathetic ac-tivity, increased sodium and water retention, va-soconstriction of blood vessels and hypertension, which creates angiotensin toxicity (40). Results of UKPDS-34 (41) study have shown that intensive treatment of metformin is followed by reduction in the complications of diabetes by 32%, mortali-ty caused by diabetes by 42% and myocardial in-farction by 39%. Results (UKPDS-33) (30) of the studies have demonstrated that reducing HbA1c to 0.9% is followed by decrease of microvascu-lar complications by 25%, and reduction of heart attack by 16%. The results confirm the theory that chronic hyperglycemia plays an important role in the pathogenesis of micro-and macrovascular complications. All three studies confirm that in-tensive metabolic control should be implemented. In the UKPDS-39 study (32) it was found that



good control of blood pressure reduces micro and macrovascular complications. Good control of blood pressure had a better effect than good blood glucose control. Reducing blood pressure values of below 144/82 mmHg reduced the risk of mor-tality associated with diabetes from 32% to 56%. Search of the STEN studies have confirmed that intensive therapy significantly lowers all parame-ters of metabolic control in patients with diabetes, and thus reduced mortality from CHD and total mortality(42). Results Serum Uric and Cardiovas-cular mortality study (43) showed that increased levels of uric acid in serum was an independent risk factor and was significantly associated with risk of cardiovascular disease in both sexes.

In conclusion, hyperglycemia greater than 6.1 mmol/ L is a trademark or essential component of glucose intolerance within the Mets. Multiple risk factor of CHD is the Mets, who through multiple toxicity (AFLIGHT) leads to atherotrombosis and its complications.

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Corresponding author Hajder M. University Clinical Center Tuzla, Internal clinic, Department of Endocrinology, Bosnia and Herzegovina, E-mail: [email protected]



Abstract

Introduction: MRI of rectum is a new, non-ionised, easily tolerable procedure which does not require use of sedation or analgesics. MRI is considered to be the best diagnostic modality so far, with the best ability to show contrasts in soft tissues. It enables MRI to stage tumour phases of rectum, as well as differentiating malignant tumo-urs from their benign precursors – polyps, which largely determines therapeutic approach

Patients and methods: 20 patients were inclu-ded in the study, out of whom 9 (45%) female, and 11 (55%) male patients The study included only those patients in whom endoluminal polypoid tu-mour proliferation was found by endoscopy, for the purpose of differentiating of their PH characte-ristics. All patients were examined by rectal MRI on 1,5 T equipment.

Results: Results of the statistical analysis, whi-ch used Non-parameter test of gamma correlation, show higher incidence of malignant PH result in male patients (p=0,004). The difference in distri-bution according to benign or malignant lesions was statistically insignificant (p=0,71), and also the size of the tumour was not statistically si-gnificant in correlation to the age (p=0,33). It is understandable that the MRI method was more precise in detection of malignancy in tumours of bigger size. MRI was a significantly more sensi-tive method in differentiating malignant changes (p<0,001), and it highlt correlated with the type of PH results.

Conclusion: MRI is a new diagnostic modality showing a high level of accuracy in differentiating polypoid lesions after an unspecified endoscopic result. MRI is a significantly sensitive method of

differentiating malignant changes and it highly correlates to the type of PH results.

Key words: MRI, rectum, polypoid tumour, adenoma, adenocarcinoma

Introduction

MRI of rectum is a new, non-ionised, easily to-lerable procedure which does not require use of sedation or analgesics. MRI is considered to be the best diagnostic modality so far, with the best abili-ty to show contrasts in soft tissues. It enables MRI to stage tumour phases of rectum, as well as dif-ferentiating malignant tumours from their benign precursors – polyps, which largely determines therapeutic approach. Since colorectal carcinoma (CRC) is the third in the scale of frequency (af-ter lungs, prostate and breast cancers), and about 40-50% of all CRCs are diagnosed in the rectum, therefore MRI of the rectum is becoming massi-vely important. (1). MRI of the rectum started to be used during 90s of the last century, on equip-ment with lower magnetic field using endorectal coil. Nowadays, its application is being used less frequently, after a new MRI technology has been introduced using much advanced gradient systems as well as high resolution surface systems of coils, thus enabling a high spatial resolution and a big FOV, allowing not only for a more detailed eva-luation of intestinal walls, but also of the surroun-ding structures and mesorectal fascia. (2). MRI sequences for abdominal imaging nowadays are much faster and have much higer temporal and spatial resolution. The wall of a normal rectum shows high intensity of the signal coming between water and a muscle on T1W sequence, and shows

mrI of rectum – a new diagnostic modality in differentiating malignant from benign lesions Amela Sofic, Nedzad Sehovic, Serif Beslic, Besim Prnjavorac, Damir Sofic

Radiology Clinic at KCUS (Clinical Centre of Sarajevo University) Sarajevo, Bosnia and Herzegovina

632



moderately higher intensity of the signal in com-parison to the surrounding fat tissue. On T2W se-quence, three lines can be distinguished, namely: internal hyperintensity line, which corresponds to mucosa and submucosa; then hypointensity one, to moderate intensity one, which corresponds to muscularis propria; and the third one, outer hy-per intensity one, showing perirectal fat tissue. Thus MRI is enabled to stage tumour processes (staging). On the T2W sequence, the rectal wall shows the intensity of the signal between fat and water. After an iv application of Gadolinium con-trast medium, the rectal wall is seen as a brightly, intensively high signal line. There have been re-ports describing it as minimal enhancement; some other reported it as enhancement of even 100%. Hamm et al described that on the contrast T1 W series, opposite to muscularis propriaa, the muco-sa shows an early and strong increase of intensity. Perirectal fat tissue is seen as hyper intensity line, with a suppression of fat as an area of hypointens-ity. Normal thickness of the colon wall is 3-4 mm, although most authors state that it should be less than 5 mm (3).


20 patients were included in the study, out of whom 9 (45%) female, and 11 (55%) male pati-ents. Average age of patients was 60,55 years (SD 12,5). The study included only those patients in whom endoluminal polypoid tumour proliferation was found by endoscopy, for the purpose of diffe-rentiating of their PH characteristics. All patients were examined by rectal MRI on 1,5 T equipment (Siemens Erlangen, Germany) after 180 ml of the ultrasound gel had been effortlessly applied into the cleansed rectum. A routine protocol for pelvis was used, containing T1 three planes (axial, coro-nal and saggital planes), T2 axial and saggital pla-nes, as well as bipolar (axial and saggital planes using surface body coil, in a natural state, as well as after the iv application of Gadolinium contrast medium (Magnevist). Duration of the examinati-on, including a planning stage, was 25-30 minu-tes. Postprocessing analysis was done on Syngo software programme. Results of MRI evaluation were compared with PH results after polypectomy or resection of the rectum. A statistical analysis of

Table 1Number Age Sex Location of the rectum size MRI result PH result Type of PH

1 38 F middle third 2,5 cm Benig Benig Villous adenoma

2 66 F middle 2 cm Benig Benig Tubulovillous adenoma

3 58 M lower 3 cm Malig Malig Adenocarcinoma

4 74 M middle 2,5 cm Malig Benig Tubulovillous adenom a

5 49 M lower 2,3cm Malig Malig Adenocarcinoma6 68 M middle 2,4 cm Malig Malig Adenocarcinoma7 71 F middle 3,1 cm Malig Malig Adenckarcinoma8 69 F upper 3,5 cm Malig Malig Adenocarcinoma9 68 M middle 3,2 cm Malig Malig Adenocarcinoma10 74 M lower 2.3 cm Malig Malig Adenocarcinoma11 64 F upper 2 cm Benig Benig Villous adenoma12 78 M middle 4 cm Malig Malig Adenocarcinoma13 42 M middle 3,2 cm Malig Malig Adenocarcinoma14 45 F middle 3,1 cm Malig Malig Adenocarcinoma15 65 F middle 2,8 cm Malig Malig Adenocarcinoma16 75 F upper 3,5 cm Malig Malig Adenocarcinoma17 63 M upper 5 cm Malig Malig Adenocarcinoma18 45 F middle 2,2 cm Malig Malig Adenocarcinoma19 47 M middle 2,9 cm Malig Malig Adenocarcinoma20 52 M lower 2,7 cm Malig Malig Adenocarcinoma



the data was done by using Non-parameter test of gamma correlation, which is the applicative met-hod for our quantity of samples.

Results

As it can be seen from Table 1, all 20 patients in our sample had a PH confirmation of results. In 4 patients a benign tumour was noted, whilst it was a malignant tumour in 16 patients. From 16 mali-gnant lesions, 62.5% (n=10) was found in male patients, 37.5% (n=6) in female patients. Avera-ge age of patients with malignant lesions was 51 years of age, although it could be said that most of them were in their sixties or seventies. The yo-ungest patient was 49, and the oldest 78.The PH result in all malignant tumours was Adenocarci-noma 100% (n=16). It could be said that the Ade-nocarcinoma was most frequently located in the middle third of the rectum 50,25% (n=9), then on the lower third, or 25% (n=4), and the least frequ-ently located on the upper third 18,75% (n=3).MRI found 4 benign lesions without the inflitra-tion of the wall, which had been confirmed by the PH result, and they were: 2 tubulovillous adenoma on the middle third of the rectum, then 2 villous adenoma, out of which one was on the middle, and the other on the upper third. Out of total of three benign lesions, 3 were found in women, and 1 in a man. Only in case of one male patient, a tubulo-villous lesions, size 2.5 cm, located in the middle third of the rectum, was pre-diagnosed by MRI as a malignant lesion. The MRI was significantly a more sensitive method in detecting malignant changes (p<0,001), and it greatly correlated with PH result. Out of total of 20 rectal lesions, MRI di-fferentiated 19 of them as malignant and obtained the accuracy of 95%.

Results of the statistical analysis, which used Non-parameter test of gamma correlation, show higher incidence of malignant PH result in male patients (p=0,004). Taking into account MRI dis-crepancy in defininf a type of the lesion in regar-ds to PH in one male patient in our quantity of samples (n=20), while calculating correlation of the sex and description of the PH result, it can be noted that statistically there no significant diffe-rences. The difference in distribution according

to benign or malignant lesions was statistically insignificant (p=0,71), and also the size of the tu-mour was not statistically significant in correlati-on to the age (p=0,33). It is understandable that the MRI method was more precise in detection of malignancy in tumours of bigger size. MRI was a significantly more sensitive method in differenti-ating malignant changes (p<0,001), and it highlt correlated with the type of PH results. Although one could expect that bigger tumours are detected in older patients, this analysis did not demonstrate any statistical significance (p=0,33). The size of all lesions, regardless of PH results, ranged from 2 cm to maximum 4 cm, with no significant diffe-rence regarding the sex or age of patients.

a

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b

cFigure 1. a) Polyps b) middle third of the rectum c) rectosygmoid

a

b

cFigure 2. Carcinoma of a), b), c) upper third of the rectum



a

b

cFigure 3. Carcinoma of a), b) middle and c) lo-wer third of the rectum

Discussion

Every year, in countries of the European Com-munity, some 130,000 of new patients with colo-rectal carcinoma are diagnosed, and about 140,000 in the USA. However, it is interesting to note that mortality due to colorectal carcinoma has been re-duced in the USA; nevertheless, its incidence is still on a mild increase. It is considered that in the deve-loped countries around 4.6% of men and about 3.2 % of women will be affected by colorectal carci-noma during their lifetime, with the proportion of the incidence and mortality between men and wo-men being 1,05 : 1 (for the rectal carcinoma 1,4:1). (4) In view of its patohistological composition, a CRC is an Adenocarcinoma, which in 98% grow as exophytic, ulcerous and stenosing form. (5) Po-lyps are benign mushroom-like growths which are in 95% adenoma, and they represent precursors in the incidence of a CRC, which grow as peduncular or cecyl form. Adenoma are divided in three sub-types according to hystological criteria: tubulous, tubulovillous, and villous. Villous adenoma are bigger than the other two, and show a larger degree of displasia. They can be most frequently found in the area of the rectum and rectosygmoid, although they can appear anywhere. They have been noted for a bigger possibility to transform into a malig-

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nant tumour. (6) Mehdi Cadi /2005/ states the data that in France, an average development of genetical alteration of a small adenoma into carcinoma is 10 – 15 years. (7)

In our study, all malignant lesions were, accor-ding to the PH result, Adenocarcinoma. Out of 100%, there were 62.5% in male, and 37.5% in female patients. Most patients were in their sixties or seventies. All benign lesions were adenoma (3 tubulovillous and 1 villous), and female patients dominated 3:1. Author Ms Božović from Serbia mentions the most frequent distrivution of the car-cinoma in the rectum, in respect to other segments of the colon, with a slight prevalence in male pa-tients. (8). According to American surgeon, Dr Spratt (in 1960), polyps are most frequently de-tected in patients between 61 and 70 years of age, and carcinoma in the period from 51 to 70 years of age (9). A recent study by Brener (in 2007) from Germany revealed distribution according to sex of those affected by carcinoma of colorectal region, with 56,1% men, and slightly less frequent in wo-men - 43,9%. In the same study, the distribution according t age, showed that it was most frequent in patients between 70 and 79 years of age, fo-llowed by those between 60 and 69 years of age. (10) Although UICC divided rectal carcinomas in the upper, middle and lower thirds, in the present literature there are no published data regarding in-cidence of rectal carcinoma according to such cla-ssification. (11). Our research shows that the most frequent location in the middle third of the rectum - 50,25%, then in the lower third - 25%, and the least frequency is in the upper third - 18,75%. We obtained a high 95% accuracy of MRI in differen-tiating polypoid lesions in the PH results.

At present, research of evaluation of MRI in staging of rectal carcinoma is becoming increasin-gly important, and the accuracy of the results vary from the examination technique to the stage of the tumour. (12,13,14,15,16,17) Therefore, regarding the accuracy of MRI for T4 stage, it reaches even 100%, where for T3 stage with infiltration of me-sorectal fascia it reaches even 97%. Introduction of standardisation in application of MRI technique and intepretation of images it can be expected that better and more standardised results will be obtai-ned in the future (18).

Conclusion

MRI of rectum is showing a high level of accu-racy in differentiating polypoid lesions after an un-specified endoscopic result. MRI is a significantly sensitive method of differentiating malignant chan-ges and it highly correlates to the type of PH results.

References

1. Colorectal cancer. Article Last Updated: July 27;2008. Available from URL:http.//wwen.wikime-dia.org/wik/colorectal-cancer.

2. Klessen C,Rogalla P,Taupitz M. Local staging of rectal cancer-the current role of MRI.Europiean radiology.2007;17(2) 379-389.

3. Hamm B., Krestin G.P., Laniado M., Nicolas V., Taupitz M. MRT von Abdomen und Becken. GeorgThiemeVerlagKG,Stuttgart.2007;137-142. Zovak Mario. Korelacija ekstranodalnih depozita i kliničkopatoloških faktora u bolesnika operiranih zbog mucinoznog kolorektalnog karcinoma.Doktor-ska disertacija, Sveučilište u Zagrebu. Article Last Updated:Jan 13;2007. Aviable from URL:http://medlib.mef.hr/3591

4. M.Mušanovic,N.Obralić.Onkologija.Bošnjački In-stitut .2001; 281-284.

5. Brkić T,Grgić M.Colorectal Carcinoma.Medicus. 2006;15:89-97.

6. Mehdy Chadi. CT colonography(virtual colonosco-py).Guerbet .2005;12:5.

7. Vesna Mihajlović-Božović.Risk factors for colorec-tal cancer. Arch Oncol 2004;12(1):45-9.

8. John S. Spratt, Jr., Lauren V. Ackerman, and Carl A. Moyer. Relationship of Polyps of the Colon to Colonic Cancer. Ann Surg.1960:151(2):278.

9. H.Brenner, J.Chang-Claude, C.M. Seiler, T. Stür-mer and M. Hoffmeister. Potential for Colorec-tal Cancer Prevention of Sigmoidoscopy Colo-noscopy: Population-Based Case Control Study Cancer Epidemiology Biomarkers & Prevention. 2007;16,:494- 496.

10. Sobin LH, Wittekind C International Union Aga-inst Cancer (UICC). TNM classification of mali-gnant tumours: Wiley, New York.2002



11. Bissett IP, Fernando CC, Hough DM et al. Identi-fication of the fascia propria by magnetic resonan-ce imaging and its relevance to preoperative asse-ssment of rectal cancer. Dis Colon Rectum.2001; 44(2):259–265.

12. Torricelli P, Lo Russo S, Pecchi A, Luppi G, Cesi-naro AM,Romagnoli R. Endorectal coil MRI in lo-cal staging of rectal cancer. Radiol Med (Torino) 2002;103:74–83.

13. Brown G, Radcliffe AG, Newcombe RG, Dalli-more NS, Bourne MW, Williams GT. Preoperative assessment of prognostic factors in rectal cancer using high-resolution magnetic resonance ima-ging. Br J Surg.2003; 90(3):355–364.

14. Akin O, Nessar G, Agildere AM, Aydog G. Preope-rative local staging of rectal cancer with endorec-tal MR imaging.Comparison with histopathologic findings. Clin Imag 2004;28:432–438.

15. Tatli S, Mortele KJ, Breen EL, Bleday R, Silver-man SG.Local staging of rectal cancer using combined pelvic phasedarray and endorectal coil MRI. Magn Reson Imag 2006;23:534–540.

16. Piippo U, Pääkkö E, Mäkinen M, Mäkelä J.Local staging of rectal cancer using the black lumen magnetic resonance imaging technique. Scand J Surg. 2008;97(3):237-42.

17. Taylor F.G.M, Swift R.I, Blomqvist L, Brown G. A Systematic Approach to the Interpretation of Preoperative Staging MRI for Rectal Cancer.AJR 2008;191:1827-1835.

Corresponding author Amela Sofic, Radiology Clinic at KCUS, Clinical Centre of Sarajevo University, Bosnia and Herzegovina, E-mail: [email protected]

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Abstract

Introduction: Hypothiroidism and hyper-thyroidism are followed by cardiovascular struc-tural and functional changes which entirely wit-hdraw after timely treatment with l-thyroxin and tireostatics.

Cardiovascular changes caused by hyperthyro-idism and hypothyroidism withdraw to a great de-gree upon therapy with l-thyroxin and tireostatics, e.g upon achivement of euthyroidism.

Aims of research: To demonstrate changes of blood pressure caused by hyperthyroidism and hy-pothiroidisam by analyzing values of blood pre-ssure to check cardiovascular changes caused by hyperthyroidism and hypothyroidism by compa-rative methodology upon achivement of euthyroi-dism by medicament therapy.

Patients and methods: One hundred patients took part in the study: 50% of them suffering from hypothiroidism; 50% with hypothyroidism, being of either sex, aged over 14, hospitalized at the Clinic of Endocrinology, Diabetes and Metabolic Deseases, Clinic for Heart Deseases and Reuma-thism, or observed at the their ambulances over the period 1997-2003. Patients suffering from sub-clinical hypo and hyperthyroidism took part in the study as well. All patients have undergone physical examination- measure of blood presure and laboratory tests, thyroid hormonal status, be-fore being treated by l-thyroxin and tireostatics, and after achievement of euthyroidism. Patients

suffering from central and secondary hyperthyroi-dism, those who have record of rheumathoid fever in their personal anamnesis were excluded from the study.

Results: Hypothyroidism was associated with significant decrease of means values blood pressure after at least six months therapy with levothyroxin . Hyperthyroidism was associated with decrease of values blood pressure, after at le-ast six months therapy with antithyroid drugs.

Conclusion: Our study showed particularly statisticaly significant improvement in disfunction of thyroid gland and consequential cardiovascu-lar changes after treatment of medications, but not satisfactory by clinical aspect.

Key words: blood presurae, hypothyroidism, hyperthyroidism, l-thyroxin, antithyroid drugs.

Introduction

Stroke and minute volume of the heart is redu-ced in hypothyroidism. Convergent arterial hyper-tension occurs. Systolic blood pressure is reduced, while diastolic is increased, due to peripheral va-soconstriction, i.e. increase in peripheral resistan-ce. Diastolic hypertension occurs secondarily, after the change in systemic resistance combined with the increased blood volume (1,2,3,4,5).

Heart work is increased in hyperthyroidism, as expected, due to the increased oxygen requi-rement in peripheral tissues and increased blood

Comparison of arterial blood pressure values in dysfunction of thyroid gland before and after the therapyIsmana Surkovic1, Ismet Suljevic2, Azra Kudumovic3

1 Clinic of Endocrinology, Diabetes and Metabolic Deseases, University Clinical Centre Sarajevo, Bosnia and Herzegovina,2 Clinic of Anestesiology, University Clinical Centre Sarajevo, Bosnia and Herzegovina,3 General Hospital, Sarajevo, Bosnia and Herzegovina,



flow in skin, muscles, brain, thyreoidea and kid-neys. Tachycardia is not reduced during sleep; it is refractory to digitalis preparations as well. Diver-gent arterial hypertension occurs. Systolic blood pressure is increased due to the increased stroke volume, and diastolic is normal or reduced, due to the reduced peripheral resistance.

Structural and functional cardiovascular chan-ges occur in hypo- and hyperthyroidism, whi-ch completely regress in the achieved euthyroid condition, after early therapy with l-thyroxine and thyreostatics (1,3,6,7).

Objective of the study

To demonstrate cardiovascular changes in hypo- and hyperthyroidism, using the analysis of arterial blood pressure values. To examine rever-sibility of cardiovascular changes in hypo- and hyperthyroidism after medicament therapy, in the achieved euthyroidism, by comparative method.

Methods

The study involved 100 randomly selected patients (random sample method): 50 with hy-pothyroidism and 50 with hyperthyroidism, aged over 14, both genders, hospitalized at the Clinic for Endocrinology, Diabetes and Metabolism Di-seases, Clinic for Heart Diseases and Rheumati-sm, or observed through their counseling. The study also involved the patients with subclinical hypo- and hyperthyroidism. The patients were observed in the period from the year of 1997 to 2003. Hormone status of thyroid gland was deter-mined for all patients, as well as physical exami-nation of arterial blood pressure value before the ordinance of medicament therapy, regardless of the previous duration of dysfunction (l-thyroxine for hypothyroidism and methimazole or propylthi-ouracil for hyperthyroidism), and after at least six months of medicament therapy.

Secondary and central hypo- and hyperthyroi-dism and the patients whose anamnesis reveals the history of rheumatic fever and congenital heart de-fect were excluded from the study. The study is clinical, comparative, retrospective.

Analytical method was used in the study and the work techniques were:

Determination of hormone status of thyroid gland (FT4, T4, FT3, T3, TSH) was carried out using the radioimmunoassay and immunometric method. Reference ranges (Ref. r.) TSH 0.3-4.2, FT4 11.4-21.3, FT3 3.2-7.4, T4 70-180, T3 1.3-3.3.

Physical examination of arterial blood pressure value. Arterial blood pressure value (mmHg) was measured by a mercury manometer, in the pati-ents’ lying position.

Statistical evaluation

The data were evaluated by statistical analysis using the computer program SPSS, by adequate statistical analyses of the data collected (t-test, correlation…). All tests and analyses are conside-red statistically significant, on the level of signifi-cance of p<0.05. The results obtained will be pre-sented on charts.

Results

The main value of TSH in hyperthyroid pati-ents 0.05+/- 0.05

The main value of TSH in hypothyroid pati-ents 15.67+/- 10.31

In the test of independent samples, the values of systolic blood pressure differ, but not signifi-cantly, in hyper- and hypo- conditions, in the test and retest P=0.7, P(r)=0.9.

Arithmetic means of systolic blood pressure – test in hyperthyroid patients is 146.22, with stan-dard deviation of 28.24.

Arithmetic means of systolic blood pressure – test in hypothyroid patients is 148.40, with stan-dard deviation of 33.29.

Arithmetic means of systolic blood pressure – retest in hyperthyroid patients is 137.20, with standard deviation of 21.241.

Arithmetic means of systolic blood pressure – retest in hypothyroid patients is 137.80, with stan-dard deviation of 26.84.

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Chart 1. Change of the value of systolic blood pressure before and after the therapy, by groups.

The values of systolic blood pressure – test are slightly higher in hypo- than in hyper-, but with hi-gher tendency towards a decrease than the tenden-cy towards a decrease in systolic blood pressure in hyperthyroid patients (Chart 7).

The values of systolic blood pressure in both groups of patients are within the normal range both before and after the therapy, with insignifi-cantly higher values in hypo-, as well as insignifi-cant therapy effect (Chart 8).

