Frequency and characteristics of side effects associated with antidepressant drugs

2 Bosnian Journal of Basic Medical Sciences 2 (1-2) 2002.

EDITORIAL BOARD

Editorial in chief:Zuli} Irfan

Deputy editor:Selak Ivan

Secretary:Emina Naka{-I}indi}

Technical editor:Selvi} Faruk

Members:Dilberovi} FarukHadòvi} SafetHuski} JasminkoJadri}-Winterhalter MiraKulenovi} HuseinMihaljevi} MilenaNikolin BrankoPleho AmirPotkonjak DubravkaSinanovi} OsmanSu{i} Husein

ADVISORY BOARD:

Muji} Muzafer, PresidentBerberovi} LjubomirGruji}-Vasi} JelaHadòvi} SabiraHamamdì} MuhidinKonjhodì} FarukLoga SlobodanMulabegovi} NedàdNikulin AleksandarPuji} Zdravko

Contents:

EDITORIAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

FREQUENCY AND CHARACTERISTICS OFSIDE EFFECTS ASSOCIATED WITHANTIDEPRESSANT DRUGS . . . . . . . . . . . . . . . . . . . . . . . . . . .5(Jasna Kusturica, Irfan Zuli}, Svjetlana Loga-Zec,Ismet Ceri}, Slobodan Loga, Elvedina Kapi})

ISOLATION AND IMMUNOCHEMICALCHARACTERIZATION OF HUMAN CYSTITIS C . . . . . . . . . . .12(Lejla Begi}, Selma Berbi})

FREQUENCY OF THE ANTIPSYCHOTICTHERAPY ACUTE SIDE EFFECTS IN THETREATMENT OF ACUTE PSYCHOSIS . . . . . . . . . . . . . . . . . . .16(Svjetlana Loga-Zec, Irfan Zuli}, Nedàd Mulabegovi},Ismet Ceri}, Slobodan Loga, Saida Fi{ekovi},Jasna Kusturica and Sanja Kro{njar)

ASSESSMENT OF CHRONIC NEUROPSYCHOLOGICALEFFECTS OF MERCURY VAPOUR POISONING INCHLORAL-ALKALI PLANT WORKERS . . . . . . . . . . . . . . . . . . .29(Pranji} N, Sinanovi} O, Karamehi} J, Jakubovi} R)

ASSESSMENT OF HEALTH EFFECTS INWORKERS AT GASOLINE STATION . . . . . . . . . . . . . . . . . . . .35(Pranji} N, Mujagi} H, Nurki} M, Karamehi} J, Pavlovi})

MITOCHONDRIAL MEDICINE - A KEY TOSOLVE PATHOPHYSIOLOGY OF XXI CENTURY DISEASES .46(Emina Kiseljakovi}, Radivoj Jadri}, Sabaheta Hasi},Lorenka Ljuboja, Jovo Radovanovi}, Husein Kulenovi}and Mira Winterhalter)

MATHEMATICAL METHODS FOR QUANTIFICATIONAND COMPARISON OF DISSOLUTION TESTING DATA . . . .49(Edina Vrani}, Aida Mehmedagi}, Sabira Hadòvi})

FREQUENCY OF RH PHENOTYPES IN RELATIONTO THE OUTCOME OF PREGNANCY IN THE TWOGROUPS OF PREGNANT WOMEN . . . . . . . . . . . . . . . . . . . . .53(Amira Redì}, Fatima Begi})

POSTTRAUMATIC STRESS DISORDER(PTSD) AND CO-MORBIDITY . . . . . . . . . . . . . . . . . . . . . . . . . .57(Ifeta Li~anin, Amira Redì})

THE NEW TRENDS IN THEOPHYLLINE THERAPY . . . . . . . . .62(Aida Mehmedagi}, Edina Vrani}, Sabira Hadòvi},Milena Pokrajac, Jela Mili}, Bakir Mehi}, Hasan @uti},Abdulah Konjicija)

TRANSCRANIAL DOPPLERSONOGRAPHY AS DIAGNOSTIC METHOD . . . . . . . . . . . . . .66\elilovi}-Vrani} Jasminka

INSTRUCTIONS FOR PREPARATION OF MANUSCRIPTS . . .71

UDRU@ENJE BAZI^NIHMEDICINSKIH ZNANOSTIFBIH SARAJEVOASSOCIATION OF BASIC MED-ICAL SCIENCES FBIH SARAJEVO

Predsjednik: Prof. Dr. Irfan Zuli}

Adresa redakcije:Glavni i odgovorni urednik:Prof. Dr. Irfan Zuli}Medicinski fakultetInstitut za farmakologijuêkalu{a 90 SarajevoTel./Fax: 441 813, 441 895

3Bosnian Journal of Basic Medical Sciences 2 (1-2) 2002.

Editorial

We would like to inform our readers that the Association of Basic Medical Sciences published the second edition of“Bosnian Journal of Basic Medical Sciences”.

The Association of Basic Medical Sciences includes several Societies (Anatomists, Physilogists, Biologists,Pharmacologists etc.). This Association was founded during the war aiming to gather all scientists who stayed inSarajevo and continued their work under the most difficult conditiones. This is an excellent opportunity for young sci-entists to publish the results of their research.

We would kindly ask our readers to inform other collegues and associates about the Association and to offer them amembership.

All observations, suggestions and proposals made by our readers are wellcome and will improve the quality of theJournal.

We are quite confident that the following editions will offer more results of the research performed during the war espe-cialy about the stress influence on sensitive biological mechanisms.

We do hope that the scientists from whole Bosnia and Herzegovina will take part in the publishing of further editionsof our Journal.

Editorial Board

Sarajevo December 2002.

UDRU@ENJE BAZI^NIH MEDICINSKIH ZNANOSTI FEDERACIJE BOSNE I HERCEGOVINEASSOCIATION OF BASIC MEDICAL SCIENCES OF FEDERATION OF BOSNIA & HERZEGOVINA


Apstrakt

Depresija je jedno od naj~e{}ih hroni~nih oboljenja, zakoje se procjenjuje da }e do 2020. god., biti vode}iuzro~nik radne nesposobnosti i drugih ìvotnih tegoba.Depresivni sindrom se zbog toga mora ozbiljno shvatiti ilije~iti.

Tretman depresije podrazumjeva postizanje potpuneremisije, {to nije jednostavno posti}i, jer ve}ina pacije-nata prekida primjenu lijeka prije vremena. Jedan odnajve}ih razloga za prijevremeni prekid terapije je poja-va neugodnih neèljenih efekata.Cilj studije je bio istraìti dinamiku, incidencu i karak-teristike naj~e{}ih neèljenih efekata antidepresivnihlijekova primjenjenih u pacijenata sa depresijom, tokomhospitalizacije na ènskom odjeljenju Psihijatrijskeklinike Klini~kog centra u Sarajevu. Rezultati su pokazali da je tretman bio efikasan u ve}inepacijenata. Inhibitori ponovnog preuzimanja serotonina(SSRI) su imali manje izraène neèljene efekte u odno-su na tricikli~ne antidepresive (TCA). Naj~e{}i neèljeniefekti TCA (amitriptilin) su bili poreme}aj akomodacije,tahikardija, suho}a usta, tremor i sedacija, a najja~eizraèni suho}a usta, tremor i tahikardija. Neèljeni efek-ti SSRI (fluoksetin, fluvoksamin) su bili blagog inten-ziteta, naj~e{}e u vidu mu~nine, tahikardije, znojenja isuho}e usta.

Abstract

Depression is among the most common of chronic healthproblems. WHO report predicts that depression will bethe leading cause of disability in the industrial world bythe year 2020.

To be successful, treatment for the patients sufferingfrom depression must be continued until complete recov-ery, but most patients do not stay on their antidepressantmedication long enough. One of the most frequent rea-sons for break down is appearance of unpleasant sideeffects. In this study we followed up dynamics of the character-istic side effects of antidepressant therapy, with the majorgoal to assess their frequency and characteristics. Thesample was all female patients taking antidepressantdrugs in the Department of Psychiatry of Clinical Centreof University in Sarajevo.