Chart 2. Change of the value of systolic arterial blood pressure before and after the therapy.

Diastolic blood pressure

In the test of independent samples, the values of diastolic blood pressure significantly differ in hyper- and hypo- conditions, in the test P=0.016, and do not significantly differ in the retest P(r)=0.59.

Chart 3. Change of the value of diastolic blood pressure before and after the therapy, by groups.

Arithmetic means of diastolic blood pressure – test in hyperthyroid patients is 83.70, with stan-dard deviation of 12.69.

Arithmetic means of diastolic blood pressure – test in hypothyroid patients is 91.30, with standard deviation of 17.78.

Arithmetic means of diastolic blood pressure – retest in hyperthyroid patients is 80.0, with stan-dard deviation of 9.147.

Arithmetic means of diastolic blood pressure – retest in hypothyroid patients is 81.10, with stan-dard deviation of 11.39.

Therapy effect on diastolic blood pressure is si-gnificant in both groups: in hyper- P=0.006 and in hypo- P=0.000.

The values of diastolic blood pressure are hi-gher in hypo- than in hyper-, but with higher tendency towards a decrease than the tendency towards a decrease in diastolic blood pressure in hyperthyroid patients.

The average value of diastolic blood pressure is significantly higher in hypo- than in hyper- and slightly higher in relation to the normal range; the-rapy effect was statistically significant and there is a clinical improvement in hypo-. Normal range was registered in hyper-, both in the test and retest.



Chart 4. Change of the value of diastolic blood pressure before and after the therapy.

Discussion

Average values of systolic blood pressure are within the normal range in both groups in our study, both in the test and retest; the value is sta-tistically insignificantly higher in hypo- (148.40 +/-33.3) than in hyper- (146.22+/-28.2); therapy effect was statistically significant and the avera-ge value in retest was more decreased in hypo- (137.80+/-27) than in hyper- (137.20+/-21.2).

The average value of diastolic blood pressu-re is significantly higher in hypo- (91.30+/-17.8) than in hyper- (83.70+/-12.7) and slightly higher in relation to the normal range; therapy effect was statistically significant and there is a clinical im-provement in hypo- (81.10+/-11.4). Normal range was registered in hyper-, both in the test and retest (80+/-9).

Fabio Monzani et al. 2001 were examining a group of patients with subclinical hypothyroidism and the average value of systolic blood pressure (b. p.) of 115.3 +/-7.1; 6 months after the therapy, it increased to 117.3+/- 6.8.They were examining a group of patients with subclinical hypothyroidi-sm and the average value of diastolic b. p. of 75.3 +/-4.2; 6 months after the therapy, it decreased to 74.8+/- 7.2 (1).

Vitale et al.: Myocardial Evaluation by TD in Subclinical Hypothyroidism J.Clin Endocrinol Metab, September 2002.87 (9):4350-4355 were examining a group of patients with subclinical hy-pothyroidism and the average systolic blood pre-

ssure (b. p.) of 118.7+/-16.5.They were examining a group of patients with subclinical hypothyroidi-sm and the average diastolic b. p. of 73.2+/-8.9 (2).

Volzke H et al. 2009.concluded that subclinical hyperthyroidism is not associated with changes in blood pressure, pulse pressure or incident hypere-tension (8).

Said Gazibegović et al. 2009. reported that the-re was no difference in mean values of diastolic pressure before and after the treatment, however systolic pressure and heart rate declined signifi-cantly after the treatment. (p<0.0001)(9)

P. Iglesias at al. Concluded that normotensive hyperthyroid patients exhibit higher ambulatory systolic blood pressure throughout 24h than nor-motensive euthyroid subjects. Control of hyper-thyroidism decreases ambulatory sistolyc blood pressure values. Mean nocturnal fall in blood pre-ssure is comparable in normotensive hyperthyroid patients and control subjects (10).

Conclusion

The value of systolic blood pressure in our pa-tients is within the normal range in dysfunction of thyroid gland.

Diastolic blood pressure is within the normal range in hyperthyreosis, and higher in hypothyre-osis; statistically significant therapy effect is clini-cally satisfactory.

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References

1. Monzani F, Di Bello V, Caraccio N, et all. Myocar-dial dysfunction in Hypothyroidism. Department of Internal Medicine, University of Pisa School of medicine, 56126 Pisa, Italy, JCE & M. 2001; Vol. 86.No. 3: 1112.

2. Vitale G, Galderisi M, Lupoli G.A et al. Myocardial Evaluation by TD in Subclinical Hypothyroidism. J Clin Endocrinol & Metab, September 2002; 87 (9): 4351.

3. Stephen.A Falk.S. Thyroid Disease: Endocrinology, Surgery, Nuclear Medicine, and Radiotherapy, Li-pincott-RavenPublishers, Philadelphia, 1997.384.

4. Imaizumi M, Akahoshi M, Ichimaru S, et al. Su-bclinical Hypothyroidism and heart Disease: J.Clin:Endocrinol.Metab. July 2004; 89(7): 3369.

5. Obuobi K, Smith J, Evans L.M, et al. Hypothyro-idism and Central Arterial Stiffness, J,Clin Endo-crinol metabol. October 2002; 87(10): 4662-4666.

6. Sgarbi JA. The effects of early antithyroid therapy for endogennous subclinical hyperthyroidism in cli-nical and heart abnormalities. J.Clin. Endocrinol. Metab 2003; 88: 1672-1677.

7. Braverman L.E, Pearce E.N, Boston, USA. Su-bclinical hyperthyroidism: Thyroid International 5-2001 Merck KgaA, Darmstadt, Germany, 2001; 3-8.

8. Vozke H, ittermann T, Schmidt CO, Dorr M, John U, Wallaschfski H, Stricker BH, Felix SB, Rettig R.: Subclinical hyperthyroidism and blood pressure in a population-based prospective cohort study. Eur J.Endocrinol.2009.Oct; 161(4):615-21.Epub 2009 Jul 6.

9. Gazibegović S, Jakubović- Čičkušić A, Kušljugić Z, Baraković F: Treatment of subclinical hyperthyro-idism significantly reduces the rate and symtomps of associated cardiovascular abnormalities, Acta Med.Sal 2009; 38(1):30-35.

10. Iglesias P, Acosta M, Sanchez R, Fernandez-Reyes M.J, Mon C, Diez J.J. Ambulatory blood pressure monitoring in patients with hyperthyroidism befo-re and after control of thyroid function. Clinical Endocrinology.Volume 63 Issue 1, Pages 66-72. Published Online:15 Jun 2005.

Corresponding author Ismana Surkovic, Clinic of Endocrinology, Diabetes and Metabolic Deseases, University Clinical Centre Sarajevo, Bosnia and Herzegovina e-mail: [email protected]



Abstract

The presence of hazardous chemicals in labo-ratory workplaces has raised concerns about their potential negative effects on fertility, pregnancy outcomes and birth defects in offspring. Occupa-tional exposure to certain laboratory chemicals may directly affect the outcome of pregnancy, such as spontaneous abortion, stillbirth, pre-term birth, small-for-gestational age and birth weight and may also interact with fetal development, re-sulting in health effects in the offspring that ran-ge from congenital birth defects, neurobehavioral disorders at young age and even cancer in older age. In general, the reproductive toxins are hazar-dous chemicals which can affect the reproductive health before or after conception by chromosomal damage (mutagen) and by many different negati-ve effects on fetuses (teratogen). These chemicals can seriously affect a developing embryo or fetus because the course of human development from conception to adulthood is extremely complex. Because of the complexity, there are numerous opportunities for “things to go wrong.” Between conception (the union of sperm and egg) and bir-th, human life advances from a single-cell zygote to an infant capable of living outside the womb. During this period, complex and rapid changes are normal, from the molecular level through all the biochemical and physical processes that de-termine the course of development. Cell division, migration, differentiation and apoptosis all must occur in the correct sequence in the correct spa-tial orientation, coordinated through a large num-ber of control and signaling systems. Because of the complexity and speed of development and the high rate of growth through the prenatal period, this stage of development has a special set of vul-

nerabilities to environmental exposures that are not seen at any other time. It is essential that labo-ratory workers are guaranteed and provided with the maximum protection to prevent adverse repro-ductive health consequences. A timely recognition of the impact of hazardous agents, elimination of exposures in the workplace and education of labo-ratory workers are very important in this direction.

Key words: laboratory chemicals, reproducti-ve health, prenatal development

Introduction

Today many talk about the impact of various factors on health. Mode, working conditions and working and living environment are closely linked and can cause various acute and chronic damages to certain organs and organic systems and the or-ganism as a whole. Reproductive system is more sensitive than the other organ systems. Reproduc-tive health, in the broadest sens, the possibility that people have a normal, healthy offspring, can be damaged in many ways. The factors that have negative impact on the reproductive health may act and be expressed before conception, during in-trauterine fetal development and after the birth of a new individue, during its postnatal life.

Prior to conception, the menstrual cycle can be disturbed in females and fertility as well as sexual potency in males, which can lead to infertility or reduced fertility of a couple. Genetic damage in male and female sex cells can be transmitted to the offspring with the result of disease or develo-pmental disorders, but can also lead to stillbirths and abortion. Damages during intrauterine develo-pment occur by transmission of hasardous agents through placenta from mother’s body to offspring

negative effects of laboratory chemicals on the reproductive healthAlicelebic Selma

Institute of Histology and Embryology, School of Medicine, University of Sarajevo, Bosnia and Herzegovina

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(eg drugs, chemicals, viruses) or by the direct ac-tion on the offspring (e.g. ionizing radiation) and can lead to stillbirth, spontaneous abortion and, if the offspring survives, to different diseases and congenital anomalies of the fetus. Toxic factors can be transmitted to the baby through breast milk or brought home on the parents working clothes.

The main characteristics of prenatal development

The effects of the environmental hasardous factors mostly depend on the developmental sta-ge in which they operate and, in order to preserve reproductive health, essential knowledge of basic stages and characteristics of development is nece-ssary.

Individual human development begins at con-ception or fertilization, the process during which a male gamete or sperm (spermatozoon) unites with a female gamete or oocyte (ovum). Sexual cells, gametes, start to develop during fetal life, even though they do not mature until puberty. Toxic su-bstances from the environment can damage germ cells and can so jeopardize fertility of an adult in-dividual or interferre with its offspring health.

The course of human development from con-ception to adulthood is extremely complex. A huge number of biochemical, physical and organi-sational processes must be precisely coordinated to assure proper development, maintain health and avoid disease. Because of the complexity, there are numerous opportunities for “things to go wrong”.

To have the knowledge of the crucial stages in human development, from the moment of concep-tion, and even those processes that precede it is necessary for a better understanding of the impor-tant moments for the occurrence of disturbances of normal development under the influence of harmful effects from the environment and for the establishment and promotion of strategic plans of protection from exposure to the harmful effects of these adverse environmental factors.

The human development course is divided into two periods – the prenatal and the postnatal. The prenatal period occurs during pregnancy and in-cludes the preembrional, the embrional and the fetal period, and the postnatal period occurs after

birth and includes the period of childhood and adolescence. During the period from conception to birth the new organism develops itself from a single cell called zygote to a newborn capable of living outside the mothers body. Regarding the complexity and speed of development and growth of the prenatal period, it is the most vulnerable of the developmental periods.

All the numerous different processes, that pro-perly coordinate in space and time, are based on the cell’s function and their changes. Some of the most incredible phenomenons is that from the one cell, zygote, develop more than 200 morphologi-cally and functionally different cells. These basic developmental processes occur at the same time and intermingle each other. They are determined by genetic potential, environmental factors and regulatory factors (hormons, growth factors, cyto-kines, ions).

One trillion cells, contained in the adult human organism, originate from the one cell, zygote, by the process of proliferation. Rapid cell division is the primary driver of development and that is why embrional cells have a very short cell cycle, ge-nerally. A shorter cell cycle, implying more rapid metabolic and control processes, generally makes cells more vulnerable to possible toxic effects.

Fertilized oocyte, of 0.0015 mg and invisible by the naked eye, develops by prenatal growth to a newborn of about 3.200g weight and about 50cm in length. Characteristics of cell growth are do-ubling of the DNA, synthesis of specific proteins and other essential factors. Growth is regulated by genetic factors, the concentration of nutrients and oxygen, and regulatory mechanisms that involve a numerous growth and neuroendocrine factors.

Induction is a process during which a group of cells through signaling molecules influence the se-cond group of cells to differentiate into morpholo-gically and functionally specific types of cells. The diversity of the cells is achieved by differentiation. They are different allthough they have the same genome, due to different expression activity of in-dividual genes. Cell migration implies the move-ment of the whole cell or of its parts. Cells reco-gnize each other, assemble, shift or move away by specific molecules. An important developmental process that allows the formation of tissues and organs is apoptosis, the programmed cell death.



The process is regulated by genes and takes place under the influence of internal and external factors that lead to cell death. Extraordinary complexity of all these developmental processes provides a wide range of possibilities for adverse impacts to influence and to disturb the formation.

Speed and diverse nature of processes that occur during critical developmental periods expla-in their particular sensitivity.

Prenatal development is commonly divided into three periods: preembryonic - the first two weeks after fertilization, embryonic - from 3rd to 8th week and fetal - from 9th week to the birth.

In the first two weeks of development, during the preembryonic period, zygote divides many ti-mes, implants in the uterus wall and moulds in a simple embryo. Exposure to harmful factors in this period usually leads to death of the embryo, and lethal harmful effects result in spontaneous abortion. To determine that there existed a pre-gnancy is possible only with biochemical tests, for example, a positive pregnancy test.

Three germ layers develop during embryonic period: ectoderm, endoderm and mesoderm. By their differentiation, through the processes of hi-stogenesis, morphogenesis and organogenesis, develop the body cavities, the nerve tube, all or-gans and organ systems and body shapes. Histo-genesis is the process of creating a tissue during which certain cells differentiate into morphologi-cally distinct cells of epithelial, connective, musc-le and nervous tissue and produce an appropriate extracellular matrix. Morphogenesis is a series of processes that involve coordinated creation of internal organs with the precise plan of their spa-

tial organization and the formation of the exter-nal appearance of the body. Organogenesis is the process of morphological and functional develo-pment of organs. Embryonic period is the most sensitive period of development during which or-gans are the most sensitive just at the time of their intensive development (Table 1).

During the fetal period grows the fetal body mass occurs, but also fine morphologic, functional and biochemical processes that lead to the esta-blishment of the almost complete structure and function of organs and their training for the fun-ction outside the mother’s body after the birth. In this period, decreases gradually the sensitivity of the fetus.

Factors that damage reproductive health

Many chemical substances (for example, pe-sticides, gases, metals), different physical factors (radiation, vibration, atmospheric pressure) and biological agents (viruses, bacteria, fungi, parasi-tes) can significantly affect conception, pregnancy course and outcome and can damage reproductive health of people and their ability to have normal, healthy offspring, but for the reproductive health it is particularly important to have the knowledge of mutagens and teratogens.

Mutagens are physical and chemical agents that lead to structural changes in genes, mutations. When these changes occur in the somatic cells of an organism, they remain there only as a change in that particular body and can not be transmitted further to the offspring, whereas mutations that

Table 1. Critical periods in some organs prenatal development Body System Especially Sensitive Development up to …

Central nervous system/Brain 4th to 8th weeks Postnatal, through to adulthoodHeart 5th to 9th weeks 12th week

Upper limbs 6th to 10th weeks 12th weekEyes 6th to 10 weeks Term

Lower limbs 6th to 10th weeks 12th weekTeeth 9th to 11th weeks TermPalate 9th to 11th weeks 16th week

External genitalia 9th to 11th weeks TermEars 6th to 11th weeks 13th week

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occur in germ cells are hereditary and are tran-smitted from generation to generation. The most famous physical mutagen is ionizing radiation (gamma and x-rays), while the chemical known mutagens are arsenic, ethidium bromide and al-kylating agents (e.g., dimethyl sulfate).

Teratogens are biological, chemical and physi-cal agents that interfere with the normal embryonic development and thus lead to congenital malfor-mations or fetal death. Teratogens differ from mutagens in the way that there must be present a developing fetus. Damage of the fetus (embryo) is most likely to occur early in pregnancy, during the first 8 - 10 weeks. Teratogens may produce congenital malformations or death of the fetus

without inducing damage to the pregnant woman. Chemicals with proven teratogenic effects or ge-nerally adverse effects on reproductive health are dibromochloropropane, lead, ethylene oxide, anti-mony, carbon disulfide, polychlorinated bipheno-ls (PCBs), nitrous oxide, formaldehyde, ethylene dibromide(1).

Adverse effects before and during conception

Chemicals can affect the organism before it was conceived by effecting its germ cells. Germ cell development starts before birth and continu-es almost during the whole individual life. During

Table 2. Reproductive health damages of occupational exposure to chemicals

Chemical agents Pregnancy outcomes(maternal exposure)

Birth defects(maternal exposure)

Semen quality(paternal exposure)

Lead Low birth weight Neural tube defects Reduced sperm countMercury Spontaneous abortionOrganic solvents Spontaneous abortionTetrachloroethylene Spontaneous abortion Cleft lip/palateGlycol ethers Spontaneous abortion Reduced semen quality

Dibromopropane Menstrual disturbances,spontaneous abortion Neural tube defects Reduced semen quality

Ethylene oxide Pre-term birth,spontaneous abortion Cleft lip/palate

Anaesthetic gases Spontaneous abortionAntineoplastic drugs Spontaneous abortion

Pesticides Reduced sperm count Azoospermia

Ethylenedibromide Neural tube defects,cleft lip/palate Reduced quantity and quality

Carbon sulfide Reduced quantity and quality

Table 3. Disorders of men and women fertility occurred after chemicals exposure in workplace Chemical agents Maternal exposure Paternal exposure

Lead + +Mercury +Toluene +Aliphatic hydrocarbons +Aromatic hydrocarbons +Tetrachloroethylene +Glycol ethers + +Ethylene oxide +Anaesthetic gases +Pesticides + +



this long period there are many possibilities to damage very sensitive germ cells. Therefore, the effects of harmful factors on them can lead to in-fertility or reduced fertility of a man or a woman. Thus, dibromochloropropane, used as a pesticide, fumigant, and nematocide leads to azoospermia and infertility of workers who use it (2,3). Decre-ased fertility was found in women whose mothers smoked cigarettes during pregnancy (4). Tables 2. and 3. show a negative impact on fertility and re-productive health damages occurred after peoples exposure to chemicals in the workplace (5).

It is known that many factors from the envi-ronment can lead to such damage of germ cells that result in an increased incidence of malignan-cies, particularly leukemia, in the offspring of ex-posed persons (6-8). The connections between malignant diseases of the offspring whose fathers were exposed to benzene and various other harm-ful components are summarized on Table 4.

A connection between the appearance of ma-lignant diseases in the offspring of women who were exposed to various harmful factors in the workplace before conception (6,7,9) or both be-fore and after conception (10) is found. Data from the literature is shown in Table 5.

Researches on animals clearly confirm that the exposure to harmful factors in the prenatal period can damage their future reproductive function in adult period.

For example, prenatal exposure to chemicals with a high content of chlorine (2,3,7,8-tetrac-hlorodibenzo-p-dioxin and polychlorinated bip-henol-169) was associated with reduced sperm production in rats (11,12) even after a single dose (13). The toxicity of lead, hexachlorobenzene, cyclophosphamide, and many other chemicals was also proven in the experimental animal ova-ries (14-18).

Experiments on animals confirm the observa-tion in humans that germ cell exposure to harm-ful factors at work during their development in-creases the risk for the occurrence of malignant disease in their offspring, as well (19). Genetic studies suggest possible mechanisms that lead to the emergence of these consequences.

Thus, it was observed that smoking and ex-posure to pesticides and products of combustion increases the occurrence of sperm aneuploidy (20,21) and chemical changes of sperm DNA whi-ch cause mutations by acting on fertility and heal-th of the offspring (22). DNA can also be altered

Table 4. Association Between Preconception Exposures in Men and Cancer in Offspring

Exposure Dose to Father Exposure Period Cancer ThatDeveloped in Child

Benzene6 Not quantified Preconception LeukemiaDiagnostic Not quantified Preconception Leukemia

X-rays7

Ionizing > 100 mSv 6 months prior to conception Leukemia/non-Hodgkin’s

Radiation7,8

(milliSievert) lymphoma> 100 mSv Lifetime preconception Leukemia> 10 mSv 6 months prior to conception Leukemia1-5 mSv Preconception Acute lymphoblastic leukemia

Metals6 Not quantified Before birth HepatoblastomaWood dust7 Not quantified Preconception Leukemia

Table 5. Association Between Preconception Exposures in Women and Cancer in Offspring

Exposure Dose to Mother Exposure Period Cancer ThatDeveloped in Child

Food industry7 Not quantified Preconception or prenatal Leukemia/non-Hodgkin’s lymphomaMetal dusts,

petroleum products,paints, pigments6 Not quantified Preconception or prenatal Hepatoblastoma

Radiation9 Not quantified Preconception Leukemia/non-Hodgkin’s lymphoma

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by toxic activity during preconceptional period in female germ cells, for example, alcohol leads to disruption of chromosome duplication, resulting in aneuploid embryos (23).

It is known that different effects of various environmental factors are possible during con-ception, or shortly after conception. Increased number of spontaneous abortions and congenital anomalies has been noted in populations in whi-ch fathers were exposed to anesthetic gases, lead, mercury, organic solvents, pesticides, marijuana and tobacco (24-29). Preterm birth and lower bir-th weight could be associated with fathers’ occu-pational exposure to lead, pesticides and organic solvents (27,30-32). Even four to five years after mother’s exposure to polychlorinated biphenyls in food, these substances are transferred through the placenta from the mother’s body to the child and cause the reduction of its birth weight (33). Also, lead stored in bone tissue of women after previous exposure, may be mobilized and transferred du-ring pregnancy through blood and placenta to the offspring and may damage it (34,35).

Adverse effects during prenatal development

During early embryonic development a huge number of different genes are permanently ac-tivated and inactivated in a specific order which provides many opportunities for harmful factors to act. Today, it is considered that the harmful effects of environmental factors on the gene are “responsible” for the emergence of one fourth to one half of all developmental disorders (36). Also, it has been proven that ionizing radiation and mer-cury during the prenatal development can disturb the normal migration of neurons and thus lead to developmental disorders of nervous system (37). The normal developmental process of gradual dif-ferentiation of undifferentiated cells to those with very specific and complex shapes and functions is under the control of processes of signal transduc-tion inside and between cells. This is another in a series of sensitive points during the development in which may manifest toxic effects of various ad-verse effects from the environment and it is well known that undifferentiated cells are significantly more vulnerable than differentiated cells. Chemi-

cals which are proven to damage the specific types of undifferentiated cells are ethanol (38), manga-nese (39), nicotine (40) and dioxin (41).

Harmful effects of environmental factors during prenatal development can lead to early embryo-nic death, congenital malformations, slowed fetal growth, fetal death during late fetal development, complications of pregnancy and premature birth.

Early embryonic death which is clinically mani-fested as a spontaneous abortion can be caused by a wide range of different harmful factors from the environment. It was found that excessive pregnant women’s intake of coffee and smoking increase risk of miscarriage (42,43). Increased number of spon-taneous abortions was noted in pregnant women who were exposed to the harmful effects of cyto-statics, organic solvents and pesticides in the wor-kplace (25, 44-47). Exposure to organic solvents in the workplace before or during pregnancy leads to an increased risk of congenital malformations of the fetus (48) but leads also to an increased risk of preeclampsia in later stages of pregnancy (49). Numerous studies suggest an association between smoking during pregnancy and fetal intrauterine growth retardation as well as early child birth (50) but, paradoxically, it appears that smoking reduces the risk of preeclampsia (51).

Most of the damages that occur in the later sta-ges of prenatal development under the influence of harmful factors from the environment are expressed after birth in the form of functional disorders of or-gans and organic body systems, but not in the form of malformations or intrauterine growth retardation (52). Pathological processes that lead to these dis-orders begin to act at the time of exposure, but their consequences are not evident until the child is born. Thus, exposure of the fetus to neurotoxins such as lead, mercury and polychlorinated biphenyls, from the middle to the end of prenatal development, is associated with behavioral disorders that appear la-ter in child’s life (53-55).

Researches on animals and in humans clearly in-dicate the existence of the delayed effects of exposu-re to various harmful factors from the environment that occur during early development. However, de-tection and recording of latent and long-term nega-tive impact on general health, including the repro-ductive health, in the broadest sense, is extremely difficult and it is necessary to do an extensive and



long-term study in the future of a huge number of factors on a large population sample.

Acknowledgement

Invited plenary lecture presented at the 2nd Congress of Association of Laboratory and Sani-tary Technicians in Bosnia and Herzegovina with International Participation, Neum, May, 13-17, 2009.

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Corresponding author Alicelebic Selma Institute of Histology and Embryology, School of Medicine, University of Sarajevo, Bosnia and Herzegovina E-mail: [email protected]

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Abstract

Background: The management of chylothorax can be extremely difficult because of the tremen-dous diversity seen in its magnitude and etiology. The crucial decision in the management is how long to continue with a trial of conservative tre-atment.

Objective: To make a simple algorithm in the treatment of chylothorax based on literature data and own experience.

Patients and Methods: All patients with chylothorax at the Clinic for Thoracic Surgery-UCC Sarajevo between January 1990 and January 2010.

Results: Over the past twenty years were tre-ated 15 patients. The average age was 27.5±4.6 (range 6 to 73) years. Male/female ratio was 1.5:1. In one young girl chylothorax was a consequence of lymphangiomatosis. Trauma was the cause of chylothorax in 13 patients: blunt thoracic injury in 2 patients; penetrating chest injuries were the cau-se in 3 cases and iatrogenic chylothorax was pre-sent in 8 patients. In one case the cause was a M. Hodgkin. Average daily loss of pleural contents was 613.07 ± 217.77 (range 250 to 2300) ml. Tho-racotomy was done in 33.3% (5/15) of patients. From a total of 13 drained patients, drainage was successful in 69.2% (9/13) cases. Thoracocentesis was successful only in one patient. The average length of hospitalization was 19.33 ± 8.77 (range 10 to 43) days. Mortality was zero.

Conclusions: If conservative therapy is not successful after two or three weeks surgical tre-atment is necessary and efficacious. The thoracic duct is explored by a full thoracotomy on the side of effusion. It is readily seen during operation if a mixture of milk and cream or olive oil is given to the patient a few hours before surgery. We hope

that the presented algorithm can be a good base landmark in the diagnosis and treatment of chylot-horax.

Key words: chylothorax, conservative trea-tment, surgical treatment

Introduction

Accumulation of chylous fluid in pleural space is quite a rare phenomenon which confirms the to-tal number of presented patients in most of articles published in the literature (1,2,3,4,5,6). The funda-mental mechanism behind chylothorax is leakage of chyle into the pleural space. This generally im-plies disruption of the thoracic duct in the majority of instances. Contemporary, trauma to the thoracic duct is the most common mechanism of chylotho-rax (1,3,4,5,6,7). The commonest cause is thoracic surgery, particularly involving dissection of the mediastinum .During the first half of the twenti-eth century mortality due to untreated chylothorax ranged from 50-100% (1). Loss of large amounts of fats, proteins and other nutritional ingredients may cause difficult inanition . The patient descri-bed by Little and colleagues lost more than 500 l of chylous liquid for a period of 18 months and was cured using conservative treatments(1). Expectati-on of spontaneous closure of thoracic lymph fistu-la with the use of conservative treatment is often hard and exhausting for both the patient and the surgeon (1,3,5,6,7,8). The initial step in the succe-ssful treatment of this problem was the report of Lampson in 1948 which is described transthoracic ligation of ductus thoracicus (1). This has shown that surgical treatment can be very effective with clear indications. Since then made further impro-vements in both operational and in the conserva-tive treatment (2,3,5,6,7,9,10,11). Key decision in

simple algorithmic approach to the treatment of chylothoraxSafet Guska, Ilijaz Pilav, Safet Musanovic

Clinic for thoracic Surgery, University Clinical Center Sarajevo, Bosnia and Herzegovina



treating these patients is timely determination of the indications for surgery (1,2,5,7,11,12). Good knowledge of anatomy and physiology of ductus thoracicus is a basic prerequisite for understan-ding the origin of chylothorax, consequences and the application of appropriate forms of treatment. An outline of etiology, clinical investigations and management of this rare condition is presented stressing that therapy must be chosen and modi-fied with consideration for the cause and clinical response of each case. Based on published reports and on personal experience will be made simple diagnostic and therapeutic algorithm that should be the guide to coping with this disease.


The paper retrospectively analyzed all cases with chylothorax treated at the Clinic for Thora-cic Surgery-UCC Sarajevo between January 1990 and January 2010. Etiological factors and diagno-stic procedures (physical examination, biochemi-cal, bacteriological and citopatological analysis of pleural fluid and limphography) are investigated thoroughly. Applied therapeutic methods were:

- Hygienic-dietary regime (reduction or cessation of oral food intake and total parenteral nutrition)

- Thoracocentesis - Chest closed drainage with use of active

suction and - Surgery (thoracotomy at the side of

effusion with oversewing of leakage site).

The amount of drained pleural content was re-gularly measured. Duration of chylous fistula is shown in the number of days from the beginning to the end of the conservative treatment or to set the indications for surgery. It was described deter-mination of the date for surgery, type of surgery and the final outcome. In order to define the exact site of the chylous fistula preoperatively were gi-ven orally to patients scrambled ice cream in the milk or olive oil.

Results

Over the past twenty years at the Clinic for Thoracic Surgery UCC-Sarajevo were treated 15 patients with chylothorax. The average age was 27.5±4.6 years (the youngest patient was 8 and the oldest 73 years old). Male/female ratio was 1.5:1 (9 men and 6 women). Table 1 shows the causes of chylothorax in our patients. Table 1. Etiology of chylothorax

I Congenital Lymphangiomatosis 1 II Trauma Blunt injuries 2 Open penetrating injuries 3 Iatrogenic injuries (during surgery) Removal of the posterior mediastinal tumor 1

Esophageal surgery 4 Cardiac surgery 3III Neoplasm Morbus hodgkin 1

Total : 15

Various forms of trauma as the most common causes of chylothorax are established in 86.7% (13/15) of patients. In relation to the type of trau-ma as the most common cause of the disease are different surgical procedures established in 61.5% (8/13) of cases. Operations on the esophagus ca-used 30.8% (4/13) of cases with chylothorax, followed with cardiac operations which caused chylothorax in 23,1% (3/13) of patient and in one case (7,7%) chylothorax developed after removal of posterior mediastinal tumor . Penetrating chest injuries were the cause of chylothorax in 23,1% (3/13) patients. Blunt thoracic injury was the cause of chylothorax in 15.4% (2/15) of patients. In one young girl chylothorax was a consequence of rare systemic lymphoblastic malformations (lymphan-giomatosis) with a relatively benign clinical cour-se. In one case the cause was a malignant disease (M. Hodgkin).

In all patients, together with the registration of clinical signs and radiological studies final diagnosis of chylothorax is set on the basis of the following diagnostic procedures of pleural fluid:

654



- typical appearance of milky pleural content that is odorless, established in all patients, was of crucial diagnostic value in setting up the possibility of doubt on the chylothorax,

- bacteriological analysis and culture of pleural content remained sterile (chylous content is sterile and bacteriostatic)

- biochemical analysis of pleural content showed the presence of elevated values of triglycerides (>110mg/dl) and cholesterol/triglycerides ratio was less than the 1.

- citopatological analysis were uncharacte-ristic

In two patients was done lymphografy and it fai-led to show localization of lymph fistula, but lymp-hografy helped to clarify the etiology of chylothorax (in one patient the findings pointed to the systemic

lymphoblastic malformations and in the other one on the nature of malignant disease of the lymphatic system). All patients were subjected to restrictions of per oral food intake with simultaneous intravenous nutritional compensation. Regardless of the cause of the disease an average daily loss of pleural contents was 613.07 ± 217.77 (range from 250 to 2300) ml. Table 2. shows implemented treatment modalities.

Regardless of the cause of chylothorax thora-cotomy was done in 33.3% (5/15) patients. From a total of 86.7%(13/15) drained patients, as well as the definitive modality of treatment, drainage was successful in 69.2% (9/13) cases.

Pleural puncture as modality of treatment was successful only in one patient.

The average length of hospitalization was 19.33 ± 8.77 (range from 10 to 43) days. Intrahos-pital mortality was zero.

Table 2. Treatment modalities in relation to the etiology of chylothoraxThe average daily loss of

chylous contentModality oftreatment

The duration of hospitalization

ETIOLOGY N° of ml PP* PD* T* N° of daysCONGENITAL Lymphangiomatosis 1.patient 570.6±34.3 + + 43TRAUMABlunt injuries 1.patient 650.3±60.8 + + 31 2.patient 0.00±0.0 + + 11Penetrating injuries 1.patient 970.5±87.4 + + 15 2.patient 0.00±0.0 + + 12 3.patient 460.5±55.7 + 14Iatrogenic injuries Mediastinal surgery 1.patient 270.5±70.7 + 10 Esophageal surgery 1.patient 620.5±45.8 + 17 2.patient 460.6±34.8 + 19 3.patient 330.8±76.8 + 16 4.patient 820.8±50.6 + 22 Cardiac surgery 1.patient 470.5±20.9 + 23 2.patient 950.7±60.8 + 18 3.patient 670.5±60.7 + 13NEOPLASM Morbus Hodgkin 1.patient 730.5±80.6 + 26

PP=Pleural puncture*;PD=Pleural drainage*;Thoracotomy*



Discussion

In the past, issue of chylothorax was extreme-ly rare (1,2,3,6,7,8,9). Today, with the increasing incidence of thoracic injuries and the number of surgeries on mediastinal structures chylotorax appears much more frequently (1,2,4,5,7,8,9,10).

Thoracic injuries and cardio-thoracic and esop-hageal operations were the most common cause of chylotohrax established in 86,7%(13/15) of our cases.

Among traumatic chylothoraces, iatrogenic causes constitute the majority (1,3,5,7). Thoracic surgery constitutes the majority of iatrogenic cau-ses (1,3,5,8,9,10). Esophageal resection is perhaps the most common iatrogenic cause of chylothorax (2,3,6). The proximity of the thoracic duct to the esophagus, presence of collateral channels, and highly variable course leads to its injury during esophageal resection. In our series chylotoraks appeared in 30.8% (4/13) of cases after surgery on the esophagus. Other surgical procedures in the vicinity of the thoracic duct, such as cardiac surgery and operating procedures in the posterior mediastinum, can inadvertently damage the thora-cic duct (2,3,6,8,9,10). Cardiac operations caused chylothorax in 23,1% (3/13) of our patient and in one case (7,7%) chylothorax developed after re-moval of posterior mediastinal tumor.

Based on literature, chylothorax as a result of penetrating and blunt thoracic trauma is rare (1,3,5,8,10,11,12). In our patients, penetrating chest injuries were the cause of chylothorax in 23.1% (3/13) of patients while blunt thoracic injury has been identified as the cause in 15.4% (2/15) of cases.

Loss of large amounts of fats, proteins and other nutritional substances led to a serious state of extre-me inanition (2,4,6,7,9,10). Very important therape-utic step makes a better understanding of correction of nutritional status with the possibility of applying the complete parenteral nutrition, which allows the extension of conservative treatment and maintained good general condition of the patient and makes him/her capable to submission for further active treatment modalities (2,3,4,5,6,7). Dietary regime, which involves cessation of oral food intake and intravenous compensation of all nutritional ingre-dients was carried out in all of our patients. This

regime is applied immediately after diagnosing chylothorax and represents an essential and unavo-idable basis of the total treatment regardless of the following applied therapeutic methods.

With available diagnostic methods can be rela-tively easy to prove the existence of chylothorax if anyone is thinking of this option. Characteristic milky appearance of pleural content (no need to replace it with pus), which is odorless and steri-le; in which the biochemical tests may show in-creased values of triglycerides; when cholesterol/ triglycerides coefficient is less than 1; with non-specific citopatological findings provide a secure diagnosis of chylothorax (1,2,3,4,5,6,7,10). Afore-mentioned facts were also the basis of the diagno-sis of chylotorax in our patients.

Spontaneous closure of thoracic lymph fistula is very unpredictable (1,3). Lampson’s report from 1948 years which is described ligation of ductus thoracicus has set an initial step in the successful surgical treatment of this problem. Since then, furt-her progress has been made both in operational and in the conservative treatment (1,2). The main pro-blem in treating these patients is a timely decision on the termination of unsuccessful conservative tre-atment and timely access to surgery (1,2,3,4).

Intermittent pleural puncture rarely lead to sa-tisfactory results (1,3,6,7,8,12). It was applied in 2 of our cases, of which at one result was satisfactory (M.Hodgkin), and in another one, it was necessary to do surgery (lymphangiomatosis). In a patient who suffered from M.Hodgkin we achieved a satis-factory result after 5 pleural punctures with an ave-rage removal of 730.5±80.6 ml (range 240-1900) of chylous content, it lasted 26 days, after that the pa-tient is addressed to oncologists to continue already started chemotherapy. Another case is ten years old girls with lymphangiomatosis in whom we could achieve only temporary stabilization with multiple pleural punctures and later the surgery was made. Preference should be given to a continuous active pleural drainage that allows a permanent and com-plete reexpansion of the lungs which can contribute to the closure of lymphatic fistula (1,2,3,4,11,12). Pleural drainage was applied in 13 of our patients and in 69.2% (9/13) cases led to healing of chylot-horax. This method was successful in all patients with iatrogenic chylothorax and in one patient with penetrating thoracic trauma.

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Opinions about the length of the application of previously mentioned conservative treatment met-hods are different and the criteria for termination of unsuccessful conservative treatment are mixed (1,2,34,6,7). Based on available literature and on the basis of our own experience, we think that the-se conservative measures do not need to conduct more than 3 weeks, with a daily loss of chylous fluids larger than 500 ml. In our patients, regardle-ss of the cause of the disease, an average daily loss of contents was 613.07 ± 217.77 (range from 250 to 2300) ml.

The next step in the treatment of chylothorax is surgical closure of chylous fistula (1,2,3,4,6,7). Surgical closure of the fistula was applied in our 5 cases (a young girl with lymphangiomatosis, two patients with blunt and two patients with pe-

netrating thoracic trauma) with fully satisfactory results. Thorax was opened on the side of the chylous effusion. Preoperative oral giving scram-bled ice cream in milk or olive oil allowed easier identification of chylous fistula, which was then oversewn or ligated. The average length of hos-pitalization was 19.33 ± 8.77 (range from 10 to 43) days. For years, the mortality due to untreated chylothorax was between 50 to 100% (1,2) but to-day, thanks to a better understanding of anatomy, pathophysiology and improved treatment options the mortality had declined considerably (we did not have any lethal outcome).

Numerous literature data and own experience were used for the creation of a given algorithm (Figure 1).

Figure 1. Simple algorithmic approach to treatment of chylothorax



Conclusion

Accumulation of chylous content in pleural cavity is quite rare phenomenon illustrated by the fact that we had only 15 cases during the last twen-ty years. Spontaneous closure of thoracic lymph fistula is very unpredictable and crucial decisions in the treatment of this condition refer to the dura-tion of applied conservative treatment. Conserva-tive treatment includes suspension of per oral food intake and use of parenteral nutrition with intermi-ttent pleural puncture or continuous active pleural drainage. Preference should be given to pleural drainage. We believe that with conservative trea-tment should stop if there is a daily loss of chylous fluid of more than 500 cc for a period of 3 weeks after that it is necessary to do the operation if the-re are no contraindications for it. Thoracotomy is usually performed on the side of effusion with pri-or oral giving mixed milk cream or olive oil. We believe that the presented algorithm can be a good base landmark in the diagnosis and treatment of chylothorax.

References

1. Andreieshchev S A, Miasoiedov S D, Bul’ba M V, Driuk M F, Chernukha L M, Vakhnenko L M. Surgi-cal treatment of persisting chylothorax. Klin Khir. 2008 Sep ; (9):5-9.

2. Nair S K., Petko M, Hayward M P. Aetiology and management of chylothorax in adults. Eur J Cardi-othorac Surg 2007;32:362-369.

3. Townshend A P, Speake W, Brooks A. Chylothorax. Emergency Medicine Journal 2007;24:332-345.

4. Platis IE. Nwogu CE. Chylothorax. Thorac Surg Clin. 2006 Aug;16(3):209-14

5. Ghrairi H. Cheikh Rouhou S. Ammar J. Hantous S. Hammami S. Hamzaoui A. Idiopathic chylothorax: usefulness of etiologic survey and the follow-up. Tunis Med. 2006 Oct;84(10):663-5.

6. Doerr CH, Allen MS, Nichols FC, et al. Etiology of chylothorax in 203 patients. Mayo Clin Proc Ro-chester 2005;80:867–71.

7. Vaz MA, Fernandes PP. Chylothorax. J Bras Pneu-mol. 2006;32 Suppl 4:S197-203.

8. Worthington M, Groot, MD, Gunning A, Oppell UV. Isolated thoracic duct injury after penetrating chest trauma. Ann Thorac Surg 1995;60:272-274.

9. Riquet M, Le Pimpec Barthes F, Badia A. Chylot-horax . Presse Med. 2002 Mar 30;31(12):548-55.

10. Riquet M, Badia A. Surgery for chylothorax.Rev Pneumol Clin. 2004 Apr;60(2): 104-8.

11. Taveira-DaSilva A M. Steagall WK. Moss J. Lymphangioleiomyomatosis. Cancer Control. 2006 Oct; 13(4):276-85.

12. Burgess S. Harris M. Dakin C. Borzi P. Ryan C. Cooper D.Successful management of lymphangio-matosis and chylothorax in a 7-month-old infant.J Paediatr Child Health. 2006 Sep; 42 (9): 560-2.

Corresponding author Safet Guska, Clinic for thoracic Surgery, University Clinical Center Sarajevo, Bosnia and Herzegovina, email: [email protected]

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Abstract

Background: Many apparent advantages of the magnetic resonance imaging (MRI) in esta-blishing diagnosis of lumbar disc herniation are counter parted by its relatively high cost and spar-se availability in developing countries. Thus, a significant portion of patients are still subjected to lumbar disc surgery based solely on computed tomography (CT) findings.

Aim: The aim of this study was to compare diagnostic characteristics of afore mentioned ra-diological tests (CT and MRI) and to investigate if the choice of diagnostic test influences outcome of discectomy.

Methods: Basic demographic, clinical and radiological variables were evaluated in a group of 70 patients operated on for disc herniation of whom 30 were operated based on MRI findings and the remainder were operated based on CT scan alone. Outcome was assessed using Visual Analogue Scale (VAS) and Roland-Morris (RM) scale 6 months postoperatively and correlated to the type of neuroradiological examination. Basic diagnostic characteristic of the two diagnostic mo-dalities (MR and CT) were compared.

Results: The type of radiological investigati-on was shown to be statistically poor predictor of outcome after microdiscectomy. Even though MR scan was more sensitive in detecting disc extrusi-

on than CT (sensitivity of 100% versus 65%, res-pectively), the presence of preoperative MR scan did not influence the outcome.

Conclusion: We conclude that although the presence of preoperative MR scan does not influ-ence outcome, higher sensitivity and specificity in detecting disc extrusions and superior ability to detect nerve root compression warrant an intro-duction of MR scan prior to any disc surgery.

Key words: lumbar disc herniation, discecto-my, outcome, magnetic resonance, computed to-mography

Sažetak

Uvod: Relativno visoka cijena i slaba dostu-pnost magnetne rezonance (MR) baca u sjenu njene prednosti u dijagnostici lumbalne diskus hernije. Stoga se u zemljama u razvoju veliki broj pacijenata operira zbog diskus hernije na osnovu nalaza kompjuterizirane tomografije (CT).

Cilj: Cilj ove studije je da uporedi dijagnostič-ke karakteristike spomenutih radioloških modali-teta ( CT i MR) i da ispita da li izbor radiološkog testa utiče na ishod diskektomije.

Metode: Osnovne dmografske, kliničke i ra-diološke varijable su zabilježene u skupini od 70 pacijenata operiranih zbog lumbalne diskus herni-je, od kojih je 30 operirano na osnovu magnetne

diagnostic characteristics of neuroradiological tests in lumbar disc herniationDIJagnosTIČke karakTerIsTIke nEurOrAdIOLOŠkIH TEsTOVA kOd LumbALnE dIskus HErnIjEMirza Moranjkic1, Zlatko Ercegovic1, Mirsad Hodzic1, Harun Brkic1, Farid Ljuca2

1 Department of Neurosurgery, Clinical Center Tuzla, Bosnia and Herzegovina2 Department of Physiology, Medical Faculty, University of Tuzla, Bosnia and Herzegovina



rezonance, a 40 na osnovu CT-a. Ishod je procije-njen upotrebom dvije ljestvice; Visual Analogue Scale (VAS) i Roland-Morris (RM) ljestvice 6 mjeseci nakon operacije i koreliran sa tipom radi-ološke pretrage. Određene su i upoređene osnovne dijagnostičke karakteristike CT-a i MR.

Rezultati: Tip preoperativno načinjene neuro-radiološke pretrage se pokazao lošim prediktorom ishoda mikrodiskektomije. Iako je MR bila senzi-tivnija u detekciji ekstruzije diska nego CT ( sen-zitivnost od 100 %, nasuprot senzitivnosti od 65 %), prisustvo MR snimka prije peracije nije utica-lo na ishod mikrodiskektomije.

Zaključak: Iako tip preoperativne neuroradio-loške pretrage ne utiče na ishod mikrodiskektomi-je, veća senzitivnost i specifičnost magnentne re-zonance i njena superiorna sposobnost detekcije kompresije nervnog korijena nameću potrebu za MR snimkom prije svake operacije diskus hernije.

Ključne riječi: lumbalna diskus hernija, disk-sktomija, ishod, magentna rezonanca, kompjuteri-zirana tomografija.

Introduction

Magnetic resonance (MRI) depicts tissues with a digital matrix representing shades of gray, de-pending on the intensity of the radio waves ema-nating from the tissue. Due to its relatively high cost, sparse availability in developing countries and inability to subject patients with metallic fo-reign bodies to MRI a relatively large proporti-on of patients are still being operated for lumbar disc herniation based solely on CT findings [1]. Many lumbar disc herniations can be visualized on computed tomography (CT) that represents the method of choice if MR is not available or not to-lerated by patients. In general, surgery is indicated based on CT scan alone if there is a clear corre-lation between history, neurological examination and CT scan. Several recent multicenter studies compared diagnostic value of CT, CT myelograp-hy and MRI revealing similar sensitivity and spe-cificity of the CT scan as compared to the MRI while noting that both methods are superior to CT myelography. Aforementioned studies were based on correlation of the CT and MR findings with operative findings and failed to make any conclu-

sion regarding outcome after surgery in respect to the choice of radiological study [2, 3, 4].

The aim of this study was to compare diagno-stic characteristics of afore mentioned radiological tests and to investigate if the choice of diagnostic test influences outcome of discectomy.


The study was conducted prospectively and en-compassed 70 patients operated on for lumbar disc herniation, 30 of which had been subjected to MR scan prior to surgery. Specific inclusion criteria at enrollment were radicular pain (below the knee for lower lumbar herniations, into the anterior thigh for upper lumbar herniations) and evidence of nerve-root irritation with a positive nerve-root tension sign (straight leg raise–positive between 30° and 70° or positive femoral tension sign) or a corresponding neurologic deficit (asymmetrical depressed reflex, decreased sensation in a derma-tomal distribution, or weakness in a myotomal distribution). Additionally, all participants were surgical candidates who had undergone advanced vertebral imaging showing disk herniation (pro-trusion, extrusion, or sequestered fragment) at a level and side corresponding to the clinical symp-toms. Patients with multiple herniations were in-cluded if only one of the herniations was consi-dered symptomatic ( if only one was planned to be operated on). Exclusion criteria included prior lumbar surgery, cauda equina syndrome, scoliosis greater than 15°, segmental instability (10° angu-lar motion or 4-mm translation), vertebral fractu-res, spine infection or tumor, inflammatory spon-dyloarthropathy, pregnancy, comorbid conditions contraindicating surgery, or inability/unwillingne-ss to have surgery within 6 months. Neuroradio-logical work-up encompassed either CT or MRI scan of the lumbar spine. Scans were analyzed by neuroradiologists and again by neurosurgeons. Measures used in the study were:

- Roland-Morris (RM) Low Back Pain and Disability Questionnaire

- Visual Analogue Scale (VAS)

Patients were required to asses their level of pain and functional disability using afore menti-

660



oned scales preoperatively and 6 months after the surgery. All patients were operated by neurosur-geon and the procedure performed was standard open microdiscectomy. Due to the fact that our data were primarily ordinal numbers (acquired by RM test) and continuous values (obtained by VAS scale) unparametric methods were used for cal-culating statistical significance. Wilcoxon’s test and its version for unpaired variables Mann-Whit-ney test were used for calculating the difference among groups.