The treatment with antidepressants was efficient in mostof the patients. A major advantage of SSRI over TCAwas less pronounced side effects. The most intensive sideeffects of TCA (amitriptyline) were dry mouth, tremorand tachycardia while the most frequent side effectsincluded blurred vision, tachycardia, dry mouth, tremorand sedation. Side effects of SSRI (fluoxetine/fluvoxam-ine) were mild, and the most frequent were nausea,tachycardia, swelling, dry mouth.

Introduction

Depression is an affective disorder, known from anticperiod. It is characterized by the mood changes as pri-mary clinical manifestation, which can lead to the otherserious mental health changes. Depression is among the most common of chronic healthproblems, also associated with societal costs higher thanany other chronic diseases, especially in terms ofpatients’ severe limitations in daily functioning and well-being1.It is estimated that one in five women and one in fifteenmen can expect to develop depression during their life-time.

Without treatment, 10 to 15% of people suffering fromsevere depressive disorder commit suicide. With treat-ment, the majority of patients with this illness will recov-er.

Depression high morbidity rate can also be seen in a factthat, antidepressant Prozac® is on the 3rd place of thetop selling 200 drugs in 19992.A recent World Health Organization report predicts thatdepression will be the leading cause of disability and pre-mature death in the industrial world by the year 20203.

The American Psychiatric Association has publishedstandardised criteria for the depressive disorders diag-nosing (table 1).

Frequency and characteristics of side effectsassociated with antidepressant drugs

Jasna Kusturica, Irfan Zuli}, Svjetlana Loga-Zec, Nedàd Mulabegovi}, Slobodan Loga, Elvedina Kapi}Institute of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of SarajevoDepartment of Psychiatry of Clinical Centre, University of Sarajevo Bosnia & Herzegovina


There are several approaches to the treatment of depres-sion depending on the severity of the condition and therisks to the patients, but in any cases it includes psy-chotherapy and antidepressive therapeutic agents.Untreated depression recovers within 6-12 months, withvery high relapse rates. The use of antidepressantsapproximately doubles the chance that a depressedpatient will recover within 2-4 months.

The best way to evaluate scientifically the effectivenessof therapy is to conduct a randomised controlled clinical

trial. Here are the results of the few randomised con-trolled clinical trials that have been done (table 2). Forthe less severely depressed patients antidepressant drug,placebo pill with supportive visits to a physician, inter-personal and cognitive behavioural therapy were equallyeffective; for the severely depressed patients antidepres-sive therapy was highly effective while cognitive thera-py was ineffective. There is a surprising lack of any scientific research doneon the effectiveness of psychoanalysis and psychothera-py in the treatment of major depression3.

A. Five (or more) of the following symptoms have been present during the same 2-week period and represent achange from previous functioning; at least one of the symptoms is either (1) depressed mood or (2) loss of interestor pleasure.

1. Depressed mood most of the day, nearly every day;2. Markedly diminished interest or pleasure in all, or almost all, activities most of the day, nearly every day;3. Significant weight loss (when not dieting) or weight gain, or decrease or increase in appetite nearly every

day;4. Insomnia or hypersomnia nearly every day;5. Psychomotor agitation or retardation nearly every day (not merely subjective feelings of restlessness or

being slowed down)6. Fatigue or loss of energy nearly every day;7. Feelings of worthlessness or excessive or inappropriate guilt (which may be delusional) nearly every day

(not merely self-reproach or feelings of guilt about being sick);8. Diminished ability to think or concentrate, or indecisiveness, nearly every day;9. Recurrent thoughts of death (not merely fear of dying), recurrent suicidal ideation, or a suicide attempt.

B. The symptoms cause clinically significant distress or impairment in social, occupational, or other importantareas of functioning.

C. The symptoms are not due to the direct physiologic effects of a substance or a general medical condition.

D. The symptoms do not meet criteria for a mixed episode and are not better accounted for by bereavement.

Table 1 Criteria for major depressive episode

Table 2 Result of the clinical trials on the therapies of severe major depression

Treatment Effectiveness

Antidepressive drug5 good

Interpersonal psychotherapy5 fair

Cognitive therapy5 unknown

Electroconvulsive therapy6 good

Anticonvulsant therapy, lithium (for prevention)7 good

Antianxiety medication8 poor

Antipsychotic medication3 unknown

Psychoanalytic psychotherapy3 unknown

Family therapy3 unknown

Group therapy3 unknown


Various drugs have been developed and used in depres-sion treatment. As the neurobiology of mood disordersand the mechanism of action of antidepressants continueto be elucidated, there has been a shift in emphasis fromchanges in neurotransmitter release and metabolism tothe regulation of gene expression and neuroprotection9. Antidepressive therapy usually takes 2-4 weeks beforeany significant improvement is obtained, and 2-6 monthbefore maximal improvement appears. To be successful, treatment for the patients sufferingfrom depression must aim not to the partial improvementof illness but to the complete remission (virtual elimina-tion of symptoms) and recovery of life quality, accordingto an expert panel comprised of guidelines U.S. MentalHealth Association (NMHA), Nov. 30th, 2001, in NewYork City. Antidepressant therapy should not be with-drawn before there have been 4 -5 symptom-free month(table 3).

Most patients do not stay on their antidepressants longenough for it to be effective. One study found that only25% of patients started on antidepressants by their fami-ly physician stayed on it longer than one month10. One ofthe greater reasons for break down is appearance of theunpleasant side effects. They occur early in the treatment(while improvement is often delayed), produce bed com-pliance and patients refuse the drug therapy before com-plete recovery11. Most frequently side effects are relatedto their mechanism of action. Antidepressants interferewith activity of many neurotransmitters, on peripheraland central site of receptors12. But, individual classes ofantidepressants are distinguished by their side-effect pro-files.

Method

Study was epidemiological, prospective, and ran-domised. In this study we followed up dynamics of 16 differentsymptoms (dry mouth, constipation, dizziness, tremor,blurred vision, anxiety, sedation, nausea, dyspepsia,vomiting, headache, urinary disturbances, palpitations,

sweating, myalgia, skin rashes), which are the character-istic side effects of antidepressive therapy, with majorgoal to asses their frequency and characteristics.The sample was all female patients taking antidepressivedrugs in the Department of Psychiatry of Clinical Centreof University in Sarajevo, during the period of fivemonths.The was done evaluation is done by the measuring ofbody weight, pulse rate in sitting and standing position,blood pressure in sitting and standing position, HAMDscale, scale of appetite, carbon hydrate needs scale andadverse effects scale.All parameters were taken one day before the antidepres-sive therapy introduction, than every following week andon the last hospitalisation day.

Results and discussion

The sample was 27 patients who meet criteria ICD-10 forsevere depressive episode, among 120 patients hospi-talised in the Department of Psychiatry during the fivemonth-period.

Graph 1 Diagnosis of depressive disorder

Patients were divided into two groups depending on anti-depressants.Average daily dose of amitriptyline was 75 mg (18patients), fluoxetine 30 mg (8) and one patient was tak-ing fluvoxamine in daily dose of 300 mg (table 4).

Table 3 Common duration of antidepressive therapy

Table 4 Antidepressants and average daily dosage

Group of antidepressants Antidepressants Average daily dose Number of patients

Group I Tricyclic antidepressants (TCA) Amitriptyline 75 mg 18

Group II Selective serotonin reuptakeinhibitors (SSRI)

FluoxetineFluvoxamine

30 mg300 mg

81

Amitriptyline was effective in 72.3% patients, and fluox-etine/fluvoxamine was effective in 78% patients.Lonnqvist and Armstrong reported similar results of anti-depressive effective treatment in 65-75% of all patientswith depression13, 4.

Hamilton Rating Scale for Depression (HAM-D17) hasbeen used for 4 decades as the “gold standard” instru-ment to assess the severity of depression and response totherapy in clinical researches. The maximum possiblescore for HAM-D is 52, in practice very few patients’core is above 35. Most people have depression score 14or more14, 15.