Results

70 patients were subjected to microdiscecto-my in our department between January and June 2008. 36 (51, 4%) of which were male. The mean age at presentation was 45, 8 years (SD + 9, 39), ranging from 22 to 65 years. Gender specific age distribution is depicted in the figure 1.

Figure 1. Gender specific age distribution

Table 1. depicts the distribution of various disc herniation types according to CT and MRI.

Correlation between CT findings and operative findings is depicted in table 2.

Correlation between MR findings and operati-ve findings is depicted in table 3.

Table 1. Disc herniation type according to CT and MRIFinding

/TestExtrusion

N %Protrusion

N %Other

N %Total

N %CT 26 37,10 13 18,60 1 2,50 40 57,00MR 17 24,30 11 15,70 2 2,80 30 43,00

Total 43 60,50 24 34,20 3 5,30 70 100,00

Table 2. Correlation between CT findings and operative findingsOperative finding*

CT findingExtrusion**N %

ProtrusionN %

OtherN %

TotalN %

Extrusion 24 40,00 1 2,50 1 2,50 26 65,00Protrusion 2 5,00 6 15,00 5 12,50 13 32,50

Other 0 0,00 0 0,00 1 2,50 1 2,50Total 26 65,00 7 17,50 7 17,50 40 100,00

*Correlation coefficient r = 0, 0692; p = 0, 3363**Sensitivity 65, 00%; specificity 42, 80% (in detecting extrusion)

Table 3. Correlation between MR findings and operative findingsOperative finding*

MR findingExtrusion**

N %ProtrusionN %

OtherN %

TotalN %

Extrusion 11 36,60 5 16,70 1 3,30 17 56,60Protrusion 1 3,40 7 23,30 3 10,00 11 36,60

Other 0 0,00 0 0,00 2 6,60 2 6,80Total 12 40,00 12 40,00 6 20,00 30 100,00

* Correlation coefficient r = 0, 6015 p = 0, 1486**Sensitivity 100, 00%; Specificity 72, 20% (in detecting extrusion)



Comparison of CT and MRI diagnostic charac-teristics is summed up in table 4.Table 4. Comparison of CT and MRI diagnostic characteristics

Test type Sensitivity* Specificity*CT 65,00% 42,80%MR 100,00% 72,00%

* Sensitivity and specificity apply to the ability to detect extrusion intraoperatively

Figures 2 and 3 depict ROC curves for CT and MRI, respectively.

Figure 2. CT ROC curve

Figure 3. MRI ROC curve Outcome of surgery, defined as difference

between preoperative and postoperative VAS and RM values denoting recovery is presented in table 5. Level of confidence is presented in the last co-lumn. Recovery is defined as a percentage of VAS and RM value reduction on the control examinati-on 6 months following surgery as compared to the preoperative values.

Table 5. Outcome of surgeryPre op Post op Difference (%) p

VAS 76,38 22,65 53,73 < 0,0001RM 14,04 5,87 8,17 < 0,0001

Table 6. Correlation of the type of neuroradiological examination with outcome MR

Pre op Post op Diff p CT

Pre op Post op Diff pDiff

pVAS 74,56 24,00 50,56 <0,0001 77,75 21,65 56,1 <0,0001 0,3671RM 14,03 5,63 8,4 <0,0001 0,9527

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Discussion

Most patients in our study had a neuroradiolo-gical (CT or MRI) signs of disc extrusion, which correlates well with operative findings as graded by annular competence, meaning that there was no statistically significant difference between ope-rative findings and CT or MR findings in terms of disc herniation type. It was shown that MR is more sensitive and more specific in detecting extrusion than CT (100% sensitivity of MRI, as opposed by 65% sensitivity of CT). Early studi-es investigating a role of MR imaging in the tre-atment of lumbar disc herniation [5] revealed an excellent sensitivity and specificity of MR ima-ging in detecting annular competence and thus in detecting type of disc herniation. Investigating the sample encompassing 17 patients with total of 19 disc herniations, using 0, 5 T MR Scanner Gre-nier et al. [5] correlated operative findings with imaging findings. They revealed that the posterior longitudinal ligament was compromised in 8 cases and intact in 11 cases intraoperatively. The lack of low-intensity signal line around the disc her-niation was the most consistent predictor of disc extrusion (without false positive or false negative results). The presence of low-intensity signal line excluded annular disruption. Total test sensitivity in this series proved to be 100%, whilst total spe-cificity reached 78%. More recent studies failed to confirm afore mentioned results. The study con-ducted by Silverman [6] in 1995. correlated 3 MR features with operative findings: the presence and integrity of low- signal intensity line around the disc herniation, disc size to spinal canal size ratio and the presence of free discal fragment. The aut-hor concluded that MR parameters are not reliable predictors of annular competency. Weiner and Pa-tel [7] reached similar conclusions in 2008. Their study revealed the MR sensitivity of 72% and spe-cificity of 68%. In 2004. Pfirrmann [8] developed a grading system for nerve root compression on MR imaging, derived from the specimen of over 250 subjects. MR imaging features correlated well with operative findings (r = 0.86). Sensitivity and specificity of CT proved to be similar to those of MRI in most studies. In the study designed to compare radiological evaluation of spiral CT with MRI in patients with suspected herniated discs 57

patients with lumbosacral radicular syndrome un-derwent spiral CT and 1.5 T MRI. For detection of herniated or bulging discs, no significant diffe-rence in interobserver agreement was noticed (CT kappa 0.66 vs. MRI kappa 0.71; p50.40). For root compression, significantly better interobserver agreement at MRI evaluation (CT kappa 0.59 vs. MRI kappa 0.78; p50.01) was noticed. In the cases without disagreement, CT differed from MRI in 6 discs (3.5%) and in 3 nerve roots (0.7%). The authors concluded that for radiological evaluati-on of lumbar herniated discs, there is no evidence that spiral CT is inferior to MRI. For evaluating lumbar nerve root compression, spiral CT is less reliable than MRI [9].

Our study failed to reveal significant relation between the type of preoperative neuroradiologi-cal examination (CT vs. MRI) and outcome (p= 0, 3671 for difference in outcome between patients with MR and those with CT according to VAS sca-le; p= 0, 9527 according to RM scale). Very few studies investigated the influence of preoperative neuroradiological study on outcome. Several aut-hors investigated MR findings in relation to outco-me in conservatively treated patients [10]. Several studies correlated MR findings with the disc her-niation propensity to recur and revealed that some MR features are predictive of disc herniation re-currence. Dora in 2005. retrospectively compared preoperative MR findings in 30 patients exhibiting reherniation after surgery and 30 patients without reherniation 2 years upon surgery. Reherniation risk decreased by 3,4 times with each disc dege-neration level [11].

Conclusions

Although our study failed to reveal statistically significant relation between the type of neuroradi-ological examination (CT vs. MRI) and outcome after herniated disc surgery, MR proved to be su-perior in detecting the type of disc herniation and the degree of nerve root compression. Since the degree of neural compression is usually the deter-mining factor in decision making process MR is prerequisite to any disc herniation surgery.



References

1. Albeck MJ, Hilden J, Kjaer L, Holtas S, Praestholm J, Henriksen O et al. A controlled comparison of myelography, computed tomography and magnetic resonance imaging in clinically suspected lumbar disc herniation. Spine 1995; 20(4):443-8.

2. Jackson RP, Cain JE Jr, Jacobs RR, Cooper BR, McManus GE. The neuroradiographic diagnosis of lumbar herniated nucleus pulposus: II. A compari-son of computed tomography (CT), myelography, CT-myelography, and magnetic resonance ima-ging. Spine 1989; 14(4):1362-7.

3. Kent DL, Haynor DR, Larson EB, Deyo RA. Dia-gnosis of lumbar spinal stenosis in adults: a metaa-nalysis of the accuracy of CT, MR, and myelograp-hy. Am J Roentgenol 1992; 158(3):1135-44.

4. Kido D, Mushlin A, Thornbury J, Littenberg B, Rot-henberg R A meta-analysis of imaging technologi-es in lumbar disk herniation. Med Decis Making 1990; 10:331.

5. Grenier N, Greselle JF, Vital JM, Kien P, Baulny D, Broussin J et al. Normal and disrupted longitudinal ligaments correlative MR and anatomic study. Ra-diology 1989; 171:197-205.

6. Silverman C, Lenchik L, Shimkin P, Lipow K. The value of MR in differentiating subligamentous from supraligamentous disc herniations. AJNR 1995; 16: 571-9.

7. Weiner BK, Patel R. The accuracy of MRI in the detection of Lumbar Disc Containment. Journal of Orthopaedic Surgery and Research 2008; 3:46.

8. Pfirrmann CW, Dora C, Schmid MR, Zanetti M, Hodler J, Boos N. MR Image–based Grading of Lumbar Nerve Root Compromise due to Disk Her-niation: Reliability Study with Surgical Correlati-on. Radiology 2004; 230:583–8.

9. Rijn JC, Klemetso B, Reitsma M, Bossuyt P, Hul-smans, Peul C et al. Observer variation in the eva-luation of lumbar herniated discs and root compre-ssion: spiral CT compared with MRI. The British Journal of Radiology 2006; 79:372–7.

10. Choi SJ, Song JS, Kim C, Shin MJ, Ryu DS, Ahn JH et al. The Use of Magnetic Resonance Imaging to Predict the Clinical Outcome of Non-Surgical Treatment for Lumbar Interverterbal Disc Herni-ation. Korean J Radiol 2007; 8:156-63.

11. Dora C, Schmid MR, Elfering A, Zanetti M, Hod-ler J, Boos N. Lumbar Disk Herniation: Do MR Imaging Findings Predict Recurrence after Surgi-cal Diskectomy. Radiology 2005; 235:562–7.

Corresponding author Mirza Moranjkic, Department of Neurosurgery, Clinical Center Tuzla, Bosnia and Herzegovina, E-mail: [email protected]

664



Abstract

Introduction: Basal hyperprolactinemia and occult hyperprolactinemia (OHP) are associated with menstrual disorders, defective luteal phase, disorders of sexual function, infertility and habi-tual abortions.

The aim of this study was to assess the levels of basal and occult serum prolactin (PRL) and go-nadotropin in infertile women and women with habitual abortions.

Subjects and methods: Prospective study in-cluded 67 subjects with basal HyperPRL and OHP with established primary or secondary infertility and habitual abortions, aged 16 to 40 years. The control group was comprised of 7 healthy fertile women with normal levels of prolactin, aged 18-40 years. Fasting serum prolactin (PRL), FSH, LH, estradiol (E2) and progesterone (P) were me-asured in both, the study and the control group du-ring the mid luteal and mid follicular phase. The level of PRL was measured at the basal value and 30 minutes after administering 200 μg of TRH.

Results: Hyperprolactinemic infertile women had significantly (P<0.001) increased levels of

PRL, reduced levels of LH and E2 (P<0.01) du-ring the mid-follicular phase in comparison with the control group. In mid-luteal phase HyperPRL women had significantly elevated levels of PRL (P<0.001), decreased LH (P<0.01) FSH and E2 (P<0.05) compared to the control group. In hyper-prolactinemic women TRH test was followed by a significant increase of PRL30 (P<0.01), compared to the basal PRL, but ranged from 50 - 70 ng / ml. In women with occult hyperprolactinema (OHP) TRH test was followed by a significant increase of PRL30 (p>0.001), compared to the basal PRL, but the increase was greater than 100ng/ml. In the control group TRH test was followed by a signifi-cant increase of PRL30 (p>0.01), compared to the basal PRL, but the increase was less than 50ng/ml.

Conclusions: This research showed that the basal HyperPRL (basal PRL values>10 ng / ml, 30 PRL during TRH test between 50-70 ng / ml) and OHP (basal PRL <1O ng / ml, PRL30> 70 ng / ml during TRH test) are significant causal factors of infertility and habitual abortion.

Key words: Hyperprolactinemia, occult hyper-prolactinemia, Infertile women, Serum Prolactin, LH, FSH, Estradiol, Progesterone.

basal Hyperprolactinemia, Occult Hyperprolactinaemia and Gonadotropins secretion in infertile women bAzALnA HIPErPrOLAkTInEmIjA, OkuLTnA HIPErPrOLAkTInEmIjA I sEkrECIjA gonaDoTroPIna u nePloDnIH Žena Hajder E.1, Hajder M.2, Zukic E.3, Samardzic R.4

1 University Clinical Center Tuzla, Gynecology and Obstetrics clinic, Bosnia and Herzegovina,2 University Clinical Center Tuzla, Internal clinic, department of Endocrinology, Bosnia and Herzegovina,3 Tuzla Health Center with Outpatient clinic, Bosnia and Herzegovina,4 Cazin Health Center with Outpatient clinic, Bosnia and Herzegovina.



Sažetak

Uvod: Bazalna hiperprolaktinemija i okultna hiperprolactinemia (OHP) su udružene sa men-strualnim poremećajima, defektnom luteinskom fazom, poremećajima seksualne funkcije, neplod-nošću i habitualnim pobačajima.

Cilj ovog istraživanja je da se procijene razine bazalnog i skrivenog serumskog prolaktina (PRL) i gonadotropina u neplodnih žena i u žena sa habi-tualnim pobačajima.

Ispitanice i metode: Prospektivnom studijom je obuhvaćeno 67 ispitanica sa bazalno HyperPRL i OHP u kojih je utvrđena primarna ili sekundarna neplodnost I ili habitualni pobačaji starosne dobi od 16 do 40 godina. Kontrolnu grupu su činile 7 zdravih fertilnnih žene sa normalnom razinom prolaktina, starosne dobi od 18-40 godina. Ispita-nicima i kontrolnoj skupini su mjereni natašte se-rumski prolaktin (PRL), FSH, LH, estradiol (E2), progesterone (P) u srednjoj folikularnoj i srednjoj lutealnoj fazi. Razina PRL je mjerena bazalno i u 30.-oj minuti nakon apliciranja amp TRH 200 micg.

Resultati: HyperPRL neplodne žene imale su signifikantno (P<0.001) povišenu razinu PRL, smanjenu razinu LH i E2 (P<0.01) u toku sredi-ne folikularne faze u komparaciji sa kontrolnom skupinom. U srednjoj lutealnoj fazi HyperPRL žene imale si signifikantno povišenu razinu PRL (P<0.001), smanjen LH (P<0.01), FSH and E2 (P<0.05) u odnosu na kontrolnu skupinu. U hyper-prolaktinemičnih žena nakon TRH testa uslijedio je signifikantan porast PRL30 (P<0.01), u odnosu na bazalni PRL, ali je iznosio od 50- od 70 ng/ml. U žena sa okultnom hiperprolaktinemijom (OHP) nakon TRH testa uslijedio je signifikantan porast PRL30 (p>0.001), u odnosu na bazalni PRL, ali je porast bio veći od 100 ng/ml. U kontrolnoj skupi-ni nakon TRH testa uslijedio je signifikantan po-rast PRL30 (p>0.01), u odnosu na bazalni PRL, ali je porast bio manji od 50 ng/ml.

Zaključci: Rezultati istraživanja su pokazali da su bazalna HiperPRL (vrijednosti bazalnog PRL >10 ng/ml , PRL 30 tokom TRH testa iz-među 50-70 ng/ml) i OHP( bazalni PRL<10 ng/ml, PRL30> 70 ng/ml tokom TRH testa) značajan uzročni faktor u neplodnosti i habitualnim poba-čajima.

Ključne riječi: bazalna hiperprolaktinemija, okultna hiperprolaktinemija, neplodne žene, se-rum prolaktin, FSH, LH, estradiol, progesteron

Introduction

Hyperprolactinemia is a common endocrine disorder in women of reproductive age with a pre-valence of about 30% of women with infertility and habitual abortions (1,2). HyperPRL is usually associated with menstrual dysfunction, defective luteal phase, galactorrhea, amenorrhea, sexual function disorders, infertility and abortions (3). Basal and occult HyperPRL affects hipotalamo-pituitary-ovarian system and results in the failure of foliculogenesis and weakened oocite maturati-on, which can be regulated by inhibiting prolactin as with bromocriptine (4,5). HyperPRL is associ-ated with hypogonadism and disorders of gonadal and steroid secretion (6). Arafah et al. suggest that pituitary adenomas with partial hypopituitarism due to compression of pituitary stalk have a mild or moderate HyperPRL due to the lack of prolactin inhibitory factor (PIF) (7). About 25% of women with PCOS have elevated basal LH and prolac-tin (8). Endometriosis is associated with infertili-ty and is estimated to affect 10-15% of women of fertile age (9). PRL secretion could be the cause of infertility in women with endometriosis, a higher level of PRL effects the folliculogenesis and ooci-te maturation (10). It has been shown that patients with endometriosis and normal basal PRL have a PRL disorder during the TRH test (11). Infertile women with galactorrhea and HyperPRL often have primary hypothyroidism. In 25% of infertile women, hyperprolactinemia is caused by hypot-hyroidism (12). The aim of this study was to asse-ss the levels of basal and occult serum gonadotro-pin and prolactin in infertile women and women with habitual abortions.

Study group

A prospective study included the 67 infertile women with primary and secondary infertility and habitual abortions between the ages of 18-40 years in the period between 2006 and 2009. The patients

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were a subject of endocrinologycal and gynecolo-gical assasment made in the outpatient clinics of University Clinical Center Tuzla and in the fertili-ty centers. Criteria for inclusion in the study were: primary and secondary infertility, habitual abor-tions, menstrual cycle disorder, defective luteal phase, prolactin> 10 ng / ml, and OHP> 50 ng / ml with male factor excluded (normospermia). In-fertile women with uterine anomalies, tubal factor and male factor infertility were excluded. Patients with basal HyperPRL were grouped according to the etiological cause: tumor-dependent Hyper-PRL, HyperPRL with PCOS, endometriosis with HyperPRL, hypothyroidism with HyperPRL, hy-popituitarism with HyperPRL. TRH test was con-ducted on patients who had PRL <10ng/ml. The control group consisted of the ovulatory women with normal menstrual cycle, no abortions, ages 16-40 years. The same parameters were determi-ned in both the study and the control group.

Definition

HyperPRL is defined as the PRL> 10 ng / ml, PRL30 after TRH test > 50 ng / ml (13). Infertility is defined as the inability to conceive after 1 year of regular sexual intercourse. Habitual abortions are defined as three or more miscarriages without live births. PCOS is defined by Rotterdam diagno-stic criteria (14), Endometriosis was diagnosed laparoscopically. The extent of the disease was staged according to the revised American Fertility Society classification (15). Hypothyroidism was diagnosed by positive antibodies (anti-TPO, anti-Tg), higher level of TSH and reduced level of T3 and T4. For patients with PRL> 100 ng / ml ma-gnetic resonance imaging scan (MRI) was done. Pituitary microadenomas are defined as being less than 10 mm in diameter, and macroadenomas are >10 mm visualized using MRI.

Hormonal assay

Prior to the TRH infusion patients were at rest and were submitted to an 8 hrs fast. An intrave-nous catheter was placed in the antecubital vein 30 minutes before sampling, 200 mcg of TRH

were administered intravenously. Collections of samples were made at -15’, 0’, and after 30 mi-nutes. All samples were centrifuged for separati-on of plasma, which was frozen at -20C. Hormo-nal levels of LH, FSH, Oestradiol, Progesterone and Prolactin were determined from blood sam-ples obtained from each subject after overnight fasting in the early follicular phase (3rd-5th day of the menstrual cycle) and the mid-luteal pha-se. The reference values that were used are: LH, 5-20 mIU; FSH, 5-20 mIU/ml; and prolactin 2.5-20 ng/ml. E2, Pg, FSH, LH, Prolactin were determined by using the Wallace Fluroimmuno-assay, on Delfia Fluorometer. The original Del-fia kits (Turku, Finald) for individual hormones were used.


Mean and standard errors were calculated. Student’s t – test was applied for the comparison of hormonal levels in fertile and infertile women. Significance level was set as P<0.05.

Results

From a total of 69 (100%) infertile women with basal HyperPRL involved in this study, pituitary adenoma was proven in 22 (31.88%), of which 16 (23.18%) proved to be microadenomas and 6 (8.69%) macroadenomas. Non-tumor functional hyperprolactinemia had been proved in 47 exami-nees (68.12%), PCOS in 14 (20.28%), hipopitui-tarism in 13 (18.84%), endometriosis in 6 (8.69%) and hipopituitarism with adenomas in 2 exami-nees (2.89%). OHP was registered in 12 (17.39%) of the infertile women who had a regular basal prolactin. (Table 1.)

Infertile women with basal hyperprolactine-mia had significantly increased prolactin level (P<0,001), decreased LH and E2 (P <0.01) levels during follicular phase compared with control gro-up. In luteal phase these women had significantly increased prolactin level (P <0,001), decreased LH (P <0.02) and progesterone (P <0.01) levels com-pared with control group. The results show that the fertile hyperprolactinemic women had signifi-



cantly (P <0,001) increased prolactin level, decre-ased LH and E2 (P <0.01) level during follicular phase compared with normoprolactinemic group. In luteal phase these women had significantly in-creased prolactin level (P <0,001), decreased LH (P <0.01), FSH and E2 (P <0.05) levels compared with normoprolactinemic group. (Table 2.)

In infertile women with anovulatory phe-notype of PCOS mean basal PRL (P <0.001), LH (P <0.01), E2 (P <0.05) were significantly higher, while there was no significant difference in the level of FSH in comparison with the control gro-up. PRL 30 during the TRH test was significantly

higher (P <0.05), compared to the basal PRL but the increase was less than 70 ng / ml. (Table 3.) In infertile women with endometriosis mean basal PRL (P <0.001), was significantly higher, while there were no significant differences in gonado-tropin and estradiol in comparison with the control group. PRL 30 during the TRH test was signi-ficantly higher (P <0.05) compared to the basal PRL, but the increase was less than 70 ng / ml. (Table 3.) In infertile women with hypothyroidism mean basal PRL (P <0.001) was significantly hi-gher, while there were no significant differences in gonadotropin and estradiol in comparison with the

Table 1. Differential diagnosis of 69 infertile women evaluated for hyperprolactinemiaDiagnosis Number Percentage (%)

Tumors total 22 31,88Microadenoma 16 23,18Macroadenoma 6 8,69Functional hyperprolactinemia total 47 68,12Polycistic Ovary Syndrome 14 20,28Hyporthyreosis 13 18,84Endometriosis 6 8,69Hypopituitarism 2 2,89Occult hyperprolactinemia 12 17,39Total 69 100

Table 2. Level of serum PRL, LH, FSH, E2 and P in hyperprolactinemia groups in follicular and luteal phase

Follicular phase PRL fertile PRL infertile PRL normal

PRL (ng/ml) 16,4±0.6 *c 17,2±0,8 c 8,5±0,6LH (IU/ml) 4,6±0,6 b 4,4±0,2 b 8,1±0,9FSH (IU/ml) 4,6±0,7 3,8±0,6 5,1±0,8E2 (nmol/l) 0,06±0,02 b 0,07±0,01 b 0,14±0,003P (ng/ml) 1,2±0,42 1,8±0,3 1,1±0,2Luteal phasePRL (ng/ml) 17,0±0,7 c 16,7±0,8c 8,7±0,6LH (IU/ml) 5,6±0,6a 6,1±0,7 a 9,8±0,4FSH (IU/ml) 2,9±0,5a 3,2±0,6 6,3±1,5E2 (nmol/l) 0,17±0,04b 0,3±0,02a 0,4±0,2P (ng/ml) 9,6±4,6 5,2±1,4b 12,1±2

Note: Parameters are expressed as mean ± SD, LH=luteinizing hormone, FSH=follicle stimulating hormone. E2=estradiol. Significant values p<0.05, Significace values ap<0,05; bp<0,01; p<0,001; for hyperprolatinemic vs. control, PRL=prolactin

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control group. PRL 30 during the TRH test was significantly higher (P <0.05), compared to the ba-sal PRL, but the increase was less than 70 ng / ml. (Table 3.)

In infertile women with partial hypopituitari-sm mean basal PRL (P <0.001) was significantly higher, while LH, FSH and estradiol (P <0.05) was lower in comparison with the control group. PRL 30 during the TRH test was significantly hi-gher (P <0.05), compared to the basal PRL, but the increase was less than 70 ng/ml. (Table 3.) In infertile women with OHP, basal prolactin values were normal and less than 10 ng/ml, TRH test was followed by a significant increase of PRL30 (8.6 ±2.9 vs. 107.1 ±1.1 ng/ml, p <0,001) compared with basal prolactin, whereas there was no signifi-cant difference in the values of gonadotropin and estradiol in relation to the control group. (Figu-re 1.) In the control group, basal prolactin levels were normal, and the TRH test was followed by a significant increase in PRL30 (7.6 +2.9 vs. 36.4 +3.1 ng / ml, p<0.01) compared to the basal level of PRL. (Figure 1.) In HyperPRL infertile women there was a significant increase (24.6 +4.6 vs.51.4 +6.1 ng/ml, p <0.05) of serum PRL30 levels in the 30th minute following the TRH test, compared to the basal level of PRL, but the level of PRL was less than 70 ng/ml. (Figure 1.)

Figure 1. Basal serum prolactin and prolactin 30 minutes after TRH administration in patients with hyperprolactinema, occult hyperprolactinemia and normal prolactin groups. Significance values: a P<0.05, bP<0.01,cP<0,001, for PRL30 vs.basal prolactin.

Discussion

Hyperprolactinemia is extremely common associated disorder in women of reproductive age and is present in one third of infertile women (1,2). The role of prolactin in the gonad dysfun-ction is now widely acknowledged and symptoms depend on the level of serum prolactin, and in-clude amenorrhea with or without galactorrhea, anovulation, infertility and abortions in women of reproductive age (3). Hyperprolactinemia is the result of high circulating levels of prolactin, which acts on the hypothalamus-pituitary-ovarian

Table 3. Levels of serum PRL, LH, FSH, E2, P and serum prolactin (30’) after thyrotrophin releasing homone (TRH) in all hyperprolactinemic groups and normal group during the follicular phase

Clinical manifestation of hyperprolactinaemia

(n)(31)

PRL (0’)(ng/ml)

PRL (30’)(IU/ml)

E2(IU/ml)

LH(nmol/ml)

FSH(ng/ml)

PCOS anovulatory (9) 31,2±1.1c 39,6±4,6 ∆a 221.5±14.6*a 13,6±1*b 4.9±1.1Hypopituitarismus (2) 23. ±13.1*c 48.6±5.2 ∆a 120,1±10.6*a 1.2±1. *a 1.3.9±0.1*a

Endometriosis Infertile (4) 25,6±3,8*c 64,7±16,1∆b 266±18,1 4,8±0,1 4.7±1.2Hypothyreoidism (13) 44.3±2.8*c 56±14.6 ∆a 119.2±16,7*a 2,4±11*a 1.2±0.6 *a

Normal (5) 7,5±2, 35,6±8,6∆c 150,1±19.6 6,4±1.1 6.5±1.5Note: Parameters are expressed as mean ± SD, LH=luteinizing hormone, FSH=follicle stimulating hormone. E2=estradiol. PRL=prolactin. Significant values p<0.05 , Significant values: *a p<0.05, *b p<0.01, *c p<0,001, for hyperprolatinemic vs. control; ∆aP<0.05, ∆bP<0.01, ∆cP<0.001, for PRL30 vs.basal prolactin.



axis and causes disregulation of gonadal secretion and changes the positive feedback mechanism at the hypothalamic and pituitary level resulting in hypogonadotropic hypogonadism, infertility and miscarriages (6). Symptoms depend on the levels of prolactin: PRL> 100 µg/L is associated with amenorrhea, galactorrhea and hypogonadism; PRL levels between 50-100 µg/L is associated with oligomenorrhea; mild/moderate hyperpro-lactinemia with PRL up to 50 µg/L was associated with a short luteal phase and habitual abortion (3). OHP with basal prolactin values of less than 10 ng/ml causes abortions, up to 15% of all aborti-ons are the consequence of OHP (5). The results of this study showed that in infertile women with primary, secondary and habitual abortions, infer-tility mean PRL was > 10 ng/ml, and PRL30 du-ring the TRH test > 50 ng / ml. Basal HyperPRL or occult hyperprolactinemia affects hypothalamo pituitary ovarian system and result in the failure of folliculogenesis and oocite maturation, which can be regulated by inhibiting prolactin as with bro-mocriptine (4,5). It is possible that prolactin has biological functions such as endocrine, immune and osmoregulatory (16,17). Blank et al. conclude that bromergon not only modulates endocrine sy-stem, but also has an immunological role. Several studies have reported that occult hyperprolactine-mia induces luteal insufficiency, galactorrhea and reproductive dysfunction (5,18). These disorders including prolactin luteal dysfunction have prima-rily been treated with bromocriptine and achieved good control (4,5). It is generally accepted that the serum prolactin level ≥ 14-15 ng / ml. is a crite-ria for HyperPRL. But to differentiate between basal prolacinemia and occult hyerprolacinemia, criteria for the upper limit of normoprolactinemia is 10ng/ml, and PRL 30 in the 30th minute after TRH test is 86 ng / ml (5). Aisaka et al. proposed diagnostic standard for OHP to be as PRL30 ≥ 70ng/ml (borderline: 50 - 70ng/ml), and bromo-criptine administration was effective not only in cases of OHP, but also in cases of hyperreactivitiy of LH (so-called endocrinological PCOD) (13). Various other studies gave different results beca-use of diverse etiological factors of hyperprolacti-nemia. In hyperprolactinemia caused by tumors basal levels of PRL depend on the size of the tu-mor, microadenomas give values of PRL of up to

200 ng/ml, and macroadenomas have levels gre-ater than 200ng/ml, and it is this hyperprolactine-mia that is accompanied with hypogonadotropic hypogonadism. Results of this study showed that the value of prolactin in anovulatory phenotype of PCOS was high and amounted to 24 ±2.6 ng/ml and is not accompanied with hypogonadotropic hypogonadism, even with elevated LH secretion. In PCOS hyperestrogenemia is boosting the se-cretion of LH, which acts on the ovarian stroma and produces androgens which results in anovu-lation. Hyperprolactinemia is often detected in in-fertile patients with ovulatory disorders, such as defective luteal phase and polycystic ovaries (4,5). Results of other studies have shown that the PRL values were between 10 and 35 ng/ml (19,20,21). Differences in the results of various studies, were the result of some authors determining the total level of prolactin in all three phenotype forms of PCOS and the other authors determining the le-vel of prolactin only in anovulatory phenotype of PCOS. Ovulatory-PCOS has significantly lower estradiol, LH and PRL than anovulatorny-PCOS (22). The results of this study showed that in in-fertile women with endometriosis there was mo-derate hyperprolactinemia present with prolactin of 25 ng/ml and normal gonadotropic secretion. Endometriosis is associated with infertility and is estimated to affect 10-15% of women (9). Hormo-nal alteration of prolactin secretion could be the cause of infertility in patients with endometrio-sis. (23), and also effect the folliculogenesis and oocite maturation (10). Nevertheless, some aut-hors have shown that some of their patients with normal basal prolactin in fact have a disorder of prolactin after TRH stimulation test. Patients who have LPD or infertility, although having normal basal prolactin, prolactin shows excessive reacti-vity to TRH stimulation (24). Cunha-Filho et al. state that the disorder of prolactin secretion and low levels of estradiol in infertile patients with en-dometriosis are associated with dysfunction of hy-pothalamus-pituitary-ovarian axis and are the ca-use of infertility (11). Lima et al. have found that prolactin and oestradiol in infertile women with endometriosis grade III/IV amounted to 28.9 ng/ml, and was higher than in endometriosis grade I/II, which amounted to 23 ng/ml. PRL level was significantly higher than in fertile women without

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endometriosis. Differences in the level of prolac-tin in studies were caused by the degree of endo-metriosis, because mild endometriosis is associa-ted with hypoestrogenemia, while severe endome-triosis is associated with hyperestrogenemia (25). This study also found mild/moderate HyperPRL present in infertile women with hypothyroidism with the values of prolactin 45 ng/ml. Because of the lack of T4 hormone, the TSH and TRH are se-creted in an attempt to raise peripheral hormones, and result in HyperPRL due to TRH stimulation. Avasthi et al. found that the infertile women with hypothyroidism had PRL elevated above 25ng/ml in 46% of cases, and that the hypothyroidism was the cause of 25% cases of HyperPRL (12). Also the results of this study showed that the partial panhypopituitarsm with inactive pituitary adeno-ma (pituitary stalk phenomenon) had an elevated basal prolactin and OHP associated with hypogo-nadotropic hypogonodism. PRL is elevated beca-use inactive adenoma compresses the handle, and interferes with the activity of PIH which results in hyperprolactinemia. Baha et al. have also shown that patients with macroadenoma and partial hy-popituitarism have moderate hyperprolactinemia (7). Arafah et al. suggest that pituitary adenomas with partial hypothyroidism due to compression of pituitary stalk block portal blood flow and as a result cause mild and moderate hyperprolactine-mia due to the lack of inhibition with PIF.

In conclusion, mild and moderate HyperPRL and OHP are the result of microadenomas, whi-le functional HyperPRL is associated with PCOS, endometriosis, hypothyroidism and panhypopitu-itarism (pituitary stalk phenomenon). In practice, clinicians should always consider the diagnosis of HyperPRL and OHP because they are a signifi-cant causal factors of infertility, habitual abortion, menstrual and sexual disorders.

References

1. Corenblum B. Disorders of prolactin secretion. Eds, L. J. Copeland, J. F. Jarrell, J.A. McGreger. Text book of Gynecology. Philadelphia, Pennsylva-nia. W.B. Saunders company. 1993; 447-467.

2. Rebar RW. Advances in the Management of hyper-prolactinemia. ASRMs 53rd Annual Meeting, Cin-cinnati. 1997.

3. Serri O, Chik C L, Ur. E, Ezzat S. Diagnosis and management of hyperprolactinemia, CMAJ 2003; 169:575-581.

4. Katz E, Adshi EY. Hyperprolactinemic disorders. Clin Obstet Gynecol 1990;33:622-639.

5. Asukai K, Uemura T, Minaguchi H. Occult hyper-prolactinemia in infertile women. Fertil Steril 1993; 60:423-427.

6. Uilenbroek J, et al. Effect of prolactin on follicu-lar oestradiol production in the Rat. J Endocrinol, 1984; 102: 245-250.

7. Arafah B M, Nekl K E, Gold R S, Selman W R. Dynamics of prolactin secretion in patients with hypopituitarism and pituitary macroadenomas. Jo-urnal of Clinical Endocrinology and Metabolism. 1995; 80:3507-3512.

8. D’Hooghe TM, Hill J. Novak’s Gynecology, Infer-tility, Philadelphia. 2002, Lippincott Williams & Wilkins.

9. Olive D L, Schwartz L B. Endometriosis. N. Eng. J. Med. 1993. 328:1579-1767.

10. Cahil D J, Hull M G R. Pituitary ovarian dysfun-ction and endometriosis Hum. Reprod. Update. 2000; 6:56-66.

11. Cunha-Filho J S, Gross J L, Lemos N A, Dias E C, et al. Prolactin and growth hormone secretion af-ter thyrotrophin-releasing hormone infusion and dopaminergic (DA2) blockade in infertile patients with minimal/mild endometriosis. Human Repro-duction 2002; 17:960-965.

12. Avasthi K, Kaur J, Gupta S, Narang P A. Hyper-prolactinemia and its correlation with hypothyroi-dism in infertile women. The Journal of Obstetrics and Gynecology of India 2006; 56:68-71.



13. Aisaka K, Kaneda S, et al. A study with diagnostic standard of occult hyperprolactinemia (OHP) and the effect of bromocriptine administration. Nippon Naibunpi Gakkai Zasshi 1993; 69:1017-1027.

14. The Rotterdam ESHRE/ASRM-sponsored PCOS Concensus Work-shop Group 2004 Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycistic ovary syndrome (PCOS). Human Reproduction, 19, 41-47.

15. ASRM. Revised American Society for Reproducti-ve Medicine classification of endometriosis: 1996. Fertil Steril 1997; 67: 817-821.

16. Buskila D. The possible role of prolactin in au-toimmunity. Am J Reprod Immunol 1991;26:118-122.

17. Reber P M. Prolactin and immunomodulation. Am J Med 1993; 95: 637-44.

18. Blank M, et al. Bromocriptine immunomodulation of experimental SLE and primary antiphospholi-opid syndrome via induction of nonspecific T su-ppressor cells. Cell Immunol 1995;162:114-122.

19. Hajder M , Hajder E, Insulin sensitivity and free androgen index in women with polycystic ovarian syndrome. Healthmed 2009; 3:513-519.

20. Kauffman RP, et, al.Endocrine i metabolic diffe-rences among phenotypic expressions of polycistic ovary syndrome accoring to the 2003 rotterdam concenssus criteria.

21. Papaleo E, Doldi N, et al. Cabergoline influen-ces ovarian stimulation in hyperprolactinaemic patients with polycystic ovary syndrome. Human Reproduction 2001; 16:2263-2266.

22. Murdoch A P, Dunlop W, Kendall-Taylor P. Studies of prolactin secretion in polycistic ovary syndrome. Clinical Endocrinology 1986; 24:164-175.

23. Cunha-Filho JS, Gross JL, Lemos NA, Brandelli A, Castillos M, Passos EP. Hyperprolactinemia and luteal insufficiency in infertile patients with mild and minimal endometriosis. Horm Metab Res 2001; 33: 216-220

24. Kostal M, Tosner J (1997) The influence of latent hyperprolactinemia on the levels of LH, FSH, E2 and T in the midfollicular phase of the cycle. Arch. Gynecol. Obstet. 1997; 359:65-68.

25. Lima A P, Moura M D, Rosa e Silva A A M, Pro-lactin and cortisol levels in women with endome-triosis. Braz J Med Biol Res 2006; 39:1121-1127.

Corresponding author Hajder E., University Clinical Center Tuzla, Gynecology and Obstetrics clinic, Bosnia and Herzegovina, E-mail: [email protected]

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Abstract

The aim of the study was to determine serum levels of matrix metalloproteinase 9 (MMP-9) and high sensitivity C reactive protein (hsCRP) in pa-tients with diagnosis of acute coronary syndrome (ACS). The study included 150 patients divided in three groups: patients with significant coronary artery disease (CAD), patients without significant coronary artery disease and patients with acute myocardial infarction (MI). Method used for de-termination of coronary artery disease significan-ce is coronary angiography, and CAD is determi-ned as significant if level of stenosis is >50%. The group without significant CAD had lower MMP-9 concentrations than group with significant CAD, witch has lower MMP-9 than group with acute MI. Difference in levels of MMP-9 concentration between groups with and without CAD is statisti-cally significant. Level of hsCRP in group with MI is significantly higher than in other two groups. There is no significant difference in hsCRP level between group of patients with significant CAD and without significant CAD. Our results demon-strate significance of MMP-9 and hsCRP level determination to help in assessing acute coronary syndrome patients in the future as a biomarker of plaque instability.

Key words: matrix metalloproteinase 9; high sensitivity C reactive protein, acute coronary syn-drome; plaque instability

Introduction

The diagnosis and management of acute coro-nary syndrome (ACS) are limited by the lack of a widely available and sensitive assay for use in assessment of coronary artery disease. The dia-gnosis of ACS in most hospitals is based on clini-cal grounds, invasive techniques such as coronary angiography. Furthermore, most hospitals do not have these specialized services and the assessment of urgency and difficultness of CAD remains lar-gely based on clinical decisions. This suggests the need for an adjunctive biochemical test that could provide diagnostic information with the ability to correlate with CAD severity.

In particular, pro-inflammatory cytokines and acute phase reactants including C-reactive protein (CRP) have been used as biomarkers and predic-tors for acute coronary syndromes (ACS) (Dane-sh, Wheeler, 2004). In parallel, pro-inflammatory cytokines such as interleukin-1 (IL-1) and tissue necrosis factor-α (TNF-α) have been shown to up-regulate matrix metalloproteinase-9 (MMP-9) (Galis, Sukhova, 1994). Matrix metalloproteinase (MMPs) are a large, tightly regulated family of zinc-endopeptidases that degrade different com-ponents of the extracellular matrix (ECM) under normal conditions, although they have also been reported in a wide range of pathological proce-sses, including acute myocardial infarction (MI). Members of the MMP family are divided into cla-

biomarkers of plaque instability in acute coronary syndrome patients

Jasmina Nurkic1, Farid Ljuca2, Midhat Nurkic3, Farid Barakovic4, Denijel Tulumovic4, Elmir Jahic3, Amra Dzankovic5

1 Department of Immunology, Policlinic for laboratory diagnostic, University Clinical Centre Tuzla, Bosnia and Herzegovina,2 Medical faculty University of Tuzla, Bosnia and Herzegovina,3 Clinic for Cardiovascular Disease, University Clinical Centre Tuzla, Bosnia and Herzegovina,4 Clinic for Internal Disease, University Clinical Centre Tuzla, Bosnia and Herzegovina,5 General hospitale, prim dr A.Nakaš Sarajevo, Bosnia and Herzegovina,



sses based on their structure or substrate specifici-ty, one of which is gelatinases MMP-9.

Proinflammatory cytokines, released as a result of acute ischemic event, enhance the expression of adhesion molecules on endothelial cells. Adhesion molecules support the invasion of polymorphonuc-lear leukocytes and monocytes to the site of the le-sion (Libby, 1995). Hemodynamic changes, injury, inflammation, and oxidative stress all appear to re-gulate MMP activity and induce MMP overexpre-ssion. MMPs are known to play an important role in the physiological and pathological remodeling of blood vessels (Woessner, 1991). More importantly, they have been postulated to be involved in the destabilization and rupture of atherosclerotic lesi-ons in unstable carotid plaques. Tissue destruction seems to be especially related to the overexpression of MMP-2 and MMP-9 (Kai, Ikeda, 1998).

Numerous prospective studies in different popu-lations have shown that high levels of CRP predict future cardiovascular events, even in apparently healthy individuals (Anzai, Yoshikava, 1997). This has led to the recent position statement by the Ame-rican Heart Association-Centers for Disease Con-trol, recommending cutoff levels of CRP less than 0.0085, 0.0085–0.025, and more than 0.025 µM (<1.0, 1.0–3.0, and >3.0 mg/liter) for low, average, and high risks for subsequent acute cardiovascular event (Pearson, Mensah, 2004).

In addition to it being a risk marker, recent data from several laboratories strongly suggest that CRP is proatherogenic and prothrombotic (Auer, Rammer, 2002).

Therefore, our aim was to study serum levels of MMP-9 and hsCRP in patients with signifi-cant CAD, patients with insignificant CAD and patients with acute MI and to determine possible correlation that will help in future risk assessment.


Patients were recruited during the period from August 2008 to January 2009. A total of 150 pati-ents from which 100 are admitted to the Clinic for Cardiovascular disease University Clinical Cen-tre Tuzla (UKC) with diagnosis of ACS and with aim to proceed coronary angiography, and 50 is admitted on Intensive Care Unit UKC Tuzla with

diagnosis of acute MI. Patients with history of chronic, malignant or acute infective disease were excludes from this study.

Demographic characteristics, as well as risk factors according to the World Health Associati-on (WHO) i.e., smoking, hypertension and total blood cholesterol, were collected for each patient. CAD severity was assessed using the coronary an-giography on group of patients admitted on Clinic for Cardiovascular disease (n=100) with criteria for significant coronary artery stenosis of stenosis >50%. Venous blood samples were drawn upon patient arrival at the hospital before any treatment.

Laboratory methods of this study were conduct on Department for immunology, Policlinic for la-boratory diagnostic University Clinical Centre Tu-zla. Plasma MMP-9 concentrations were measu-red using a commercially available enzyme linked immunosorbent assay (R&D Systems ELISA). C-reactive protein was determined by a highly sensi-tive (hs), automatic nephelometric method («Dade Behring«). Significance of the results was deter-mined based on their difference between known groups of patients.

Blood was drawn under standardized conditions before coronary angiography and stored at -80°C. From patients hospitalized because of acute myo-cardial infarction, blood sample was taken in period of 48 hours from beginning of first symptoms.

MMP-9 is determined as marker of plaque rup-ture and hsCRP, as indirect sign of chronic inflam-matory process on coronary arteries.

We analyzed MMP-9 and hsCRP values in three determined group of patients and then compare re-sults between group of patients with coronary artery disease verified by coronary angiography and group without CAD verified by same method. Same anal-yze was conduct with group of patients with IM.

Minimal detected dose of MMP-9 was 0,156 ng/ml. Values of hsCRP above 3 mg/dL point out infla-mmation, which together with patient’s history and values of other cardiovascular markers can show inflammatory process at cardiovascular system.

Resultates of coronary angiography of our pa-tients we get from Department for Interventional Cardiology Clinic for Cardiovascular Disease.

Other laboratory tests were determined by rou-tine diagnostic of acute myocardial infarction and angina pectoris during patient’s hospitalization.

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In our study we determined level of hsCRP for all patients from one blood sample taken before coronary angiography. We did just one determina-tion of hsCRP because of good experience from our previous study on levels of hsCRP in pati-ents with ACS. In that study we used good exc-luding criteria for ACS patients and results from two hsCRP levels determination two weeks apart, showed no significant difference. Excluding crite-ria are absence of maligned, chronic or acute in-flammatory disease on basis of data from patient’s clinical history.


In statistical analysis we use methods of des-criptive statistic with help of «Medcalc» Compu-ter program. For testing of statistical significance between groups we use parametric and nonpara-metric tests. The level of statistical significance was set at p=0.05.

Results

The group without CAD include 50 patients, 27 (54%) of them male, mean age of 63,50±8,54 years. The group with CAD include 50 patients, 31 (62%) of them male, mean age of 61,45±9,87 and group with MI include 50 patients, 36 (72%) of them male, mean age 60,78±12,26. Risk factor profiles are presented in Table 1.

There is no statistically significant difference in values of hsCRP between groups with and without CAD (p=0,0798).

There is statistically significant difference in levels of MMP-9 between group with CAD and group without CAD (p=4,178E-08).

There is statistically significant difference in hsCRP between group with CAD and group with IM (p=1,84E-06).

There is no statistically significant difference in MMP-9 values between group with CAD and group with IM (p=0,29).

There is statistically significant difference in hsCRP values between group without CAD and group with IM (p=8,94E-07).

Table 1. Risk factors for ACSRisk factors Group without CAD Group with CAD Group with IM

Smoking 24% 42% 36%Family history 48% 54% 16%Obesity 24% 24% 22%Hypertension 84% 80% 62%Hyperlipidemia 84% 86% 32%History of MI 10% 42% 22%History of ICV 6%

Table 2. Values of laboratory markers between three study groups Group with CAD Group without CAD Group with IM P

hsCRP (mg/L) 3,09 2,23 26,11 1,25E11MMP-9 (ng/mL) 880,82 434,94 986,12 2,68E-09

Table 3. Values of laboratory markers between patients in group with and without CAD Group with CAD Group without CAD P

hsCRP 3,09 2,23 0,0798MMP-9 880,82 434,94 4,178E-08



There is statistically significant difference in MMP-9 values between group without CAD and group with IM (p=2,17E-08).

In group without CAD highest percentage of patients have hsCRP values from 0 to 1 mg/l. In group with CAD highest percentage of patients have hsCRP values from 3 to 10 mg/l. In group with acute MI highest percentage of patients have hsCRP values higher than 10 mg/l.

In our study we also determine the number of coronary arteries with significant stenosis (>50%) and correlate it with MMP-9 and hsCRP values (Table 7).

Values of hsCRP are higher in patients with si-gnificant stenosis in three coronary artery than in patients with two, and in patients with two higher than in patients with one, but this difference is not statistically significant.

Values of MMP-9 are higher in patients with significant stenosis in three coronary artery than in patients with two, and in patients with two is

lower than in patients with significant stenosis on one coronary artery but these difference is not sta-tistically significant.

There is a statistically significant differen-ce in tested laboratory markers of inflammation between three study groups.