In group I on the first day Hamilton scale score was 24,after seven days 21. 8 (t-test p-0.001) and on the last dayit was 18. 2 (t-test p=<0.001). In group II on the first dayHamilton scale was 25. 5, after seven days 19.3 (t-test p-0.022) and on the last day it was 17. 3 (t-test p=<0.005)(graph 2). Significant difference was seen in both groups afterseven days of the therapy.

Graph 2 HAMD score

On the first day was recorded: depressive symptoms thatcaused clinically significant impairment in social, occu-pational and other functioning areas. Patients had feel-ings of worthlessness and inappropriate guilt and thoughtof death. Depressive feeling was significantly decreased,but still was there on the last hospitalisation day (scale 0-3; graph 3)

Graph 3 Depressive feeling

The first improved symptom (item on the HAM-D), afterseven days of amitriptyline treatment, was insomnia(delayed) (scale 0-2) and in SSRI group that was depres-sion feeling, work capacity/interests (scale 0-4) and retar-dation (scale 0-3). The improvement in insomnia, workcapacity and interests, retardation, anxiety and appetite inTCA group was obtained -on the last hospitalisation day(graph 4, 5, 6).

Graph 4 Insomnia (delayed)

Graph 5 Work capacity/interests

Graph 6 Retardation

Antidepressants express an influence on the body weight.Patients in group I had significant increase of appetite(p=0.009) and weight gain (average value 0. 83 kg), andin group II patients experienced weight loss (average


value 0.67kg). Anorexia has also occurred during fluoxe-tine therapy according literature data, and it is most like-ly associated with the weight loss observed in severalstudies. Anorexia has occurred in 9-15% of patients treat-ed, and occurs more frequently with fluoxetine than withother antidepressants; however, it is rarely a cause of thedrug discontinuation16, 17, 18.

In this study, all patients developed side effects.Incidence of side effects in TCA group was 7.4 on patient(7.4/ ) and 4 on patient (4/ ) in SARI group. This highincidence of side effect can be explained with mechanismof action of antidepressants. Tricyclic antidepressantsinterfere with the activity of at least five putative neuro-transmitters by several different potential mechanisms atboth central and peripheral sites, which results withadverse effects12. There is also a wide range of inter-individual sensitivity and susceptibility between patients,and very little consistent correlation between plasma con-centration and particular adverse effects.

Side effects were the most intensive (range 0 till 3) dur-ing the second week of therapy, and reduced within thethird week (graph 7).

Graph 7 Intensity of side effects

Patients in TCA group (group I), after seven days of ther-apy, experienced significantly different severity of all fol-lowing symptoms (p=< 0.001), and it was lasted throughthe whole period. A significant difference in intensity(range 0-3) was found for dry mouth (p=0.007; p=0.002),blurred vision and sedation after seven days of therapy,,while for tachycardia (p=0.014) and tremor (p=0.002) onthe last day (graph 8, 9, 10, 11, 12). None of the sideeffects where reported to necessitate additional pharma-cotherapy. Only one patient with tachycardia needed adose reduction.It is well known that all tricyclic antidepressants have aspectrum of anticholinergic activity, presenting as trou-blesome adverse effects such as dry mouth, sweating,confusion, constipation and blurred vision. The mostserious adverse effects relate to the cardiovascular sys-tem11, 12, and 19. Elderly patients are more sensitive toanticholinergic effects of TCAs and may develop confu-

sion or delirium even at moderate doses or with concur-rent use of other anticholinergic drugs; orthostatichypotension in the elderly can lead to padovi, particular-ly in patients with heart disease; TCAs lower the thresh-old for bundle branch block and complete heart block.Tricyclic antidepressants have sedative effects too, andthis may be desirable and undesirable depending on par-ticular patient’s state of apathy or agitation.Change in intensity of the following symptoms vs. pre-treatment throughout the whole period in SSRI group(group II) was not significant, probably because of thelow affinity to muscarinic and histaminic receptors(graph. 8, 9, 10, 11, 12).

Graph 8 Dry mouth

Graph 9 Tremor

Graph 10 Blurred vision


Graph 11Pulse rate in sitting and standing position (group I)

Graph 12 Pulse rate in sitting andstanding position (group II)

The most frequent side effects, observed in group I, wereblurred vision (13/18) and tachycardia (9/18) after sevendays, and dry mouth (10/18), tremor (12/18) and sedation(10/18) on the last day (graph13).

Graph 13 Frequency of side effects (group I)

Selective serotonin re-uptake inhibitors (SSRIs), com-pared with tricyclic drugs, have less pronounced anti-cholinergic effects and cause less severe cardiotoxicity.Gastrointestinal side effects are one of the major disad-vantages of SSRIs. Wernicke, Cohn and Wilcox pub-lished that the most frequent side effects, which occurredin 10-25% of the patients, were nausea (25%), nervous-ness, insomnia, headache, tremor, anxiety, drowsiness,dry mouth (14%), sweating (30%) and diarrhea17, 20.Most of these side effects occurred early in the treatmentand seldom led to the drug withdrawal.

The most frequent side effect in group II after seven daysof therapy was nausea (30%), than sweating and drymouth seen on the last day. Only one patient had inten-sive nausea, but the patient continued with the applica-tion of the same dose. None of side effects necessitatedadditional pharmacotherapy or dose reduction.

Graph 14 Frequency of side effects (group II)

Conclusion

Antidepressant’s treatment is efficient in most of thepatients. Side effects are common during the treatments.A major advantage of fluoxetine over amitriptyline is lesspronounced side effects. Every patient in amitriptylinegroup had average 7.4 side effects and 4 in fluoxetinegroup. The most intensive amitriptyline side effects weredry mouth, tremor and tachycardia whilethe most fre-quent side effects included blurred vision, tachycardia,dry mouth, tremor and sedation. The most frequent flu-oxetine side effects were nausea, tachycardia,swellingand dry mouth.We can conclude that SSRIs and TCAs are equal in effi-cacy in the treatment of depression, but SSRIs are toler-



ated better (lack the significant anticholinergic, cardio-vascular and sedative effects of TCAs), so SSRIs should

be considered as first-line agents especially in the treat-ment of older adults.

References

1. Gelder M., Gath D., Mayou R. (1993): Psychiatry. Oxford Medical Publications. 133-162;2. www.pharmacytimes.com/top200.html3. Long M.D. (1998); Major Depressive Disorder, Treatment www.mentalhealth.com/rx/p23-md01.html4. Armstrong L. (1997): Antidepressant Update. Topics in Drug Therapy, Vol 35(6);5. Elkin I., Shea M.T., Watkins J.T., Imber S.D., Sotsky S.M., Collins J.F., Glass D.R., Pilkonis P.A., Leber W.R.,

Docherty J.P. et al. (1989): National Institute of Mental Health Treatment of Depression Collaborative ResearchProgram. General Effectiveness of Treatments. ArchGen Psychiatry;46(11):971-82;

6. Piper A. Jr. (1993): Tricyclic Antidepressants versus Electroconvulsive Therapy: A Review of the Evidence forEfficacy in Depression. Annals of Clinical Psychiatry;5(1):13-23;

7. Stuppaeck C.H., Barnas C., Schwitzer J., Fleischhacker W.W. (1994): Carbamazepine in the Prophylaxis ofMajor Depression: A 5-year Follow up. Journal of Clinical Psychiatry;55(4):146-50;

8. Hubain P.P., Castro P., Mesters P., De Maertelaer V., Mendlewicz J. (1990): Alprazolam and Amitriptyline in theTreatment of Major Depressive Disorder: A Double-blind Clinical and Sleep EEG Study. J AffectiveDiss;18(1):67-73;

9. Trevor L.Y., Bakish D., Beaulieu S. (2002): The neurobiology of treatment response to antidepressants andmood stabilizing medications J Psychiatry Neurosci; 27(4):260-5.Simon G.E., VonKorff M., Wagner E.H.,Barlow W. (1993): Patterns of antidepressant use in community practice. General Hospital Psychiatry;15(6):399-408.