We analyzed relation between hsCRP and MMP-9 values in group of patients with acute myocardial infarction and conclude that there is weak positive correlation between hsCRP and MMP-9 values.

hsCRP1 MMP 9hsCRP1 1 MMP 9 0,28437426 1

Table 4. Values of laboratory markers between groups with CAD and IM Group with CAD Group with IM P

hsCRP 3,09 26,12 1,84E-06MMP-9 880,82 986,12 0,29

Table 5. Values of laboratory markers between patients in group without CAD and group with IMGroup without CAD Group with IM p

hsCRP (mg/L) 2,23 26,11 8,94E-07MMP-9 (ng/mL) 434,94 986,12 2,17E-08

Table 6. Distribution of hsCRP values by AHA classification in tree study group of patientsHsCRP (mg/L) Group without CAD Group with CAD Group with IM

0-1 38% 14% 4%1-2 26% 24% 14%2-3 4% 22% 2%

3-10 26% 30% 18%>10 6% 10% 62%

Table 7. Number of stenosed coronary artery in CAD patientsNumber of stenosed coronary artery>50% Percentage of patients in group with CAD

3 40%2 28%1 32%

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Figure 1. Correlation of hsCRP and MMP-9 values in group of patients with acute myocardial infarction

Discussion

It is today widely accepted that inflammatory processes are involved in plaque rupture prece-ding acute coronary events (Fuster, Badimon, 1992). In daily practice we met with huge number of patients which beside coronary angiography analysis that shows that there is no CAD have one of the clinical manifestations of ACS. Often pati-ents with CAD and stenosis higher than 50%, pro-ven with coronary angiography, have no expected clinical manifestation.

All patients in our study had diagnosis of ACS, which is working diagnosis for coronary angio-graphy based on patient’s clinical history, changes on ECG and values of biochemical parameters.

In our study average age of patients in group without CAD is 61-70 years (63,50±8,54), in gro-up with CAD and acute IM is 51-60 years.

In all three analyzed groups there is higher number of man then women. In a group with IM 36%, in group with CAD 31%, and 27% of man in a group without CAD. Percentage of women is highest in group without CAD and the lowest in group with IM.

Age and sex distribution of patients in our study is as expected by results of number of cli-nical studies conducted on patients with ACS or acute myocardial infarction (Ridker, Stampfer, 2001). We notice a number of patients age 40 to 60 years with ACS or IM but it does not significantly affect results of our final statistical analysis.

Many studies in last decade proved that certa-in markers of inflammation, systemic and locally, have very important role in development of athe-rosclerosis.

So far, most studies exploring elevated levels of MMP-9 and its association to coronary artery disease (CAD) have been based on a experimental or clinical design, the latter showing elevated le-vels of plasma MMP-9 in patients with CAD (Fu-kuda, Shimada, 2006).

MMP-9 is considered to be a key determinant of extracellular matrix degradation, having collagen as the main substrate. Increased concentrations and activity of MMP-9 have been observed in human atherosclerotic vulnerable plaques with high infla-mmatory activity, suggesting a role in matrix degra-dation and plaque rupture (Galis, Sukhova, 2004).

Increased expression and activity of MMPs have been identified in various pathological proce-sses, such as general inflammation, tumor metasta-sis, respiratory diseases, myocardial injury, vascu-lar aneurysms, and remodeling (Woessner, 1991).

Serum levels of MMP-9 have been reported to be elevated in patients with MI and unstable an-gina (Kai, 1998) as well as in stable angina (Noji, 2001). FINRISK retrospective study showed that MMP-9 is significantly high in persons with hi-story of MI comparing with healthy individuals (Renko, 2004).



In January 2003 associate protocol from Cen-ters For Disease Control and Prevention (CDC) and American Heart Association (AHA) point out hsCRP as marker of inflammation in estimation of risk for cardiovascular disease (Pearson, 2004).

In our study levels of hsCRP are highest in gro-up with IM as expected (26,11mg/l). In that group 62% of patients have levels of hsCRP higher than 10 mg/l.

Myocardial necrosis is reason for high hsCRP values in group of patients with IM, while mean hsCRP values in group with CAD is 3,09 mg/l which is above referent values (0-3 mg/l). Mean values of hsCRP in group without CAD is 2,23 mg/l. Values in group with and without CAD are not showing any statistically significant difference which point out the active plaque and active infla-mmatory process in both group of patients.

Based on AHA recommendation, patients in three study groups we can divide on subgroups based on hsCRP values and determine level of risk for acute cardiovascular event (IM, ICV, etc.) for those patients. Values of hsCRP in group with CAD are higher than in group without CAD but that difference is not statistically significant.

In group of patients without CAD 38% of pa-tients have hsCRP values 0-1 mg/l, which shows that there is highest percentage of patients with low risk for acute cardiovascular event. Values of hsCRP 3-10 mg/l, have 26% of patients which po-int out the fact that in this group are also highly risk patients.

Group with CAD has 30% highly risk patients, and 38% have hsCRP values lower than 2 mg/l.

The fact is that more than one third of patients in group without CAD are highly risk patients and that more than one third of patients in group with CAD are low to middle risk for acute cardiovascu-lar even, leads to conclusion that there is no signi-ficant difference in values of this marker between these two study group.

Significant effect on hsCRP values probably had a patient’s therapy. Consider high number of influence factors it is important that in group with CAD hsCRP values point out active plaque prone rupture and make risk estimation easier.

Elevation of hsCRP after acute IM or unstable angina has positive correlation with consequence of disease (Anzai, 1997).

Coronary angiography determine level of coro-nary artery lumen stenosis but it is not adequate in determination of CAD severity because we know about plaque rupture and active inflammation even when plaque don’t take more than 50% of vessel lumen (Nissen, 2001).

Because MMP-9 has characteristic of plaque rupture marker and hsCRP as marker of active inflammation of atherosclerotic plaque it is inte-resting to follow these markers values in all three study group. Values of MMP-9 are the highest in group with IM, lower in group with CAD and the lowest in group without CAD. Values of hsCRP are also highest in group with IM and lowest in group without CAD. We can see that there is no statistically significant difference in hsCRP va-lues between group with and without CAD, but difference in values of these markers is still there. Difference between these markers values between group with CAD and group with IM is statistically significant. We analyzed values of hsCRP and MMP-9 in group of patients with acute IM and conclude that there is weak positive correlation.

The importance of determination both MMP-9 and hsCRP together is probably in the fact that mechanism of there synthesis is different.

Serum level of MMP-9 correlate with number of diseased coronary artery detected by coronary angiography but statistically significant correlati-on is seen in group of patients with stenosis on three coronary artery which is more than with two or one stenosed coronary artery or with patients without CAD.

HsCRP is highly sensitive but weekly specific marker so it is very important to combine it with patients clinical data but also with values of mar-ker as MMP-9 who point out plaque rupture.

There is a variety of factors influencing hsCRP and MMP-9 values, we exclude patients with acu-te or chronic inflammatory disease or malignan-cies. But is noted significant influence of therapy that CVD patients used on study markers. In our study there is no statistically significant correlati-on between hsCRP and MMP-9 levels and used therapy.

In our study we take data from the patients about the drug therapy they use. In group without CAD 74% of patients is on therapy with ACE inhi-bitors, 54% is on therapy with beta blocators, 36%

678



on statines, and 24% use Aspirin. In group with CAD 92% of patients use medicine from ACE in-hibitors group, 68% use Aspirin, 56% statins and 54% beta blocatores. In group with MI 78% of pa-tients use ACE inhibitors, 48% beta blockers, 24% statins and 18% Aspirin.

When we consider presence of list risk factors in all three group of patients than prescribe the-rapy get on importance. Data of therapy use we get from patients which not exclude possibility of incorrect and irregular use.

Most of the patients use drugs from the group of ACE inhibitors, than beta blockers or Aspirin. Statin therapy is mostly used in group of patients with CAD same as therapy with ACE inhibitors. Statins therapies can lover collagen degradation and MMP-9 activity (Corti, Farkouh, 2002).

Clinical studies showed reduction in hsCRP level for 15-28% in period of 6 weeks statine usa-ge, independently of LDL level reduction (Ridker, 2001).

Zaman in his study recommended combination of lipid reductors and antioxidants (to help plaque stabilization by lipid reduction, stabilization of fibrose cap and reduction of MMP-9 activity in plaque), ACE inhibitors (improvement of endo-tel dysfunction) and beta-blockers (reduction of blood stream influence on fibrose cap) (Zaman, 2000).

Conclusion

Our study point out importance of hsCRP de-termination because it is marker of inflammation at atherosclerotic plaque and is important in asses-sment of plaque rupture risk in patients with ACS.

MMP-9 is marker of plaque instability and po-int out at plaque rupture.

The low but significant correlation between hs-CRP and MMP-9 is of particular interest in our findings. This implies that hsCRP and MMP-9, at least to some extent, could be markers of different physiological pathways.

It is important to correlate data about presence of other risk factors for CAD and correct data abo-ut used medical therapy with data of laboratory marker levels to get assesment of acute cardiovas-culare event, CAD and plaque rupture.

References

1. Anzai T, Yoshikava T, Shiraki H, Asakura Y, Akaishi M. Mitamura H, Ogawa S (1997) C breactive pro-tein as a predictor of infarct expansion and cardiac rupture after a first Q wave acute myocardial in-farction. Circulation;96:778-784.

2. Auer J, Rammer M, Berent R, Weber T, Lassnig E, Eber B (2002). Relation of C-Reactive Protein Levels to Presence, Extent, and Severity of Angi-ographic Coronary Artery Disease. Indian Heart J;54:284-288.

3. Beaudeux J, Giral P, Bruckert E, Foglietti M, Chapman M (2004) Matrix metalloproteinases, inflammation and atherosclerosis: therapeutic per-spectives. Clin Chem Lab Med; 42:121-131.

4. Danesh J, Wheeler J, Hirschfield G (2004) C-Re-active Protein and Other Circulating Markers of Inflammation in the Prediction of Coronary Heart Disease. NEJM;350:1387-1397.

5. Fukuda D, Shimada K, Tanaka A (2006) Compari-son of levels of serum matrix metalloproteinase-9 in patients with acute myocardial infarction versus unstable angina pectoris versus stable angina pec-toris. Am J Cardiol; 97: 175-180.

6. Fuster V, Badimon L, Badimon J (1992) The patho-genesis of coronary artery disease and the acute co-ronary syndromes (1). N Engl J Med;326:242–250.

7. Galis Z, Sukhova G, Lark M, Libby P (1994) In-creased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regi-ons of human atherosclerotic plaques. J Clin In-vest;94:2493-2503.

8. Kai H, Ikeda H, Yasukawa H, Kai M, Seki Y, Kuwa-hara F, Ueno T, Sugi K, Imaizumi T (1998) Perip-heral blood levels of matrix metaloproteinase 2 and 9 are elevated in patientes with acute coronary syn-drome. J Am Coll Cardiol;32:368-372.

9. Libby P (1995) The Molecular Basis of Acute Coro-nary Syndromes. Circulation; 91: 2844-2850

10. Loftus I, Naylor A, Bell P, Thompson M (2002) Matrix metalloproteinases and atherosclerotic plaque instability. Br J Surg;89:680-694.

11. Nissen S (2001) Coronary angiography and intra-vascular ultrasound. Am J Cardiol;87:15-20.



12. Noji Y, Kajinami K, Kawashiri M, Todo Y, Hori-ta T, Nohara H, Higashihatk T, Inaza A, Koizumi J,Takegoshi T, Mabuchi H (2001) Circulating ma-trix metaloproteinase and their inhibitors in pre-mature coronary atherosclerosis. Clin Chem Lab Med;39:380-384.

13. Pearson A, Mensah A, Hong Y, Smith S (2004). CDC/AHA workshop on markers of inflamma-tion and cardiovascular disease:application to clinical and public health:overview. Circulati-on;110:534-544.

14. Renko J, Kalela A, Jaakkola O, Laine S, Hoyhtya M, Alho H, Nikkari S (2004) Serum matrix me-talloproteinase-9 is elevated in men with a history of myocardial infarction. Scandinav J Clin and Lab Investig; 64: 255-261.

15. Ridker P, Stampfer J, Rifai N. (2001) Novel risk factors for systemic atherosclerosis: a comparison of C-reactive protein, fibrinogen, homocysteine, lipoprotein(a), and standard cholesterol scree-ning as predictors of peripheral arterial disease. JAMA; 285(19):2481-5.

16. Ridker P, Danielson E, Fonseca F, Genest J, Gotto A, Kastelein J, Koenig W, Libby P, Lorenzatti A, MacFadyen J, Shepherd J, Willerson J, Glyn R. (2008) Rosuvastatin to Prevent Vascular Events in Men and Women with Elevated C-Reactive Prote-in NEJM; 359:2195-2207.

17. Woessner J (1991) Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J;5:2145-2154.

18. Zaman G, Helft G, Worthley G, Badimon J (2000) The role of plaque rupture and throm-bosis in coronary artery disease. Atherosclero-sis;149(2):251-66.

Corresponding author Jasmina Nurkic, Department of Immunology, Policlinic for laboratory diagnostic, University Clinical Centre Tuzla, Bosnia and Herzegovina, E-mail: [email protected]

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Abstract

Traumatic head injury caused temporary dis-ruptions and final stereopsy as the most important degree of binocular vision. The aim of this pa-per is to establish whether there is a deficit in the perception of depth in patients with closed head injuries, analysis and application sensitivities tests. The study included 80 patients, aged 10 to 40 years, with closed head injuries, the main diffi-culty was the inability to sense the depth of space so they seem smaller objects. Excluded from the study were patients younger than 10 years and ol-der than 40 years, patients with neurological and ophthalmic diseases, disorders of early binocular vision and refractive errors larger than 1 Dptr. The control group consists of patients of the same age with the same disorders, who had no head injury. Statistically we followed and investigated the correlation between the control and research gro-ups of respondents in relation to the examination of convergence values stereo vision and fusion before treatment, 2 and 6 months after treatment. Comparing the correlation convergent fusion ste-reoscopic vision and values, we conclude that the-re is a statistically significant correlation at the le-vel of significance ά = 1% before treatment, 2 and 6 months after treatment. We have used the pri-sm adaptation treatment in treatment and reduced astenostoptic deficits, improving convergention fusion, deep sense of spatial perception. This type of treatment is easily feasible and should always apply there in disorder of oculomotoric functions.

Key words: perception of depth, traumatic head injuries.

Sažetak

Traumatske povrede glave izazivaju prolazne i definitivne poremećaje stereopsije kao najvaž-nijeg stepena binokularnog vida. Cilj rada je da se ustanovi postoji li deficit percepcije dubine kod pacijenata sa zatvorenim povredama glave, ana-lizom i primjenom disociranih testova. U studiju je uključeno 80 pacijenata, u dobi od 10 do 40 godina, sa zatvorenim povredama glave, glavna poteškoća je bila nemogućnost osjećaja dubine u prostoru tako da su im predmeti izgledali manji. Iz studije su isključeni pacijenti mlađi od 10 godina i stariji od 40 godina, pacijenti sa neurološkim i oftalmološkim bolestima, ranijim poremećajima binokularnog vida i refrakcionim greškama ve-ćim od 1 Dptr. Kontrolnu grupu čine pacijenti iste starosne dobi sa istim smetnjama, koji nisu imali povredu glave. Statistički smo pratili i ispitivali korelaciju između kontrolne i istraživačke grupe ispitanika u odnosu na ispitivanje stereovida i vri-jednosti konvergentne fuzije prije tretmana, 2 i 6 mjeseci poslije tretmana. Uporedbom korelacije konvergentne fuzije i vrijednosti stereoskopskog vida, zaključujemo da postoji statistički značajna korelacija na nivou značajnosti ά=1% prije tretma-na, 2 i 6 mjeseci poslije tretmana. Koristili smo prizma adaptacioni tretman u liječenju i reducirali astenopske deficite, poboljšavajući konvergen-tu fuziju, prostornog osjećaja duboke percepcije. Ova vrsta tretmana je lako izvodljiva i treba je uvijek primjenjivati tamo, gdje postoji poremećaj okulomotorne funkcije.

Klučne riječi: percepcija dubine, traumatske povrede glave.

disorder at depth perception of the traumatic head injuryRaif Serdarevic

Ophthalmology Clinic, Clinical Center University of Sarajevo, Bosnia and Herzegovina



Introduction

Traumatic head injury caused temporary and final disruptions (disturbance) of stereopsy as the most important degree of binocular vision.

Binocular parallax of an object is the angle un-der which an object seen with both eyes, optic lines, which are both eye fixed a point object. Binocular parallax difference between two points is a current or stereoscopic parallax and occurs as a result of two points that lie in different planes (6, 7).

Stereopsis to behave differently in closed head injury, depending on the region of injury, prima-rily including its qualitative part-local and global stereopsis (1, 2, 3, 4, and 5).

Determine whether there is a deficit in the per-ception of depth in patients with closed head inju-ries, analysis and application specific tests.

Materials and method

The study was conducted as prospective, clini-cal, controlled, descriptive studies, lasting two ye-ars at the Department of Ophthalmology, Universi-ty Clinical Center in Sarajevo. The study included

80 patients, aged 10 to 40 years, with closed head injuries; the main difficulty was the inability to sen-se the depth of space so they seem smaller objects. Excluded from the study were patients younger than 10 years and older than 40 years, patients with neurological and ophthalmic diseases, and disor-ders of early binocular vision and refraction errors larger than 1 Dptr. The control group consists of pa-tients of the same age with the same disorders, who had no head injury.

The methods we used to history of disease, where we are interested in time, type, means and the region of injuries, the time from injury to first symptoms, and subjective disability in the cour-se of injury, sex, occupation. We investigated vi-sual acuity at distance and near monocular and binocular. Refraction we determined with skias-copy and autorefractometry, review anterior eye segment, posterior eye segment preview pane Godmanovom lens, examination of motility and testing occulomotor balance external eye muscles, Cower-Uncower test, the Hess-Lancaster on the diplopia, we investigated the state of fusion with a prism and measuring stereo vision and compari-son with the findings of Titmus and TNO test, and we thus attempted to detect stereoscoptic depth-

Table 1. Correlation between Control and Research groups of respondents in respect to testing and values stereo vision convergent fusion before treatment, 2 and 6 months after treatment

Group No Central value The sum of the valuesRank stereo vision values before treatment for TITMUS test

ResearchControlTotal

354580

38,5742,00

1350,001890,00

Rank stereo vision values before treatment for the TNO test


7280152

71,7880,75

5168,006460,00

Rank values stereo vision 2 months after treatment for TITMUS test


364581

38,5043,00

1386,001935,00

Rank values stereo vision 2 months after treatment for the TNO test


7180151

72,2079,38

5126,006350,00

Rank values stereo vision 6 months after treatment for TITMUS test


324577

37,5940,00

1203,001800,00

Rank values stereo vision 6 months after treatment for the TNO test


7180151

63,5287,25

4496,006980,00

682



disparity before treatment, 2 and 6 months after treatment comparison study with control group, together with the calculation takes the working distance and interpupilar distance and time and work to close without fatigue.

All reviewed were classified into three groups according to treatment: before treatment, 2 and 6 months after treatment. All results were analyzed statistical tests.

Results

Of 80 patients with traumatic head injuries, ages 10 - 40 years, 53 (66.25%) were males, and 27 (33.75%) females and 80 patients in control group with out of traumatic head injuries.

Correlations

convergent fusionvalues before

treatment

convergent fusion

values 2 months

after treatment

convergent fusion

values 6 months

after treatment

rankvalues stereo vision before

treatment to test

TITMUS

rank values stereo vision before

treatment for the

TNO test

rank values stereo

vision 2 months

after treatment

for TITMUS

test

rank values stereo

vision 2 months

after treatment

for the TNO test

rank values stereo

vision 6 months

after treatment

for the TITMUS

test

rank values stereo

vision 6 months

after treatment

for the TNO test

convergent fusion values before treatment

Correlation coefSig. (2tailed)N

1,000

80

,93800080

,749,00080

1,000

35

,965,00072

,999,00036

,918,00071

,535,00232

,738,00071

convergent fusion values 2 months after treatment

Correlation coefSig. (2 tailed)N

,93800080

1,000

80

,850,00080

,999,00035

,905,00072

1,000,00036

,942,00071

,553,00132

,844,00071

convergent fusion values 6 months after treatment


,749,00080

,850,00080

1,000

80

,568,00035

,618,00072

,630,00036

,725,00071

,664,00032

,920,00071

rank values stereo vision before treatment to test TITMUS


1,000

35

,999,00035

,568,00035

1,000

35 32

,999,00035 32

,799,00026 32

rank values stereo vision before treatment for the TNO test


,965,00072

,905,00072

,618,00072 32

1,000

72

1,000

33

,931,00071 29

,715,00071

rank values stereo vision 2 months after treatment for TITMUS test


,999,00036

1,000,00036

,630,00036

,999,00035

1,000

33

1,000

36 32

,735,00027 32

rank values stereo vision 2 months after treatment for the TNO test


,918,00071

,942,00071

,725,00071 32

,931,00071 32

1,000

71 28

,798,00071

rank values stereo vision 6 months after treatment for the TITMUS test


,535,00232

,552,00132

,664,00032

,799,00026 29

,735,00027 28

1,000

32 28

rank values stereo vision 6 months after treatment for the TNO test


,738,00071

,844,00071

,920,00071 32

,715,00071 32

,798,00071 28

1,000

71

Correlation is significant at the 0.01 level (2-tailed)



Discussion

In studies of Rossetti Y. and al., G. Rode and al. (8, 9) was first used prismatic “adaptation” in the daily treatment. These authors noted that it provides long-lasting effect which should be fo-llowed. Treatment with prism spent twice daily through two weeks. The sample consisted of six patients and the authors found a significant incre-ase in spatial distance and proximity space, and these improvements were accompanied by two days after treatment, one week and five weeks af-ter treatment, with a tendency of further improve-ment, which were long kept despite the breadth of diversity of the visual spatial response. Rehabili-tation with the prism compare with other rehabi-litation procedures such as optocinetyc and vesti-bular stimulations that are not so invasive methods and can be performed on patients who do not need help hospitalization or therapist, but did not give positive results as the prism adaptation treatment.

On the basis of our research there is a statisti-cally significant difference between the values of patients in the Titmus test, 2 months after treatment, the level of significance ά = 5%, and the difference between the examined and control groups for the TNO test, 2 months after treatment was statistically significant at the 5% level , there is a statistically significant difference between the values of patients Titmus test 6 months after treatment, the level of significance alpha 10%, the difference between the examined and control groups for the TNO test, 6 months after treatment is statistically significant at the level of 5% and 1%. Comparing the correlation convergent fusion and values of stereoscopic visi-on, we conclude that there is a statistically signifi-cant correlation at the level of significance ά = 1% before treatment, 2 and 6 months after treatment.

Conclusion

Treatment and testing in neuro- ophthalmology rehabilitation, stereo vision disorders in closed head injuries are rarely implemented. Comparison sce-nes with closed head injury patients with the inabi-lity to sense the depth of the difficulties in the area, represents a new therapeutic approach to spatial changes, to improve the convergence fusion.

Prism adaptation treatment reduces astenops deficits, improving convergent fusion, deep sense of spatial perception. This type of treatment is ea-sily feasible and should always apply there, where there are disorders of oculomotoric functions.

References

1. Kerkhoff G. Rehabilitation of visuospatial cogniti-on and vision exploration in neglect: a cross over study. Rest Neurol Neurosci, 1998; 12:27-40.

2. Kerkhoff G. Restorative and compensatory herapy approaches in cerebral blidness. Rest Neuronal Ne-urosci, 1998; 8:15-22.

3. Kerkhoff G, Artinger F, Ziegler W. Contrasing spa-tial hearing deficits in hemianopia and spatial ne-glect. Neuro Report 1999; 10:3555-3560.

4. Kerkhoff G, Heldmann B. Balint Syndrom und asso-ziierte Storungen. Springer-Verlag, 1999; 70:859-869.

5. Kerkhoff G, Schindler I. Neurovisuelle Storungen Blackwell, Oxford, 1999; 313-336.

6. Rode G, Klos T, Courtois JS, Rosseti Y, Pisella L. Neglect and prism adaption: a new therapeutic tool for spatial cognition disorders. Restor Neuronal Neurosci, 2006; 24(4-6):347-56.

7. Kaufman H. Strabismus. Georg Thieme Verlag, 2004.

8. Rode G, Klos T, Courtois JS, Rosseti Y, Pisella L. Neglect and prism adaptation: a new therapeutic tool for spatial cognition disorders. Restor Neural Neurosci, 2006; 24(4-6):347-56.

9. Rossetti Y, Rode G, Piseella L, Farnea A, Li L, Boisson D, Perenin MT. Prism adaptation to a ri-ghtward optical deviation rehabilitates left hemis-patial neglect. Nature, 1998; 395(6698):166-9.

Corresponding author Raif Serdarevic, Ophthalmology Clinic, Clinical Center University of Sarajevo, Bosnia and Herzegovina, e-mail: [email protected]

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Abstract

The aim of this study was to assess the effects of antenatal corticosteroid treatment on the morbi-dity and mortality of preterm infants.