10. Meyer's Side Effects of Drugs (2000): An Encyclopaedia of Adverse Reactions and Interactions. FourteenthEdition. Ed: Dukes M.N.G. and Aronson J.K. Elsevier Science B.V.: 648-649;

11. Drug Facts and Comparisons (1998): Facts and Comparisons. 52nd Edition, St. Luis, A Wolters KluwerCompany; 1603-1671;

12. Lonnqvist J., Sintonen H., Syvalahti E., Appelberg B., Koskinen T., Mannikko T., Mehtonen O.P., Naarala M.,Sihvo S., Auvinen J. et al. (1994): Antidepressant Efficacy and Quality of Life in Depression. Acta PsychiatricaScandinavica;89(6):363-9;

13. Carrol B.J., Fielding J.M. and Blashki T.G. (1973): Depression Rating Scales: a Critical Review. Arch GenPsychiatry;28:361-366;

14. Roger M., Kennedy S., R., Bagby, R., Bakish D. J Psychiatry Neurosci 2002;27(4):235-9).15. Feighner J.P., Boyer W.F., Meredith C.H. et al. (1988): An Overview of Fluoxetine in Geriatric Depression. Br

J Psychiatry;153:105-108;16. Wernicke J.F. (1985): The Side Effect Profile and Safety of Fluoxetine. J Clin Psychiatry;46(3Pt 2):59-67;17. Product Information: Prozac(R), fluoxetine. Dista Products Company, Indianapolis, IN, 1997;18. Goodman and Gilman?s (1996): The Pharmacological Basis of Therapeutics. Ninth Edition, Ed. Mc Graw Hill;

431-446;19. Cohn J.B., Wilcox C. (1985): Comparison of Fluoxetine, Imipramine, and Placebo in Patients with Major

Depressive Disorder. J Clin Psychiatry; 46:26-31.


Abstract

Cystatin C is a natural inhibitor of the cysteine proteinas-es papain, and mammalian lysosomal cathepsins B, H, Land S. This protein is thought to serve an important phys-iological role as a local regulator of enzyme activity. Thechanges of levels of cystatin C in extracellular fluidshave shown themselves having potential clinical impor-tance.We have purified cystatin C from urine of patients withchronic renal failure by procedure using affinity chro-matography on CM-papain Sepharose, gel filtration onSephacryl S-200, and ion exchange chromatography onCM-cellulose. After isolation we obtained three inhibito-ry peaks (pI’s from 7.8 to 9.2) which represent isoformsof the same protein. These isoforms are immunologicallyidentical and differ in N-terminal sequence of the mole-cule. The form with pI 9.2 represents the intact inhibitorform, whereas the form with pI 7.8 is shortened for 8amino-acid residues at N-terminal end.Purified cystatin C pI 9.2 was used for immunization ofrabbits. Polyclonal antibodies, produced in rabbits, wereisolated from rabbit sera by affinity chromatography onProtein A Sepharose. Enzyme immunoassay (ELISA) forcystatin C is developed on the basis of purified antibod-ies. Using ELISA test we determined amount of cystatinC in urine and serum samples of patients with chronicrenal failure. The concentration of the inhibitor in theurine of these patients was approximately 100-fold morethan in normal urine. In the serum from the same patientswe found concentrations of cystatin C to be five timeshigher in comparison with the serum of healthy individ-uals. Keywords: human cystatin C, cystatin C isoforms,ELISA test, chronic renal failure.

Introduction

Lysosomal cysteine proteinases, the cathepsins, belong tothe papain family of proteinases. Under normal physio-logical conditions proteinases participate in a variety ofprocesses at tissue; they mediate intracellular and extra-cellular protein turnover, regulating the lifetime of pro-teins and other molecules critical for normal cell func-tion. Beside protein turnover, proteinases are involved inspecific processing step for smaller peptides, proenzymesand prohormones (1). The activation of cysteine pro-teinase requires acidic pH and may occur autocatalytical-

ly or by other lysosomal cathepsins, such as cathepsin D,in lysosomal vesicles, localized close to the plasma mem-brane (2). Cysteine proteinases are regulated by endogenous cys-teine proteinase inhibitors (CPIs), cystatins (3). Cystatinsuperfamily comprises families of closely homologousCPIs, stefins (family I), cystatins (family II), kininogens(family III) and non-inhibitory proteins (family IV) suchas human histidine-rich glycoprotein and α2 HS glyco-protein (4).Cystatins (family II) are single chain proteins stabilizedby two disulfide bridges, with molecular weight of about13.000. A presence of hydrophobic signal peptides atamino-terminal end of the cystatin molecule, which wasdetermined by molecular cloning, points out to extracel-lular function of that protein (5). Human cystatin C isabundant in various tissues and body fluids. The highestlevels have been determined in cerebrospinal fluid, sem-inal plasma and synovial fluid. It is the most potentinhibitor of cysteine proteinases with apparent inhibitionconstant in picomolar range (6).Changes of levels of cystatin C in extracellular fluidshave good potential for clinical use. Its level in bloodplasma has been shown to be a good indicator ofglomerular filtration rate (GFR), considerably better thanthe widely used creatinine (7, 8). Decreased levels of cys-tatin C in cerebrospinal fluid are associated with heredi-tary cystatin C amyloid angiopathy (HCCAA), an inher-ited disease characterized by deposition of cystatin Cvariants as amyloid fibrils in blood vessels, resulting indeath from cerebral haemorrhage (9). Cystatin C hasbeen suggested to play a role in several other diseasesassociated with alteration of proteolytic system, such ascancer (10, 11), inflammatory lung diseases (12), peri-odontal disease (13), multiple sclerosis (14), autoimmunediseases (15), HIV infection (16), and renal failure (17). The aim of our work was purification and characteriza-tion of cystatin C from urine of patients with chronicrenal failure, and determination of cystatin C levels inurine and serum samples of the same patients by employ-ing of ELISA method.

Materials and methods

Materials. Urine from patients with chronic renal failurewas collected 24 hours, and 1 mol/L NaCl was added aspreservative. Papain, bovine serum albumin (BSA),

Isolation and immunochemical characterization ofhuman cystitis C

Lejla Begi}, Selma Berbi}Department of Biochemistry, Faculty of Medicine, University of TuzlaShort title: Purification of human Cystatin C

Tween 20 and horse radish peroxidase were from Sigma,2,2’-Azino-Di-(3-ethylbenzthiazoline sulphonic acid)(ABTS) from Boehringer Mannheim. Sephacryl S-200Superfine, CNBr-activated Sepharose 4B, ampholines,and molecular weight standard proteins were fromPharmacia. CM-cellulose was purchased from Serva. S-carboxymethylated papain Sepharose 4B was prepared asdescribed by Barrett (18). Purification of the inhibitor. Urine was centrifuged at3000xg for 15 minutes and the supernatant was applieddirectly to a CM-papain Sepharose 4B column. Bindingof the inhibitor to the affinity column and subsequentwashing were performed at pH 8.0 with Tris/HCl buffercontaining 0.5 mol/L NaCl. The inhibitor was eluted with0.01 mol/L NaOH concentrated to 40 mL using YM-5membrane and applied to a Sephacryl S-200 column(4x100 cm), equilibrated and eluted with 0.02 mol/Lacetate buffer pH 6.1. The second papain inhibiting peakwas collected and further chromatographed on a columnof CM-cellulose (3x15 cm) using the same buffer as inthe gel filtration step. After elution with starting buffer, alinear gradient of NaCl (0-0.2 mol/L) in a total volume of400 mL of starting buffer was applied. The inhibitorypeaks were eluted and concentrated using a Diaflo YM-2membrane.Inhibitor assay. The inhibitory activity was determinedusing the conditions of papain activity assay on benzoil-DL-arginine-2-naphtylamide. Prior to the assay, a knownamount of inhibitor was pre-incubated wit the enzyme for5 min at room temperature before adding the substrate.One inhibitory unit (I.U.) is defined as the amount ofinhibitor which completely inhibits 1 µg of the testpapain.ELISA test. Antibodies against cystatin C pI 9.2, isolat-ed from human urine (17) were raised in rabbits. Micro-titre plates were coated with anti-cystatin C –IgG andincubated over night at 40C. The samples were applied inthe wells and after 2 hours of incubation at 370C the anti-cystatin C–IgG peroxidase conjugate was added. Theindicator reaction was carried out applying the substratesolution (ABTS and H2O2). The measured enzyme activ-ities residing in the individual wells were plotted againstthe logarithm of the concentration of the antigen.Other methods. SDS-polyacrilamide gel electrophoresiswas performed according to Weber and Osborne (19).Analytical gel focusing was carried out on 1mm thick 5%polyacrylamide gel plates with the ampholines of the pH2-10 range following the manufacturer’s instruction(Pharmacia). Double radial immunodiffusion test wasperformed according Ouchterlony (20). Proteins weredetermined by the modified method of Lowry (21).

ResultsThe first step of cystatin C isolation was CM-papainSepharose affinity chromatography at pH 8.0. Inhibitoryfractions from the affinity resin were concentrated and

chromatographed on Sephacryl S-200. Two inhibitorypeaks were resolved (Fig.1). The first one contained highmolecular weight cysteine proteinase inhibitors of thekininogen family. The second peak, which contained lowmolecular weight inhibitors and kininogen cleavageproducts, was further purified on a CM-cellulose column,where three papain inhibiting peaks were resolved. Thebulk of the inhibitor capacity was eluted in the third peak(Fig.2). The total of 9 mg of inhibitor protein from thispeak was obtained in several preparations from 7 litres ofurine. The peaks from ion exchange column were ana-lyzed by isoelectric focusing on polyacrylamide gels.The protein from peak 3 focused at pH 9.2, and thosefrom the minor peaks 1 and 2 focused at pH 7.8 and 8.4,respectively (not shown). Although the three purified iso-electric forms were immunologically identical (Fig.3),they differ in molecular weights. The difference was con-firmed by SDS-PAGE (Fig.4). Isoelectric variant 9.2 isthe longest form in the urine of patients with chronicrenal failure and has Mr of about 13.260, whereas theshorter pI 7.8 form of cystatin C has Mr of about 12.500.Using ELISA test we determined the levels oc cystatin Cin the urine and serum of patient suffering from chronicrenal diseases. The concentration of the inhibitor in theurine of these patients was found to be 9mg/L, which isapproximately 100-fold more than in normal urine. In theserum from the same patients we found concentrations ofcystatin C to be five times higher in comparison with theserum of healthy individuals.

Discussion

Using the described procedure we have isolated cystatinC in 3 electrophoretically pure forms. Two isoforms wereobtained in an amount sufficient to carry out their char-acterization. The intact polypeptide chain, pI 9.2, consistsof 120 amino acid residues. Cleavage of the N-terminaloctapeptide from the pI 9.2 form results in the formationof the 7.8 variant. Swiss-Prot Database (22) claims thatcystatin C precursor consists of 146 amino acid residues,where residues from1 to 26 constitute signal sequence.That corresponds with our results. The relatively highconcentration of cystatin C in cerebrospinal fluid, com-pared to plasma, indicates the production of cystatin C incentral nervous system (23). Researches of gene’s struc-ture for cystatin C and its expression, have confirmedsynthesis of that inhibitor in many tissues, but highestlevel of mRNA for cystatin C has been observed inchorioidal plexus of rat’s brain (24). The protein is prob-ably able to cross the blood brain barrier to the vascularspace, and rapidly filtered in the glomeruli. Nowadays, agreat attention is paid to cystatin C as a possible indica-tor of renal function, e.g. in cases of transplantation ofkidneys (25) or in diabetes (26). Cystatin C has beenadvocated as a better marker of glomerular filtration ratethan serum creatinine because, unlike creatinine, it is not


secreted by the renal tubule, is not affected by musclemass, and does not suffer the same problems with analyt-ical interference (27).Our results reveal that quantification of cystatin C inserum and urine by ELISA test can be used to detect andfollow pathophysiological processes during chronic renaldisease. The accurate screening of subjects for impair-ment in renal function requires either laborious collectionof timed urine samples and processing by ELISA (orsome other equally complicated test, e.g. immunotur-bidimetry), or the intravenous injection of radioactivesubstances. Thus, the development of simple serum testfor detecting mild to moderate reductions in glomerularfiltration rate would represent a major advance thatwould be of relevance to many fields of medicine.

Figure 1

Gel filtration on Sephacryl S-200 Superfine of theinhibitory fraction obtained after affinity chromatogra-phy on CM-papain Sephrose. Protein concentration(blue) is expressed (left ordinate) as absorbance at 280nm. Inhibitory activity (green) 100 µL of fractions is rep-resented as relative inhibition (right ordinate).

Figure 2

Ion exchange chromatography on CM-cellulose ofinhibitory fraction obtained in second peak after gel fil-tration. Concentration of protein (blue) is represented onthe left ordinate as absorbance at 280 nm, and relativeinhibition, determined with papain using BANA test(green) is represented on the right ordinate. Bound pro-teins were eluted with gradient NaCl (0-0.2 mol/L).

Figure 3

Double radial immunodiffusion test of purified cystatin Cisoforms pI 7.8 (I), pI 8.6 (II) and pI 9.2 (III) against anti-cystatin C antiserum (AS).

Figure 4

SDS-PAGE of the purified cystatin C isoforms. From theleft to the right: standards, cystatin C pI 9.2(I), cystatin CpI 8.6 (II), cystatin C pI 7.8 (III). The samples were pre-incubated for 30 minutes at 370C in 2% SDS containing10% µ-mercaptoethanol. Phosphorylase b, bovine serumalbumin, ovalbumin, carbonic anhydrase, soya-beanrypsin inhibitor and α-lactalbumin were used as molecu-lar weight standards.



References

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Abstract

Antipsychotic drugs produce a wide spectrum of physio-logical actions. Some of these effects differ among thevarious classes of antipsychotics. This medications haveindications in the treatment of acute psychotic disorders.The main goal of this investigation was to determine theincidence and prevalence of the neuroleptic therapy acuteside effects. The reason for this epidemiological investi-gation performing was the lack of knowledge of the exactneuroleptic therapy side effects incidence. Qualitativestudy on this problem has not been performed yet.Antipsychotic therapy side effects prevalence rateaccording to the literature data is ranging from 24% to74%. Different prevalence rate is a consequence of dif-ferent antipsychotic drug usage, different drug adminis-tration method and different side effects identification.On account of all these facts, we put the hypothesis onthe correlation between the antipsychotic therapy andoccurred side effects.Our experiment included all patients hospitalised fromDecember 31st 1999 to January 31st 2000 in IntensiveCare Unit of Biological Psychiatry Department ofPsychiatric Clinic in Sarajevo. All patients were dividedin three groups according to the applied therapy. All ofthem met ICD-10 criteria for schizophrenia (F20-29).During our study the following examinations were per-formed: psychiatric interview, BRPS, scale of sideeffects, psychophysiological tests, general clinicalimpression, scale of appetite, carbon hydrate needs scale.Psychiatric and statistical evaluations were done as well.The evaluation of our examination is showing successfulresults in all groups of patients. The improvement of psy-chopathological symptoms was insignificant. Reportedside effects were minimal with low incidence rate andrelatively high prevalence rate. Statistical tests were cal-culated from the obtained data after what the null hypoth-esis was rejected. Consequently, an alternative hypothe-sis was confirmed and it indicated that the acute sideeffects incidence and prevalence were within the range ofexpectation.Intensity of the recorded side effects was moderate tomild.On the basis of the obtained data, it has been concludedthat applied antipsychotic agents did not induce morepsychophysiological function impairments in the treated

patients. Psychophysiological functions remained inphysiological range limits and their changes were not sig-nificant.Neuroleptic therapy side effects were minimal, meaningno toxic signs or therapy discontinuations were recorded.

Keywords: schizophrenia, antipsychotic agents, acuteside effects, incidence, prevalence, intensity of sideeffects.