Materials and methods: This study enrolled 172 premature babies between 26 and 34 gesta-tional weeks of age, admitted to NICU, Pediatric Clinic, Clinical University Center Sarajevo, du-ring two years period. Babies with intrauterine growth restriction (IUGR), major congenital ab-normalities, and those born to diabetic mothers were excluded. 80 out of 172 babies were treated with corticosteroids prenatally, 92 of 172 did not receive such treatment. The severity of respiratory distress syndrome (RDS) was classified according to FiO2 requirements.

Results: Frequency and severity of RDS was significantly lower in the group of babies antena-tally treated by cortisosteroids (22/80) compared to the control group (53/92) (p<0,001). IVH-PVH and mortality rate were significantly lower in cor-ticosteroid group. There was no statistically signi-ficant difference in the occurrence of sepsis and BPD between groups.

Conclusion: Antenatal corticosteroid treatment significantly reduces the incidence and severity of RDS, IVH-PVH, and neonatal death but does not alter the risk of acquiring sepsis.

Key words: Antenatal corticosteroids, preterm babies, morbidity and mortality

Sažetak

Cilj studije je procjena efekata antenatalne kortikosteroidne terapije na morbiditet i mortalitet prijevremeno rođene djece.

Material i metode: Istraživanjem su obuhva-ćena 172 preterminska novorođenčeta gestacijske dobi 26-34 nedjelje, primljena na Odjel neonatalne intenzivne njege Pedijatrijske klinike u Sarajevu u toku dvogodišnjeg perioda. Iz studije su isključena djeca sa intrauterinom restrikcijom rasta (IUGR), major kongenitalnim anomalijama i djeca rođena od dijabetičnih majki. Antenatalni kortikostero-idni tretman je proveden kod 80 od ukupno 172 djece, dok je 92 djece rođeno od majki koje prije poroda nisu dobile kortikosteroide. Težina respi-ratornog distres sindroma (RDS) klasificairana je prema frakciji O2 u inspiratornom zraku (FiO2).

Rezultati: Učestalost i težina RDS-a je bila si-gnifikantno niža u grupi djece antenatalno tretira-ne kortikosteroidima (22/80) u odnosu na kontrol-nu grupu (53/92) (p<0,001). Intra-periventrikular-na hemoragija (IVH-PVH) i stopa smrtnosti bili su značajno niži u grupi tretiranoj antenatalnim kor-tikosteroidima. Nije nađena statistički značajna razlika u pojavi sepse i bronhopulmonalne displa-zije (BPD) među ispitivanim grupama. Zaključak: Antenatalni kortikosteroidni tretman signifikantno smanjuje učestalost RDS-a i teškog RDS-a, IVH-PVH i neonatalne smrtnosti, pri čemu ne poveća-va rizik akvirirane sepse.

The effects of Antenatal corticosteroids on neonatal morbidity and mortality EfEkTI AnTEnATALnE kOrTIkOsTErOIdnE TErAPIjE nA nEOnATALnI mOrbIdITET I mOrTALITETMaksic H, Heljic S, Dizdarevic J, Misanović V, Catibusic F, Dzinovic A.

Neonatology Department of Pediatric Clinic, University Clinical Center of Sarajevo, Bosnia and Herzegovina



Ključne riječi: antenatalni kortikosteroidi, ne-donošče, morbiditet i mortalitet

Introduction

Preterm delivery occurs in 7 to 10% of all pre-gnancies, with the rate increasing in recent years. Respiratory distress syndrome (RDS) causes si-gnificant mortality and morbidity in these babi-es. Other causes are: intraventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), broncho-pulmonary dysplasia (BPD), sepsis, patent ductus arteriosus (PDA) and retinopathy of prematurity (ROP).

RDS occurs due to inadequate production of pulmonary surfactant in premarture lung and is seen if labour occurs before 32-34 weeks of pre-gnancy. (1) RDS is known to affect 40–50% of babies born before 32 weeks. (2)

Intra-periventricular hemorrhage (IVH-PVH) is the most frequent type of intracranial hemorrha-ge and the major cause of neurodevelopmental di-sabilities in preterm babies. Factors predisposing to IVH-PVH are gestational age, RDS, hypoxia, reperfusion, lesions of blood vessels, disturbances of the cerebral blood flow.

Evidence has been available since 1972 that the antenatal administration of corticosteroids prior to preterm delivery reduces the incidence of RDS, IVH-PVH and neonatal death (3). A Cochrane meta-analysis of 18 randomized trials confirmed such benefit of antenatal steroids.(4 ) There was evidence of benefit in all major subgroups of pre-term babies, irrespective of race or gender.

Dexamethasone and betamethasone are the preferred corticosteroids for antenatal therapy. Within Crowley’s meta-analysis, betamethaso-ne and dexamethasone were found to be equally effective in preventing RDS (5,6). However, a large observational study suggested that antenatal exposure to betamethasone, but not dexametha-sone, is associated with a decreased risk of cystic periventricular leucomalacia among premature infants born at 24–31 weeks of gestation.(7,8,9) The RCOG Scientific Advisory Committee reco-mmends that betamethasone is the steroid of cho-ice to enhance lung maturation.(8) The treatment schemas shown to be effective are 2 doses of 12

mg of betametasone given intramuscularly 24 ho-urs apart or four doses of 6 mg of dexamethasone given intramuscularly 12 hours apart. A favorable outcome is expected if the delivery happens in the interval between 24 hours and 7 days, a maximum of 10 days after the administration of corticoste-roids. Whether this therapy increases the risk of either neonatal or maternal infection is not clear enough (10).


This study included 172 premature babies between 26 and 34 gestational weeks of age, ad-mitted to NICU, Pediatric Clinic, Clinical Univer-sity Center Sarajevo, during two years period. All babies in the study were divided into two groups: the experimental (corticosteroid) group (80/172) in which the babies were treated with corticoste-roids prenatally and a control group (92/172) in which the babies did not receive such treatment (non-corticosteroid group). The severity of RDS was classified according FiO2 requirements. RDS was classified into three categories: 1) mild RDS with FiO2 requirement < 0.4 or NCPAP, 2) mode-rate RDS with FiO2 requirement 0.4-0.8, and 3) severe RDS with FiO2 requirement > 0.8 (mode-rate and severe RDS required mechanical ventila-tion and surfactant replacement). IVH-PVH was diagnosed by ultrasound and classified by Papile. Sepsis was diagnosed by positive blood culture. The criterion for BPD was the dependence on oxygen for longer then 28 days.

Standard statistical parameters were used: de-termination of means and standard deviations for normally distributed variables, median and range for variables without a normal distribution, t-test for continous variables, and x2-test for categorical and dichotomous variables.

Results

A summary of patients data enrolled in this study and our results are given in next tables.

There is no statistically sigificant difference in male/female ratio between corticosteroid and non-corticosteroid group. (c2test 0,619)

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The frequency of RDS was significantly lower in corticosteroid group (22/80) compared to the non-corticosteroid group (53/92) (c2 test p<0,001). Severe RDS was significantly more frequent in the non-corticosteroid group 34/53 (64.15%) than in the corticosteroid group 6/22 (27.27%).

Discussion

We found significant decrease in RDS in the infants treated with antenatal corticosteroids, which is consisted with other published studies. Severe RDS was also significantly less frequent (p<0.001) in infants whose mothers received ste-roids before delivery. The steroids given to mot-hers passes across blood placental barrier and act upon the pneumocytes type II of lung, inducing production of surfactant and these help in preven-ting the RDS. The favorable effect on antenatal corticosteroids has been known from 1972, from studies of Liggins and coworkers (3). The official

recommendation for its application was given by American Institute for Health in 1994., after the analysis of all published data (11). A Cochrane meta-analysis of 18 randomised trials indicates that antenatal corticosteroid therapy reduces the incidence of RDS, neonatal death and intraventri-cular haemorrhage (4,11). The efficacy of neona-tal surfactant therapy is enhanced by antenatal ex-posure to corticosteroids (5). Crowley’s Cochrane review showed a statistically significant reduction in RDS in preterm babies born before 34 weeks of gestation (10,12).

Our study confirmed significant reduction of severe IVH-PVH (grade III and grade IV accor-ding Papile) in antenatally treated babies. The ef-fect of corticosteroids on fetal brain is not clear enough, but it is well known that favorable effects of antenatal corticosteroids on IVH-PVH have incomplete correlation with improvement of pul-monal morbidity. Probably these favorable effects were the consequence of cerebral blood flow sta-bilization and steroid induced maturation of vas-cular structure in germinal matrix (13,14).

Table 1. Male/female ratio in corticosteroid and non-corticosteroid groupcorticosteroid group

(n=80)non-corticosteroid group

(n=90)n (%) n (%)

male 48 (60.0) 51 (55,4)female 32 (40.0) 41 (44,6)

Table 2. Frequency and severity of RDS-a in corticosteroid and non-corticosteroid groupcorticosteroid group

(n=80)non-corticosteroid group

(n=90)RDS n n p

RDS severe 6 34 p< 0.001 signRDS moderate 5 10 p = 0.398 no sign

RDS mild 11 9 p = 0.813 no signRDS total 22 53 p< 0.001 signi

Table 3. Morbidity and mortality of the 2 groups

Characteristicscorticosteroid group

(n=80) non-corticosteroid group pn (%) n (%)

Sepsis 16 (20.00%) 22 (24.00%) p = 0.665 no signIVH-PVH (gr.III i IV) 3 (3,75 %) 20 (21,7 %) p = 0.001 sign

BPD 4 (5,00%) 7 (7,6 %) p = 0.700 no signDead 9 (11,25%) 28 (30,43%) p<0.001 sign



Our results showed that there were no signi-ficant differences in sepsis between both groups. As basic hormones, corticosteroids have an im-pact on the immune system; they stimulate natu-ral killer cells which are important components of the immunologic defense against viral infections, and conversely, inhibit the proliferation of T ce-lls. This leads to an inappropriate cellular immu-ne response. According to some reports, children prenatally treated with corticosteroids have higher incidence of infections during the first year of life (15). One randomised trial of single versus weekly courses of corticosteroids involving 502 pregnan-twomen between 24 and 32 weeks of gestation concluded that weekly courses of antenatal corti-costeroids did not reduce composite neonatal mor-bidity compared with a single course of treatment. (16,17). Driul et al. report no significant influence of prenatal corticosteroid therapy on the inciden-ce of perinatal infection (18). Similarly, our study demonstrates no significant difference in the inci-dence of infection between the experimental and control groups of newborns.

Chronic lung disease is multifactorial conditi-on, resulting from the interaction of many risk fac-tors such as prematurity, mechanical ventilation, high-concentration oxygen exposure, and infla-mmatory reactions in immature lungs. According to the literature, incidence of BPD is 5-20% de-pending on gestation and birth weight (19). In our study we found no significant difference in BPD between the studied and control groups.

Mortality in the non-corticosteroid group was significantly higher and resulted from sepsis, se-vere respiratory distress, air leak, pulmonary he-morrhage and necrotizing enterocolitis.

Conclusion

Antenatal corticosteroid therapy reduces the incidence of mortality, respiratory distress syndro-me, and intraventricular hemorrhage in preterm infants without increasing the risk of sepsis.

References

1. Slattery MM, Morrison JJ. Preterm delivery. Lan-cet 2002;360: 1489–97.

2. Chiswick M.Antenatal TRH. Lancet 1995; 345: 872–3.

3. Liggins GC, Howie RN. A controlled trial of ante-partum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature in-fants. Pediatrics 1972;50: 515–25.

4. Crowley P. Prophylactic corticosteroids for pre-term birth.Cochrane Database Syst Rev 2002; (4): CD000065.

5. Jobe AH, Mitchell BR, Gunkel JH. Beneficial ef-fects of the combined use of prenatal corticostero-ids and postnatal surfactant on preterm infants. Am J Obstet Gynecol 1993;168: 508–13.

6. Jobe AH, Mitchell BR, Gunkel JH. Beneficial ef-fects of the combined use of prenatal corticostero-ids and postnatal surfactant on preterm infants. Am J Obstet Gynecol 1993;168: 508–13.

7. NIH Consensus Development Panel on the Effect of Corticosteroids for Fetal Maturation on Perinatal Outcomes.Effect of corticosteroids for fetal matu-ration on perinatal outcomes. JAMA 1995; 273: 413–18.

8. Crowley PA. Antenatal corticosteroid therapy: a metaanalysis of the randomized trials, 1972 to 1994. Am J Obstet Gynecol 1995; 173: 322–35.

9. Baud O,Foix-L’Helias L,Kaminski M,Audibert F, Jarreau PH, Papiernik E, et al. Antenatal gluco-corticoid treatment and cystic periventricular leu-komalacia in very premature infants. N Engl J Med 1999; 341: 1190–6.

10. Royal College of Obstetricians and Gynaecolo-gists. Intrauterine Infection and Perinatal Brain Injury.

11. NIH Consensus Development Panel on the Effect of Corticosteroids for Fetal Maturation on Peri-natal Outcomes.Effect of corticosteroids for fetal maturation on perinatal outcomes. JAMA 1995; 273: 413–18.

12. Scientific Advisory Committee Opinion Paper 3. London:RCOG;2002.

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13. Neilson JP. Cochrane update: Antenatal cortico-steroids for accelerating fetal lung maturation for women at risk of preterm birth. Obstet Ginekol. 2007; 109(1):189-190

14. Abbasi S, Hirsch D, Davis J, et al. Effect of single versus multiple courses of antenatal steroids and outcome of premature infants. Am J Obstet Gyne-col 1999;181:709-17

15. Abbasi S, Hirsch D, Davis J, et al. Effect of single versus multiple courses of antenatal steroids and outcome of premature infants. Am J Obstet Gyne-col 1999;181:709-17

16. Guinn DA,Atkinson MW, Sullivan L, Lee M, Mac-Gregor S, Parilla BV, et al. Single vs weekly cour-ses of antenatalcorticosteroids for women at risk of preterm delivery: A randomized controlled tri-al. JAMA 2001; 286: 1581–7.

17. Vermillion ST, Soper DE,Newman RB. Neonatal sepsis and death after multiple courses of antena-tal betamethasone therapy. Am J Obstet Gynecol 2000; 183: 810–14.48.

18. Druil L, Furlan R, Makagno F, et al. Induction of fetal lung in prevention of hyaline membrane di-seases. Conection with neonatal sepsis. Minerva Ginecol 2003;(1):37-44

19. Bancalary E, et al. Bronchopulmonary dysplasia. Pediatr Clin North Am 1989; 33:1,

Corresponding author Maksic H, Neonatology Department of Pediatric Clinic, University Clinical Center of Sarajevo, Bosnia and Herzegovina, E-mail: [email protected]



Effect of Weak static magnetic fields on the number red blood Cells into fattening Turkey Peripheral blood samples under In Vitro and In Vivo ConditionsefekaT slaBIH sTaTIČkIH magneTskIH PolJa nA brOj ErITrOCITA u uzOrCImA PErIfErnE krvI ćurkI u Tovu u In vIvo I In vITro uVjETImAZijad Muharemovic1, Muhamed Katica2, Jasmin Musanovic3, Nejra Hadzimusic2, Sabina Catic4, Kenan Caklovica5, Dzelil Korkut6

1 Medical Faculty, University of Sarajevo, Biophysics, Bosnia and Herzegovina2 Faculty of Veterinary Medicine, University of Sarajevo, Pathological Physiology, Bosnia and Herzegovina3 Medical Faculty, University of Sarajevo, Biology and Human Genetics, Bosnia and Herzegovina4 Institute for Clinical Chemistry and Biochemistry, Clinical Center University of Sarajevo, Bosnia and Herzegovina 5 Faculty of Veterinary Medicine, University of Sarajevo, Hygiene and Food Technology Bosnia and Herzegovina6 Clinical Center of University of Tuzla, Neurosurgery, Bosnia and Herzegovina

Abstract

This study focuses on influence of non-ionizing weak static magnetic field (WSMF), on number of red blood cells (RBCs) into 20 fattening tur-key peripheral blood samples (PBSs) from British United Turkey 600 hybrids (BUT 600), under in vitro and in vivo conditions. WSMF is referred to intensities from 100nT to 0.5mT. For 8 turkeys, peripheral blood samples were placed inside sole-noids, through which constant direct current was passed, and were exposed to magnetic fields (MFs)

for 2 hours (h), with intensities: TB 41 0150,1 -⋅≈

, TB 42 0186,1 -⋅≈ , TB 4

3 0151,3 -⋅≈ ( 1B < 2B <

3B ). Peripheral blood samples of 12 other turkeys were taken after they have been exposed to WSMF

with approximately the same intensities: 1B , 2B , 3B

as in vitro case, for 2, 4 and 5 hours. Peripheral blo-od samples prior to WSMF exposure was taken as the control group. Determination of the number of RBCs was performed at the Institute for Clinical Chemistry and Biochemistry, at the Clinical Center of the University of Sarajevo within a period of one hour after taking the blood. For in vitro samples, statistical significance was recognized at p<0,05 and p<0,025 between the control samples and sam-ples that were exposed to magnetic fields 1B and 2B, respectively. There was no statistical significance between the mean value of control samples and the

mean value of samples exposed to WSMF 3B for two hours.

For measurements of the number of RBCs in the case of in vivo blood samples, the same sta-tistically significant differences of mean values p<0,05 were found between:

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- control group samples and samples exposed to a magnetic field 1B for 2 hours (-18,65 %), 4 hours (- 11,27 %) and 5 hours ( - 16,29 %),

- samples exposed to a magnetic field 2B for 2 and 4 hours, ( -5,04 %) and between samples exposed to a magnetic field 2B for 2 and 5 hours (-5,77 %), and

- control group samples and samples

exposed to a magnetic field 3B for 4 hours (-11,70 %) and 5 hours (-9,60 %).

Key words: static weak magnetic field, red

blood cells, turkey

Sažetak

Ova studija se fokusira na uticaj neioniziraju-ćeg slabog statičkog magnetskog polja, na broj crvenih krvnih ćelija u uzorcima periferne krvi 20 hibridnih ćurki British United Turkey 600 (BUT 600) u tovu, u in vitro i in vivo uvjetima. Za slabo statičko magnetsko polje se uzima opseg inten-ziteta od 100nT do 0.5mT. Uzorci periferne krvi 8 ćurki bili su smješteni unutar zavojnice (sole-noida), kroz koji je tekla konstantna istosmjerna struja, i u trajanju od 2 sata bili izloženi uticaju slabih statičkih magnetskih polja različitih in-

tenziteta: TB 41 0150,1 -⋅≈ , TB 4

2 0186,1 -⋅≈

, TB 43 0151,3 -⋅≈ . Uzorci periferne krvi osta-

lih 12 ćurki bili su uzeti nakon što su ćurke bile izložene uticaju slabih statičkih magnetskih po-lja s približno istim intenzitetima, kao i u in vitro slučajevima izloženosti u trajanju od 2, 4 i 5 sati. Uzorci periferne krvi prije izlaganja uticaju slabih statičkih magnetskih polja su uzeti kao kontrolni uzorci. Određivanje broja crvenih krvnih zrnaca je provedeno na Institutu za kliničku hemiju i biohe-miju, Kliničkog centra Univerziteta u Sarajevu u roku od jedan sat nakon uzimanja krvi.

Za in vitro uzorke, nađeno je statistički značaj-no odstupanje p <0,05 i p <0,025 između kontrol-nih uzoraka i uzoraka koji su bili izloženi uticaju slabih statičkih magnetskih polja 1B i 2B , respek-tivno. Nije uočeno statistički značajno odstupanje

između srednjih vrijednosti kontrolnih uzoraka i uzoraka koji su bili izloženi uticaju slabog statič-

kog magnetskog polja 3B . Za in vivo mjerenja, broja crvenih krvnih zrna-

ca u uzorcima krvi ćurki izloženih slabim magnet-

skim poljima 1B , 2B i 3B sa različitim trajanjima ekspozicije, iste statistički značajne razlike srednjih vrijednosti p <0,05 bile su uočene između:

- kontrolnog uzorka i uzoraka izloženih magnetskom polju

1B u trajanju 2 sata (-18,65 %), 4 sata (- 11,27 %) i 5 sati (- 16,29 %) ,

- uzoraka izloženih magnetskom polju 2B u trajanju od 2 i 4 sata, (- 5,04 %), kao i u slučaju uzoraka izloženih magnetskom polju 2B u trajanju od 2 i 5 sati (- 5,77 %), i

- kontrolnog uzorka i uzoraka izloženih

magnetskom polju 3B u trajanju od 4 sata (- 11,70 %) i 5 sati (- 9,60 %).

1. Introduction

There is a rapid development of technologies using static magnetic fields (headphones, telepho-ne speakers, medical implants, MRI …). The au-tomation medical and research instruments which generate magnetic fields are widely diffused in re-cent years, and the people are frequently exposed to it. Despite that the study of the effect of electro-magnetic fields on living organisms is a complex problem, but it is of more interest to give insight into the expected hazards and the proper ways of its use and protection. The term ‘‘weak magnetic field’’ is generally referred to the intensities from 100 nT to 0.5 mT [1]. A static magnetic field (SMF) is a force field created by a magnet or by the steady flow of electricity, for example in appli-ances using direct current (DC). Whereas electric field represents forces at the surface of molecules, cell membranes and even the whole body, magne-tic field penetrates inside the cell and influence even chemical and biochemical reactions [2].

Laboratory studies on cells and whole orga-nisms are useful for understanding interaction



mechanisms between biological tissue and sta-tic magnetic fields and can indicate what sorts of effects might be investigated in plants, animals, and humans. Research of processes by which the body regulates both its internal environment and its interactions with the external environment are exceedingly complex and requires the collaborati-on of experts familiar with a wide variety of biop-hysics, biology and medicine.

The results of these studies suggest the existen-ce of significant impact of WSMF on biological systems [3]. Recent plant and animal studies con-firm earlier findings that SMF of several mT may change membrane properties[4],[5]. Also there is evidence that the SMF with diferent inensities and exposur times:

- penetrates the living organism and act ions on all organs, altering the cell membrane potential and the distribution of ions and dipoles exert a preponderant controlling influence on the thermoregulation, metabolism and hematology in rats [6],

- induce change in hematocrit, hemoglobin, plasma fuel metabolites and tissue enzymes releases within the blood [7], [8],

- might affect gene expression and relevant functions in cells [9],

- have influence on pain reduction in mice [10],

- have influence on gradual loss of body weight, decrease glucose and total protein levels and alkaline phosphatase activity in serum, decrease of the numbers of monocytes, platelets, peripheral and splenic lymphocytes and increased the number of granulocytes in exposed mice [11],

- have influence on increasing in cell number, cell and nuclei size, number of nucleoli in the nuclei, and size of corpora allata in protocerebral neurosecretory neurons exposed pupae of the mealworm Tenebrio molitor [12],

- have influence on excitation and inhibition of neurons of the beetle Morimus funereus[13].

In some in vitro studies using cultured cell li-nes, exposure to magnetic fields with intensities

of several hundreds millitesla resulted in altered gene expression or DNA damage [14], while in others, exposure to much stronger SMF such as those used in clinical MRI (up to several tesla) did not cause any effects [15].

According to our knowledge, there are no pa-pers devoted to study the influence of WSMF on hematological parameters of turkeys. There are only a few papers dealing with research on the im-pact of electromagnetic fields on serum extracted from turkey’s gizzard [16].

A numerous hypotheses for molecular mecha-nisms of the biological effect of electromagnetic fields have been proposed. Proposed mechanisms include induced electric currents, direct effect on magnetic biological materials, effects on free ra-dicals, and excitation of cell membranes etc, but none have provided a reliable and comprehensive explanation of the experimental findings [17].

The difficulty in explaining these effects is usu-ally related to the fact that the involved energies are essentially less than the characteristic energy of chemical conversions, of the order of dozens of kT. In the literature this is known as kT para-dox. Meanwhile, in 2007. Binhi and Rubin [18] showed that biological effects of weak ELF ma-gnetic fields are not at variance with physical laws and that magnetobiological effects may be expla-ind in terms of classical and quantum physics.

2. Material and methods

2.1. Animal and exposure scenario

Analysis of the influence of different ranges of WSMF on RBCs was performed on twenty fatte-ning turkey peripheral blood samples from British United Turkey 600 hybrids (BUT 600) under in vi-tro and in vivo conditions. The animals were kept in clean and in hygienic environment at the Facul-ty of Veterinary Medicine, Department of Physio-logical Pathology. For in vitro and for in vivo sam-ples, blood samples were taken by venipuncture technique from vena brachialis directly into the tubes with EDTA as anticoagulant for RBCs.