Introduction

It is difficult to define schizophrenia. The differschizofrenia from other psychotic conditions, sometimes,is not clear. Schizohrenia is, may be, a group of relatedsyndromes rather than single disorder. Division of schiz-ophrenia into acute and chronic syndromes can be use-ful. The predominant clinical features of the acute syndromeare delusions, hallucinations and interference with (oftenknown as “positive” symptoms). The main features ofthe chronic syndrome are apathy, lack of drive and socialwithdrawal (so-called “negative” symptoms). (Jablensky,1995; Karno i Norquist, 1995). There is evidence for some degree of genetic predisposi-tion towards schizophrenia and environmental factors arealso thought to contribute. The pathophysiological mech-anism of schizophrenia is unclear and several hypotheseshave been proposed. Since antipsychotic drugs blockdopamine receptors in the midbrain it has been suggest-ed that dopaminergic system overactivity may beinvolved. Most commonly schizophrenia begins in ado-lescence and early adulthood and has an annual incidenceof about 0, 1 to 0, 5 per 1000. The prognosis for schizo-phrenia is general worse than for other psychiatric disor-ders and many patients develop a chronic illness withrepeated relapses and varying degrees of recoverybetween episodes. The best prognosis is patients in whothe schizophrenia is of sudden onset with a definite pre-cipitant and who have florid acute symptoms withmarked mood changes and who previously were welladjusted.Treatment of schizophrenia consists mainly of a combi-nation of social therapy and antipsychotic drugs. Drugtreatment should de individualized for each patient with

Frequency of the antipsychotic therapy acuteside effects in the treatment of acute psychosis

Svjetlana Loga-Zec1, Irfan Zuli}1, Ned`ad Mulabegovi}1,Ismet Ceri}2, Slobodan Loga2, Saida Fi{ekovi}2, Jasna Kusturica1 and Sanja Kro{njar3

1. Institute of Pharmacology, Clinical Pharmacology and Toxicology, School of Medicine, University of Sarajevo2. Department of Psychiatry of University Clinical Center, Intensive Care Unit3. Bosnalijek Medical Department

close monitoring. Negative symptoms tend to respondless well to drug therapy than do positive symptoms. A useful generalization is that low-potency antipsychot-ic are sedative and strongly antimuscarinic and antia-drenergic but are less likely to cause acute extrapyrami-dal symptoms than the high-potency agents with exhibita reversed pattern of adverse-effects. Sedative and mus-carinic effects usually diminish with continued use,although sedative effects may be useful for behavioralcontrol in acute illness (Martindale, 1999).Antipsychotic medications are indicated in the treatmentof acute psychotic disorders. The main goal of this inves-tigation was to determine the incidence and prevalence ofthe neuroleptic therapy acute side effects. The reason forthis epidemiological investigation performing was thelack of knowledge of the exact neuroleptic therapy sideeffects incidence. Qualitative study on this problem hasnot been performed yet. Antipsychotic therapy sideeffects prevalence rate according to the literature data isranging from 24% to 74%. Different prevalence rate is aconsequence of different antipsychotic drug usage, dif-ferent drug administration method and different sideeffects identification (Loga i Fi{ekovi}, 2000).

No systematic reporting and evaluating system for docu-menting these adverse effects has evolved. Even therecognition of an adverse effect may be difficiult in anindividual case (Meyler’s, 2000); for example, akathisiamay be difficult to differentiate from exacerbation of theillnes and often has a deleterious effect on compliance(Van Putten, 1974).

Definition of side effects:Each unexpected appearance during application of drug,diagnostical and preventiveagents in usual doses is called side effects.It is very hard to predict when side effects appear,because antipsychotic are drugs with large width ( forchlorpromazine amounts 25 to 5000 mg daily).

Evaluation of side effects:During the experiment following examinations weredone:

• psychiatric interview, • BRPS- Brief Psychiatric Rating Scale • scale of side effects,• psychophysiological tests, • global clinical impression • scale of appetite and carbon hydrate needs scale.

Scale of side effects:

• anxety• sleep• drowsnes

• dry mouth• tachycardia• blurred vision• muscle pain• sweating• tremor• vertigo• dizziness• dysuria• dyspepsia• nauzea• pruritus• constipation• vomiting

Physiological parameters:

• blood pressure (sitting and standing position), • pulse rate (sitting and standing position)• pupil’s size (normal and indirect light),• digit symbol test,• length of handwriting,• scale of appetite • carbon hydrate needs scale

Clinical global impresion (CGI):

• Severity of symptoms• Change of illnes state• Effects of treatment• Side effects

Basic Goal To determine prevalence and incidence of acute sideeffects in treatment with antipsychotic drugs during onemonth.

Secondary GoalTotal score acute side effects is reduced with regularselection and correct doses antipsychotic drug.

Taking in account all those facts, we put hypothesisbetween antipsychotic therapy and occurred side effects.

Null hypothesisAntipsychotic drugs don’ t cause side effects.

Hypothesis II ( alternative)Incidence and prevalence of recorded acute side effectswere within the expected limits foundin literature.

Hypothesis III (alternative)Only some antipsychotic cause acute side effects.



Method

Study is clinical-pharmacological, epidemiological,prospective and randomized. All parameters are takingon day starting the antipsychotics therapy and regularlyevery seven-day after. Psychiatrists in the Intensive care unit of the Departmentof Psychiatry of the Clinical Center University ofSarajevo generally used typical antpsychotics becausethey are cheaper then atypical antpsychotics.Our experiment had included all patients were hospital-ized from 31.12. 1999. to 31. 01. 2000. in the Intensivecare unit of the Department of Biological psychiatry ofthe Psychiatric Clinic in Sarajevo. Patients were dividedin three groups according to used neuroleptics. All ofthem met criteria ICD-10 for schizophrenia (F20-29).The demographic data of three group patients werehomogeneous.

Inclusion criteria:

• Patients of male and female sex, aged from 18to 65 years

• Patients these met ICD-10 criteria forschizophrenia (F20-29)

• Patients who use antipsychotic therapy

Exclusion criteria:

• Patients who collaborate• Children, pregnant and breast feeding women

Non-inclusion criteria:

• Patients without determined schizophrenicpsychosis (F20-F29).

• Patients who do not use antipsychotic therapy

All patients treated with antipsychotic agents were thesample of our investigation. The drugs applied during ourinvestigation were chosen by M. D., specialist in psychi-atry, who ordinates in Intensive Care Unit of BiologicalPsychiatry Department of Psychiatric Clinic in Sarajevo.

Instruments of research:

• Psychiatric interview• Brief Psychiatric Rating Scale-BPRS• Adverse effects scale • Psychophysiological tests• CGI


Patients were divided in three groups (25) according tothe used neuroleptics:I group- treated with single phenothiazineII group- treated with combination of phenothiazinesIII group- treated with phenotiazine and buthirophenone

Average daily doses of neuroleptics are presented in tab. 1.

The most used antipsychotic was promazine, and after itare: chlorpromazine, haloperidol andfluphenazine. The commonest combination was chlor-promazine, haloperidol and promazine, and promazinewith haloperidol.Age of patients are presented in graph 1.

Graph. 1. Age of patients

Drug Average daily doses No. of patientspromazine 244 mg 16

fluphenazine 5 mg 1

fluphenazine decanoat 25 mg/ per month 7

haloperidol 6,78 mg 8

haloperidol decanoat 50 mg/ per month 5

levomepromazine 266 mg 3

chlorpromazine 236 mg 15

thioridazine 75 mg 1

Table 1. Average daily doses of neuroleptics


The same number of patients was monitored in patientgroup aged from 30 to 39 and from 40 to 49 years (32%)while the smallest number of patients were older than 60years (4%).

Graph. 2. Sex of patients

Groups II and III had similar sex structure (88.9%,respectively 83.3% of men and 11.1%, respectively16.7% of women. Group I consisted of only of men(100%).

Graph.3. Diagnoses

Diagnoses presented in Graph 3 are: hebephrenic psy-

chosis (32%), regressive paranoid schizophrenia (28%),

acute polymorphic psychotic disorders with schizo-

phrenic symptoms (28%).

The demographic data of three group patients were

homogeneous.