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2.2. Exposure system for in VITRO samples

Magnetic field generator for in vitro samples has been desingned and constructed at the Depar-tment of Biophysics, Faculty of Medicine, Uni-versity of Sarajevo, Bosnia and Herzegovina.

Peripheral blood samples were placed in the middle of the coils to get homogenous magnetic field and held at 4°C [19]. Influencies of other magnetic fields (earth’s magnetic fields, magne-tic field of refrigerator) were neglected because its intensities were too weak [20]. The magne-tic fields for solenoids were calculated, while the magnetic susceptibility of deoxygenated red blood cells of turkeys was taken in the value of: χ (deox

RBCs) ≈ 5,928 601 -⋅ [21]. In the case of eight examined turkeys, peripheral blood samples were placed inside solenoid, through which constant direct current was passed, and they were expo-sed to the impact of magnetic fields for two ho-

urs, with following intensities: TB 41 0150,1 -⋅≈

, TB 42 0186,1 -⋅≈ and TB 4

3 0151,3 -⋅≈ . Peripheral blood samples prior to WSMF ex-

posure were taken as the control group.

2.3. Exposure system for in VIVO samples

Magnetic field generator for in vivo samples has also been desingned and constructed at the Bi-ophysics Department, Faculty of Medicine, Uni-versity of Sarajevo. Twelve other turkeys, divided into three groups, each consisting of four turkeys, were placed in the middle of the coils to get homo-genous weak static magnetic field with approxi-

mately the same intensities: 1B , 2B , 3B as in vitro case, for 2, 4 and 5 hours. After exposure perip-heral blood samples were taken from a vena bra-chialis. Peripheral blood samples, prior to WSMF exposure, were taken as the control group.

2.4. Biochemistry

The number of RBCs in the blood was deter-mined at the Institute for Clinical Chemistry and Biochemistry, at the Clinical Center of the Univer-

sity of Sarajevo by using an Architect ® CI 8200 analyzer within a period of one hour after taking the blood. All in vivo and in vitro blood samples were kept at 4°C during experiment and during transport [19].

2.5. Statistical analysis

Data were analyzed by Student’s t test. All the-se analysis were performed by home made spe-cifically designed Microsoft Excel Sample Sheet.

3. Results

To our knowledge, this investigation of the in-fluence of weak static magnetic fields on hemato-logical parameters in hybrid turkeys BUT 600 is performed for the first time in Bosnia and Herze-govina and beyond. Specificity related to the avian blood compared with human blood is that in the-ir erythrocyte exists nucleus (Figure 1). Hence, it can be easier to identify damages to the cells after exposure to weak static magnetic fields (Figure 2).

Figure 1. Turkey’s blood (nucleus can be seen in erithrocites)



Figure 2. Blood sample after WSMF exposure for 2 hours (cell without nucleus can be seen) (Photographies taken by Muharemovic Zijad)

3.1. Results IN VIVO

These results showed that exposure to WSMF originated hematological disruption, which appe-ared to be related to the exposure times. Exposu-re to WSMF (2h/day, 4h/day, 5h/day) induced a decrease number of red blood cells in almost all cases (Graphics 1, 2 and 3).

Graphic 1. Number of red blood cells for IN VIVO samples under influence of 1B magnetic field for 2,4,5 hours

Graphic 2. Number of red blood cells for IN VIVO samples under influence of 2B magnetic field for 2,4,5 hours

Graphic 3. Number of red blood cells for IN

VIVO samples under influence of 3B magnetic field for 2,4,5 hours

For in vivo measurements, the numbers of RBCs in the turkey peripheral blood samples are decre-

ased under influence of WSMF TB 41 0150,1 -⋅≈

compared with the numbers of RBCs in control samples, as follows:

– 18,65% (p< 0,05), for 2 hours exposure time,

– 11,27% ( p< 0,025), for 4 hours exposure time, and

– 16,29% (p< 0,025), for 5 hours exposure time. (Graphic 4)

It should be emphasized that the greatest decre-ase in the number of red blood cells in the turkey peripheral blood samples (-18,65%) occured by the influence of the weakest magnetic field 1B (

1B < 2B < 3B ) and with the shortest exposure time of 2 hours (Graphic 4).

Under the influence of the magnetic field

TB 42 0186,1 -⋅≈ ( 2B > 1B ) there were no stati-

sticaly significant differencies between the control

694



sample and other samples. The number of RBCs in turkey peripheral blood samples under the influ-ece of WSMF 2B showed changes in following cases:

– 5,04 % (p< 0,025), between samples that were exposed to a WSMF for 2 and 4 hours,

– 5,77 % (p< 0,05), between samples that were exposed to a WSMF for 2 and 5 hours (Graph. 4).

The number of RBCs in turkey periphe-ral blood samples under the influece of WSMF

TB 43 0151,3 -⋅≈ ( 1B < 2B < 3B ) compered with

the number of RBCs in control sample are decre-ased in cases:

- 11,70 % (p< 0,025), for 4 hours exposure time,

- 9,60 % (p< 0,025), for 5 hours exposure time. (Graph. 4).

There were no statisticaly significant differen-cies between the mean value of the control group samples and the mean value of the group samples

exposed to WSMF TB 43 0151,3 -⋅≈ ( 1B < 2B <

3B ) for two hours.

Graphic 4. Changes of number o RBCs in turkey peripheral blood samples compered with number of RBCs in the control samples, for different weak

magnetic fields 1B , 2B , 3B and diferent exposure time is shown in percents ( IN VIVO )

3.2. Results IN VITRO

The results of in vitro studies are useful for elu-cidating interaction mechanisms, and for indica-ting the sorts of effects that might be investigated in vivo. However, they are not sufficient to iden-

tify health effects without corroborating evidence from in vivo studies. Statistical significance was recognized at p< 0,05 and p < 0,025 between control group samples and samples that were ex-

posed to a magnetic fields: TB 41 0150,1 -⋅≈ and

TB 42 0186,1 -⋅≈ ( 2B > 1B ) respectively, while

there was no statistical significance between con-trol group samples and samples that were exposed

to the week static magnetic field TB 43 0151,3 -⋅≈

( 1B < 2B < 3B ) (Graph. 5).

Graphic 5. Number of red blood cells for IN VI-

TRO samples under influence of 1B , 2B and 3B magnetic fields

The number of RBCs in turkey peripheral blood samples (IN VITRO) under influnece of WSMFs:

1B and 2B ( 1B < 2B ) compared with the number of RBCs in control sample are decreased as follows:

- 7,12 % (p< 0,05), for WSMF

TB 41 0150,1 -⋅≈ with exposure time of 2

hours,- 7,28 % (p< 0,025), for WSMF

TB 41 0150,1 -⋅≈ with exposure time of 2

hours.

There was no statistical significance between the control group samples and the samples exposed to

the weak static magnetic field TB 43 0151,3 -⋅≈

( 1B < 2B < 3B ) (Graph. 6).



Graphic 6. Changes of RBCs counts in percents under the influence of the magnetic fields ( 1B

=0,105mT; 2B =0,168mT; 3B =0,315mT) for two hours (IN VITRO)

4. Discussion

We would like to stress that our pioneering re-search of the effects of weak static magnetic fields on hematological parameters in birds, especially turkeys, showed significant differences from pre-vious results obtained during the testing of red blood cells count of mice.

In Salama’s paper [7] can be seen that under the influence of the static magnetic field of 128mT intensity (which is significantly higher intensity then we used in our measurements (0,1-0,4mT)) observed increase in number of red blood cells of mice (+7%), whereas our in vivo results show a si-gnificant decrease in the number of red blood cells of (- 18, 65%) for the weakest magnetic field 1B .

These differences in results can be explained by the specificity of the investigated population (sample), because it is known that birds have a greater sensitivity to magnetic field.

Our in vivo and in vitro investigations on the influence of weak static magnetic fields with ex-posure time of two hours in the peripheral blo-od samples of fattening turkeys, show existence of the threshold level for magnetic fields with

intensity from TB 41 0150,1 -⋅≈ (0,105mT) to

TB 42 0186,1 -⋅≈ (0,168mT). It is in accordance

with the empirical results of Weintraub which is suggested that the minimum threshold for magne-tic field detection by the receptors of in vivo bio-logical targets be about 0,5mT [22], and also with our results we’ve got in researches on genotoxic impact of weak static magnetic fields on mitosis

in Allium cepa’s cells [4]. Similar results we’ve presented in our paper about the influence of the weak static magnetic fields on the haemoglobin concentration into fattening turkey periferal blood samples under in vitro and in vivo conditions [23].

Decreasing in red blood cells count could be explaned through facts that weak static magnetic fields can selectively remove calcium from cell membranes, which would reduce their stability [24].

Increase of the number of red blood cells for in vivo samples, influenced by the magnetic field 1B for a period of 4 hours, and the absence of changes

under the influence of magnetic fields 2B and 3B for exposure time of two hours, can be explained by the fact that living systems have a great capa-city to compensate the effects induced by exter-nal influences, including electromagnetic sources [25].

Finally, for better understanding of this pheno-menon, it should also take into account stress that turkeys had during the experiment, but we did not have conditions for measuring it.

5. Conclusions

Our in vitro and in vivo results show that sta-tic weak magnetic fields cause some noticeable or detectable changes in hematological parame-ters of animal models. Also, they show existen-ce of the threshold level of red blood cells for weak static magnetic fields with inensities from

TB 41 0150,1 -⋅≈ (0,105mT) to TB 4

2 0186,1 -⋅≈ (0,168mT). So, our results can be used as impor-tant information in analyses of potentially harmful or useful effects of these types of radiations to hu-mans, which appeared to be related to the intensity and exposure times of WSMF.

Undoubtedly, for the better understanding this extreme complex and multilevel phenomena, more researches are necessary, especially, to cla-rify the many mixed and sometimes contradictory results.

696



References

1. Biological effects due to weak magnetic field on plants N.A. Belyavskaya / Advances in Space Rese-arch, 2004, 34, 1566–1574

2. Magnetic field therapy: a review., Markov MS, Electromagn Biol Med, 2007;26:1–23

3. A study of magnetic field effects on fibroblasts cul-tures, Part 2: The evaluation of effects of static and extremely low frequency (ELF) magnetic fields on free radical processes in fibroblasts cultures, Bioe-lectrochem. . Kula, B., M. Drozdz Bioenerg., 1996, 39, 27–30

4. Genotoxic impact of weak magnetic fields on mitosis in Allium cepa’s cells, Musanovic J.,Muharemovic Z.,Metovic A., Filpovska-Musanovic M.; Folia Me-dica, 2009, Vol44.No-1suppl.

5. Cellular and molecular pathways of extremely low frequency electromagnetic field interactions with living systems. Tenforde TS. U: Blank M, ur. Elec-tricity and magnetism in biology and medicine. San Francisco (CA): San Francisco Press, 1993. str. 1–8.

6. Acute exposure to magnetic field depresses shive-ring thermogenesis in rat, Abdelmelek, H., S. Cha-ter, M. Sakly Biomedizinische Technik, Band 46, Ergänzungsband 2, 2001, 2, 164–166.

7. Effects of Static Magnetic Field Exposure on He-matological and Biochemical Parameters in Rats Salem Amara, Hafedh Abdelmelek, Mohamed Ben Salem, Rached Abidi and Mohsen Sakly1 Brazilian Archives of Biology and Technology, 2006, Vol.49, n. 6: pp. 889-895

8. Effects of sub-acute exposure to magnetic field on blood hematological and biochemical parameters in female rats ., Scihem, C., A. Hafedh, S. Mohsen, P.J. Mar, B.R. Khmais, Turk. J. Hematol., 2006, 23, 182–187

9. Brief exposures to weak static magnetic field du-ring early embryogenesis cause cuticular pattern abnormalities in Drosophila larvae. Ho MW et all. Phys Med Biol. 1992,; 37(5):1171-9

10. “Optimization of static magnetic field parameters improves analgesic effect in mice,” László J. et al. Bioelectromagnetics, 2007, Vol. 28, No. 8, 615-627

11. Assessment of biological changes of continuous whole body exposure to static magnetic field and extremely low frequency electromagnetic fields in mice. Hashish AH, et all., Ecotoxicol Environ Saf., 2007; 71:895-902

12. The effect of a static magnetic field on the mor-phometric characteristics of neurosecretory ne-urons and corpora allata in the pupae of yellow mealworm Tenebrio molitor(Tenebrionidae), Pe-ric-Mataruga et al., International Journal of Ra-diation Biology, 2008, Vol. 84, No. 2, Pages 91-98

13. A method for detecting the effect of magnetic fi-eld on activity changes of neuronal populations of Morimus funereus (coleoptera, cerambycidae), Todorovic et al., Bioelectromagnetics, 2007, Volu-me 28 Issue 3, Pages 238 – 241

14. Influence of a static magnetic field (250mT) on the antioxidant response and DNA integrity in THP1 cells, Salem Amara et al 2007 Phys. Med. Biol. 52, 889

15. In vitro evaluation of magnetic resonance ima-ging at 3.0 tesla on clonogenic ability, prolife-ration, and cell cycle in human embryonic lung fibroblasts, Schwenzer et al. Invest Radiol 2007a; 42:212-217

16. Effects of Non-Uniform Static Magnetic Fields on the Rate of Myosin Phosphorylation, Engstrom et al., Bioelectromagnetics, 2002, 23:475-479

17. Swanson J, Kheifets L. Biophysical mechanisms: a component in the weight of evidence for health effects of power-frequency electric and magnetic fields. Radiat Res 2006; 165: 470–478

18. Magnetobiology: The kT Paradox and Posible Solutions, V.N.Binhi, Rubin A.B., Electromagnetic Biology and Medicine, 2007, 26: 45-62

19. Effect of Temperature Storage on Hematological Parameters of Avian Turkey Blood, Hadzimusic N.,Katica M., Muharemovic Z., Musanovic J., International Journal of Collaborative Research on Internal Medicine & Public Health, Vol.2 No.5 (May 2010) pp.158-156

20. Introduction to Magnetic Materials, Second editi-on, by B.D.Cullity and C.D.Graham 2009.



21. Water Proton MR Properties of Human Blood at 1.5 Tesla: Magnetic Susceptibility, T1, T2, T*2, and Non- Lorentzian Signal Behavior William M. Spees, et all., Magnetic Resonance in Medicine, 2001, 45:533–542

22. Weak static magnetic fields reduce neuropathic pain dependet upon field strength and penetrati-on, Weintraub,M.I., Bioelectromagnetics 2005, Vol.20-9, 232

23. Effects of weak magnetic fields on the hemoglobin concentration in fattening turkey peripheral blo-od samples under in vitro and in vivo conditions, Muharemovic Z et al. Abstract book,for The First Conference of Medical biocemists and chemists, 2010, Sarajevo, BiH

24. Interaction between weak low frequency magnetic fi elds and cell membranes, Koch BCL, Sommarin M, Persson BR, Salford LG, Eberhardt JL. Bioe-lectromagnetics 2003; 24: 395–402

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Corresponding author Zijad Muharemovic Medical Faculty, University of Sarajevo, Biophysics, Bosnia and Herzegovina E-mail: [email protected]

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Objective

The purpose of this article is to show how li-festyle can improve cholesterol levels. It purports to illustrate the steps that can be taken to control cholesterol starting with awareness and following with diet, exercise, smoking cessation, stress re-duction, weight control and behavior change.

1st Lifestyle Aspect: Awareness

Awareness is the cornerstone of change. It im-plies knowing:

(1) Cholesterols levels (total, HDL and LDL) and checking them on a regular basis (yearly after 50 or earlier if there are any other cardio-vascular disease risk factors)

(2) Optimal cholesterol levels targets. Optimal cholesterol levels and current guidelines can be found on the American Heart Association website (go.heart.org).

It is also important to know that not only the LDL cholesterol goal depends on other risk factors (for example coronary heart disease and diabetes) but also that cholesterol levels vary with the following:

- Age: Cholesterol blood levels tend to increase with age.

- Weight: Overweight people are more likely to have high blood cholesterol levels. They also tend to have lower HDL levels. The location of the excess weight plays a role. A greater risk from increased cholesterol levels occurs when that extra weight is centered in the abdominal region.

- Gender: Men tend to have higher LDL and lower HDL levels than women especially before 50. After menopause, decreasing amounts of estrogen are thought to cause a rise in LDL levels.

- Genetics: Some people are genetically predisposed to having high levels of cholesterol. A variety of minor genetic defects can lead to excessive production of LDLs or decreased capacity for their removal. This tendency is often passed to offspring.

- Disease: For example, diabetes can lower HDL levels (it also increases triglycerides).

Similarly, metabolic syndrome, nephrotic syn-drome, hypothyroidism, anorexia nervosa, depre-ssion and some liver diseases can cause hypercho-lesterolemia.

Coronary heart disease in itself has the following risk factors: Family history, diet, stress, sedentary lifestyle, overweight, smoking, cholesterol (in par-ticular, pattern B with predominance of small and dense LDL), triglycerides, homocystein, lipoprote-in, fibrinogen and some pathologies.

High LDL cholesterol is responsible for 70% of heart disease, which is the leading killer of both men and women in the U.S.A. after 45. Low HDL-C is a potent independent cardiovascular risk fac-tor and potential therapeutic target. The Framing-ham study found that in healthy men and women aged 49 to 82 years, the most potent risk factor for coronary heart disease (CHD) was low HDL-C. Epidemiological data analysis show that for every 2-3% rise in HDL-C, the risk of CHD decreases by 2% in men and 3% in women independently of

Cholesterol and Lifestyle: The stanford Health Improvement Program (HIP) ExperienceYann A. Meunier,

Stanford Health Promotion Network, United States of America



LDL-C levels. This impact is similar to LDL-C re-duction. To date, one of the best clinical outcome data to support the benefits of raising HDL-C level comes from the Veteran Affairs HDL Intervention Trial (VA-HIT). The HDL Atherosclerosis Trea-tment Study (HATS) illustrates that combination therapy may provide independent and additive be-nefits for patients with CHD and low HDL-C. It is envisaged that the use of combination therapy or novel potent HDL-C raising drugs will help pro-vide irrefutable evidence that raising HDL-C is as important as lowering LDL-C for cardiovascular risk reduction.

2nd Lifestyle Aspect: Diet

You are what you eat. This old adage reflects reality as far as cholesterol is concerned. Although a fraction of cholesterol (specific to each indivi-dual) is synthetized de novo by the liver, contro-lling nutrition helps controlling cholesterol. The average US diet contains 37% of total calories as fat. The American Heart Association recommends that the proportion be reduced to 30%, yet a re-duction inferior to 10% may be needed to have a major effect on coronary artery disease risk. Three types of fat come from the diet: saturated, mono-saturated and polyunsaturated fatty acids (PUFAs) including omega-3 and omega-6 PUFAs. Diets high in omega-3 containing oils decrease the risk of sudden cardiac death. A Canadian study led by Dr. David Jenkins (St Michael hospital, Toronto) published in the Journal of the American Associa-tion showed that after 1 month of a vegetarian diet rich in vegetal sterols, soya proteins and almonds the LDL cholesterol decreased 28% i.e., as much as the group on statins. It also decreased the CRP levels (just like statins). Recommendations on healthy diet can be found on the American Heart Association website (go.heart.org). HIP recom-mends a Mediterranean-based diet and offers onli-ne and live classes, health promotion tools ([email protected]) and various educational materials ([email protected]) to help determine the best diet to control cholesterol levels.

NotePeople with a high level of C-reactive protein

(CRP) don’t receive the same beneficial reductions while on a low-fat, low-cholesterol diet as those with lower CRP levels.

3rd Lifestyle Aspect: Exercise

Five centuries B.C., Hippocrates knew that exercise is good for health. The first step is to find out how fit patients are. Online resources will give guidelines according to age and gender (for exam-ple, healthgoods.com). Regular exercise increases the HDL and decreases LDL levels. It also lowers triglyceride levels and blood pressure, reduces excess weight, improves heart and lung fitness and diminishes stress as shown on the National Institu-tes of Health website (health.nih.gov).

As a rule, to be in aerobic conditions one sho-uld be able to hold a conversation without being winded while exercising (walking, jogging, swi-mming, biking, rowing, etc), . HIP offers physi-cal activity classes, health promotion tools ([email protected]) and various educational materials ([email protected]).

4th Lifestyle Aspect: Smoking Cessation

Healthwise, smoking is a self-destructive habit. Regarding cholesterol, smoking cessation increa-ses the HDL level. Please note that Nicotine su-pply is not needed for people smoking less than 10 cigarettes per day and it is contra-indicated in case of drug interaction, in pregnant or breast-feeding women and in the adolescent. Also, Nicotine inha-lers and nasal sprays are superior to patches. It is best for patients to establish a smoking cessation plan with a physician. According to the Harvard medical school, the chance of success is higher if he/she follows the 4As guidelines, i.e., Asks the right questions, Advises the patient on the best op-tions, Assists in the patient’s journey and Arran-ges special consultations, if needed. The most su-ccessful approach has three prongs: (1) Nicotine substitutes for physiological dependence, (2) Bu-propion or Zyban* to control aggressiveness and bulimia and (3) Psychological advice for support

700



and determining the root cause. HIP offers on-line tobacco cessation classes (including a special program for tobacco chewers), health promotion tools ([email protected]) and various related edu-cational materials ([email protected]).

5th Lifestyle Aspect: Stress Reduction

Our body is not built to handle stress more than a few minutes and the flight-fight physiological state is only meant for us to face life-threatening emergencies. Reducing stress increases the HDL level. Sport, meditation, prayer, laughter, soothing music, reading, movies, yoga, Tai Chi, HeartMa-th, Mindfulness-based Stress Management, Reiki Healing, breathing techniques are all useful. HIP offers online and live stress reduction classes, he-alth promotion tools ([email protected]) and vari-ous related educational materials ([email protected]).

6th Lifestyle Aspect: Weight Control

Overweight is a handicap is many ways. Lo-osing weight increases the HDL level. One can loose weight by decreasing the caloric intake with a hypocaloric diet and/or increasing output thro-ugh aerobic exercise. HIP offers online and live weight control classes ([email protected]) and va-rious related educational materials through HPRC ([email protected]).

7th Lifestyle Aspect: Behavior Change

Most weight loss and nutrition programs do not produce results that can be maintained becau-se they do not include a behavior change compo-nent. HIP has created a 15 steps successful beha-vior change model that comprise the backbone of its various behavior change programs. This model includes 10 motivational assets that influence in-dividuals’ readiness for change and consequently their ability to achieve sustainable behavior chan-ge ([email protected]).

Conclusion

When hypercholesterolemia is not due to hepa-tic overproduction it can be controlled though the lifestyle modifications that have been described above. Encouraging and monitoring them effici-ently and widely would decrease drug use which would not only benefit patients but also reduce he-althcare costs significantly.

Corresponding author Stanford Prevention Research Center United States of America E-mail: [email protected] Website: http://shpn.stanford.edu



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STATISTIčKA ANALIZATestove koji se koriste u statističkim anaizama treba

prikazivati i u tekstu i na tabelama ili slikama koje sadrže statistička poređenja.

TABELE I SLIkETabele treba numerirati prema redoslijedu i tako ih

prikazati da se mogu razumjeti i bez čitanja teksta. Svaki stubac mora imati svoje zaglavlje, a mjerne jedinice (SI) moraju biti jasno označene, najbolje u fusnotama ispod tabela, arapskim brojevima ili simbolima. Slike također, treba numerisati po redoslijedu kojim se javljaju u tekstu. Crteže treba priložiti na bijelom papiru ili paus papiru, a crno-bijele fotografije na sjajnom papiru. Legende uz cr-teže i slike treba napisati na posebnom papiru formata A4. Sve ilustracije (slike, crteži, dijagrami) moraju biti origi-nalne i na poleđini sadržavati broj ilustracije, prezime pr-vog autora, skraćeni naslov rada i vrh slike. Poželjno je da u tekstu autor označi mjesto za tabelu ili sliku. Slike je potrebno dostavljati u TIFF formatu rezolucije 300 DPI.

KORIšTENJE KRATICAUpotrebu kratica treba svesti na minimum. Konven-

cionalne SI jedinice mogu se koristiti i bez njihovih de-finicija.

Preparation and physicochemical characterization of omeprazole:methyl-beta-cyclodextrin inclusion...

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Transcript of Preparation and physicochemical characterization of omeprazole:methyl-beta-cyclodextrin inclusion...