Group I group II group III group

α 77.25 80.89 87.58

SD 11.09 14.08 6.92

t-test

←⎯⎯⎯⎯⎯⎯⎯ -0.45 (p=0.66) ⎯⎯⎯⎯⎯⎯⎯→←⎯⎯⎯⎯⎯⎯⎯ -1.44 (p=0.17) ⎯⎯⎯⎯⎯⎯⎯→

←⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ -2.24 (p=0.04*) ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→


α 66.5 72.89 78.17

SD 12.39 17.0 12.59

t-test

←⎯⎯⎯⎯⎯⎯⎯ -0.67 (p=0.52) ⎯⎯⎯⎯⎯⎯⎯→←⎯⎯⎯⎯⎯⎯⎯ -0.82 (p=0.42 ⎯⎯⎯⎯⎯⎯⎯→

←⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ -1.61(p=0.13) ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→

Evaluation symptoms of psychopathology

Table 2. Total BRPS (0 day)

Table 3. Total BRPS (7 day)

Significant differences regarding psychopathologic symptoms were noticed on the null day between treated groups Iand III 0 (p=0.04*).


Psychopathologic symptom improvement was observedin all groups at the end of observation period with signif-icant improvement registered in group III (p = 0.03*).Psychopathologic symptom stabilisation was obtained invery short time and applied antipsychotic therapy com-pletely reached all examination goals:

• dereistic thinking reduction• alleviation of psychomotor unrest• hallucination control (Loga, 1999).

Side effects and their intensity, incidence and preva-lence are described below

Graph. 4. Anxiety-incidence

Graph. 5. Anxiety-prevalence

Graph. 6. Dyspepsia-incidence

Graph. 7. Dyspepsia-prevalence

Graph. 8. Bllured vision-incidence

Group αα SD t-testI group 0 day 77.25 11.09 1.29

(p=0.24)I group 7 day 66.5 12.39

II group 0 day 80.89 14.08 -1.09(p=0.29)II group 7 day 72.89 17.0

III group 0 day 87.58 6.92 2.27(p=0.03*)III group 7 day 78.17 12.59

Table 4. Total BPRS (Difference 0/7 day)

Graph. 9. Bllured vision-prevalence

Various antipsychotic drugs, particularly low-dose phe-nothiazines and thioxanthenes, commonly cause blurredvision secondary to their anticholinergic activity. This isprimarily a nuisance, except in the rare patient withclosed-angle glaucoma. Of more concern are two distinct types of adverse effectsin the eye, witch may be produced by various antipsy-chotic drugs: lenticular and corneal deposits and pigmen-tary retinopathy.

Non-phenothiazines appear to have minimal propensityto cause oculocutaneous reactions and may be preferredwhen these problems have occurred during treatmentwith phenothiazines, although the patients should still beclosely monitored. Pigmentary retinopathy, witch canseriously impair vision, is specifically associated withthioridazine, and has occurred more often with high andprolonged dosage (e.g. 1200-1800 mg/day for weeks tomonths) (Shah i sur., 1998).

Graph. 10. Sweating-incidence

Graph. 11. Sweating-prevalence

Antipsychotic drugs interfere with the temperature regu-latory function of the hypothalamus, and also peripheral-ly with the sweating mechanism, resulting in poikilo-thermy. This can result in either hyperthermia orhypothermia, depending on environmental temperature.Clozapine often cause a benign and transient increase inbody temperature early in treatment (Tremeau i sur., 1997).

Graph. 12. Pruritus-incidence

Graph. 13. Pruritus-prevalence


Many cutaneous reactions have reported with antipsy-chotic drugs, including urticaria, abscess after intramus-cular injection, rashes, photosensitivity or exaggeratedsunburn, contact dermatitis, and melanosis or blue grayskin discoloration. Skin rashes are usually benign.Chlorpromazine is most often implicated (incidence 5-10%)(George i sar., 2000). Sedation is commonlyadverse effects antipsychotic therapy. In mainly patientssedation cause low potention antipsychotic such as chlor-promazine, but and other antipsychotic. This side effectsis most frequently in initial phase of tretment (Davis isar.,1989).

Graph. 14. Sleep-incidence

Graph. 15. Sleep-prevalence

Graph. 16. Tremor-incidence

Graph. 17. Tremor-prevalence

Graph. 18. Dry mouth-incidence

Graph. 19. Dray mouth-prevalence

Dry mouth is a commonly reported autonomic adverseeffect of antipsychotic drugs and is seen more often withdrugs that have prominent anticholinergic properties,such as thioridazine and chlorpromazine. Dry mout ismainly a nuisance, but its persistence may promote den-tal caries, oral moniliasis, and infective parotitis. Whenfeasible, once daily administration of antipsychotic drugsat bedtime helps to alleviate the problem of dry mouth.In contrast, clozapine, a potent muscarinic, can causenocturnal hypersalivation (Szabadi, 1997).



Graph. 20. Pulse rate in standing position (differ-ence between 0 and 7th day)

Pulse rates measured in patient sitting position on the nulland seventh day did not significantly differ between allthree groups, as well as intra each group.

Pulse rates measured in patient standing position on thenull day showed marked differences between group I and

group II, as well between group I and group III.

The highest pulse values were registered in group III

while the lowest once were registered in group I.

Statistically significant intra group differences between

the null and the seventh day were not observed (table 5,

6, 7. and graph. 20).

Hypotension is the most commonly observed cardiovas-

cular adverse effects of antipsychotic drugs, particularly

after administration of those that are also potent a-

adrenoreceptors antagonist, such as chlorpromazine,

thioridazine. A central mechanism involving the lowering

of blood pressure. Antipsychotic drugs of high and inter-

mediate potency, such as haloperidol, have minimal a-

blocking effects and would be less likely to cause such

changes, (a fall of 30 mm Hg) were report orthostatic

changes, although in one report with these drugs in 27%

and 22% of cases respectively (Simpson et al., 2000). In

our investigation changed in pulse rate and blood pres-

sure were not significant.

Phisiological tests

Pulse rate in sitting and standing position

Table 5. Pulse rate in standing position (0 day)


α 89.25 104 105

SD 12.15 10.15 9.67

t-test

←⎯⎯⎯⎯⎯⎯⎯ -2.29(p=0.04*) ⎯⎯⎯⎯⎯⎯→←⎯⎯⎯⎯⎯⎯⎯ -0.23 (p=0.82) ⎯⎯⎯⎯⎯⎯⎯→

←⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ -2.66 (p=0.02*) ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→


α 90 108.22 107

SD 10.58 11.25 12.10

t-test

←⎯⎯⎯⎯⎯⎯⎯ -2.74 (p=0.02*) ⎯⎯⎯⎯⎯⎯→←⎯⎯⎯⎯⎯⎯⎯ -0.24(p=0.87) ⎯⎯⎯⎯⎯⎯⎯→

←⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ -2.49(p=0.02*) ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→

Table 6. Pulse rate in standing position (7 day)

Group αα SD t-testI group 0 day 89.25 12.15 -0.22

(p=0.84)I group 7 day 90 10.58

II group 0 day 104 12.33 -0.95(p=0.37)II group 7 day 108.22 10.29

III group 0 day 105 9.88 -0.56(p=0.59)III group 7 day 107 11.8

Table 7. Pulse rate in standing position (difference between 0 and 7th day)


Graph. 21 Pupil’s size normal light (differencebetween 0 and 7th day)

Pupil’s size (normal light)

Table 8. Pupil’s size normal light (0 day)


α 2.75 2.39 2

SD 0.5 0.55 0.62

t-test

←⎯⎯⎯⎯⎯⎯⎯ 1.12(p=0.29) ⎯⎯⎯⎯⎯⎯⎯→←⎯⎯⎯⎯⎯⎯⎯ -1.39 (p=0.18) ⎯⎯⎯⎯⎯⎯⎯→

←⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ 0(p=1) ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→


α 2.88 2.83 2.71

SD 0.25 0.56 0.49

t-test

←⎯⎯⎯⎯⎯⎯⎯ 0.14 (p=0.89) ⎯⎯⎯⎯⎯⎯⎯→←⎯⎯⎯⎯⎯⎯⎯ 0.54 (p=0.59) ⎯⎯⎯⎯⎯⎯⎯→

←⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ 0.63 (p=0.54) ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→

Table 9. Pupil’s size normal light (7th day)


(p=0.39)I group 7 day 2.88 0.25

II group 0 day 2.39 0.55 -2.87(p=0.02*)II group 7 day 2.83 0.56

III group 0 day 2 0.62 0.29(p=0.78)III group 7 day 2.71 0.49

Table 10. Pupil’s size normal light (difference between 0 and 7th day)

Pupil sizes remained the same (daylight) in all patients during the whole period of examination with the exception ofgroup II patients. Non significant pupil enlargements were observed in group I and group III patients (table 8, 9, 10.and graph. 21.)


Graph. 22. Digit symbol test(difference between 0 and 7th day )

There was no statistically significant differences intraand inter treated groups of patients during the whole investigation period, with the exceptionof group III (p= 0.05) (Table 11, 12, 13. and Graph. 22)Various antipsychotic drugs, particularly low-doses phe-nothiazines and thioxanthenes, commonly cause blurredvisions secondary to their anticholinergic activity (Shahet al., 1998). Although, in this tree group, after used ther-

apy did not changed toward blurred visions. The extend to which neuroleptic drugs impair mentalactivity is disputed. A recent study has shown that themore anticholinergic antipsychotic drugs impaired short-term verbal memory (Eitan et al., 1992). One studyshowed that schizophrenic patients who took neurolepticdrugs had superior information processing comparedwith unmedicated schizophrenic patients; the authorsclaimed that neuroleptic drugs probably do not cause,and may actually reverse, slowness of information pro-cessing in schizophrenic patients (Simpson et al., 2000).However, a substantial number of schizophrenic patientsdeclare that neuroleptic drugs slow their thinking, causethem to forget, and remove interest thinking, cause themto forget, and remove interest and motivation. Theseresponses to neuroleptic drugs are claimed to be dys-phoric and are often associated with drug inducedextrapyramidal symptoms, particularly akathisia (Stip, etal., 1996).The conducted investigation in the Department ofPsychiatric showed that the used neuroleptic therapy didnot impair cognitive functions of patients, as it can beobserved in the Digit symbol test and the test of hand-writing.

Digit symbol test

Table 11. Digit symbol test (0 day)


α 11 21.89 33.67

SD 11.58 16.80 21.72

t-test

←⎯⎯⎯⎯⎯⎯⎯ -1.17(p=0.27) ⎯⎯⎯⎯⎯⎯⎯→←⎯⎯⎯⎯⎯⎯⎯ -1.35(p=0.19) ⎯⎯⎯⎯⎯⎯⎯→

←⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ -1.97(p=0.07) ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→


α 31.25 29.56 44.33

SD 27.37 18.91 17.49

t-test

←⎯⎯⎯⎯⎯⎯⎯ 0.13 (p=0.89) ⎯⎯⎯⎯⎯⎯⎯→←⎯⎯⎯⎯⎯⎯⎯ -1.85(p=0.08) ⎯⎯⎯⎯⎯⎯⎯→

←⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ -1.13(p=0.28) ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→

Table 12. Digit symbol test (7th day)

Group αα SD t-testI group 0 day 11 11.58 -2.39

(p=0.09)I group 7 day 31.25 27.37


III group 0 day 33.67 21.72 -2.26(p=0.05*)III group 7 day 44.33 17.49

Table 13. Digit symbol test (difference between 0 and 7th day )


Graph. 23. Scale of apetite(difference between 0 and 7th day)

No statistically significant differences regarding appetitewere observed between examined groups.

Weak appetite of patients was registered in group I on thenull day. Appetite of the same group of patients was notstatistically increased (p=0.08) on the seventh examina-tion day. The very similar results regarding appetite ofpatients were registered in group II and group III.

Weight gain is a common adverse effect of neurolepticdrugs. The mechanisms is poorly understood, although aserotoninergic mechanisms has been proposed. A meta-analysis of trials of antipsychotic drugs calculated thefollowing mean weight gains in kg after 10 weeks oftreatment: clozapine 4,5; olazapine 4,2; thioridazine 3,2;sertinole 2,9; chlorpromazine 2,6; risperidone 2,1;haloperidol 1,1; fluphenazine 0,43; ziprasidone 0,04;molindone, -0,39; placebo -0,74 (Allison i sar., 1998;Kelly i sar., 1998).

Scale of appetite

Table 14. Scale of apetite (0 day)


α 4.25 3.33 4.67

SD 3.20 2.78 2.35

t-test

←⎯⎯⎯⎯⎯⎯⎯ 0.53(p=0.61) ⎯⎯⎯⎯⎯⎯⎯→←⎯⎯⎯⎯⎯⎯⎯ -1.19 (p=0.25) ⎯⎯⎯⎯⎯⎯⎯→

←⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ -0.28(p=0.78) ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→


α 5.5 3.56 5.42

SD 2.38 2.40 3.18

t-test

←⎯⎯⎯⎯⎯⎯⎯ 1.35 (p=0.20) ⎯⎯⎯⎯⎯⎯⎯→←⎯⎯⎯⎯⎯⎯⎯ -1.47 (p=0.16) ⎯⎯⎯⎯⎯⎯⎯→

←⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ 0.05p=0.96) ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→

Table 15. Scale of apetite (7 day)


(p=0.08)I group 7 day 5.5 2.38


III group 0 day 4.67 2.35 -1.27(p=0.23)III group 7 day 5.42 3.18

Table 16. Scale of apetite (difference between 0 and 7th day)


Graph. 24. CGI (7 th day)

CGI- Symptoms of illness were exceptionally severe in

all treated groups. After seven days of the treatment clin-

ically insignificant improvements were observed.Monitoring period was short and therapeutic effects wererelatively satisfying. Reported side effects were mild andmoderate.

Conclusion

According to the obtained data it has been concluded thatapplied antipsychotic therapy did not cause more psy-chophysiological function impairment in treated patients.Psychophysiological functions remained within physio-logical limits and they were not changed significantly. Neuroleptic therapy side effects were minimal, meaningno toxic signs or therapy discontinuations were recorded.

CGI

Table 17. CGI (7th day)

Group I group II group III groupα 3.0 3.4 3.42

SD 0.82 1.13 0.9

t-test⎯⎯⎯←⎯ -0.7 (p=0.49) ⎯→⎯⎯⎯

⎯⎯⎯←⎯ 0.06(p=0.95) ⎯→⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯←⎯ -0.82 (p=0.43) ⎯→⎯⎯⎯⎯⎯

Saètak

Antipsihotici posjeduju {iroki spektar razli~itih fiziolo{kih djelovanja, a ova dejstva se razlikuju ovisno od klase ukoju spada odre|eni antipsihotik. Ovi lijekovi indicirani su u lije~enju akutnih psihoti~nih oboljenja. Osnovni ciljna{eg epidemiolo{kog istraìvanja, koje je po svom dizajnu prospektivno i klini~ko-farmakolo{ko, bio je utvrditiincidenciju neèljenih efekata antipsihotika, ordiniranih kod shizofrenih bolesnika na akutnom psihijatrijskom hos-pitalnom odjeljenju, jer do danas nije ura|ena valjana studija u tom pogledu. Prevalencija neèljenih efekata izaz-vanih neurolepticima, prema literaturnim podacima, kre}e se u {irokom rasponu od 24% do 74%. Razli~ite stopeprevalencije su posljedica razli~ite primijene neuroleptika u istraìvanjima, na~ina njihove aplikacije i metodadetekcije neèljenih efekata. S tim u vezi postavljene su hipoteze o odnosu antipsihoti~ne terapije i pojave neèljenihefekata. Istraìvanje je obuhvatilo shizofrene pacijente koji su bili hospitalizirani u periodu od 31.12. 1999. do 31.01. 2000. godine na Intenzivnoj njezi odjeljenja za biolo{ku psihijatriju Psihijatrijske klinike u Sarajevu. Pacijentisu bili podjeljeni u tri grupe prema ordiniranoj terapiji. Kriteriji uklju~ivanja u pomenute grupe su iziskivali da paci-jenti ispoljavaju klini~ki utvr|enu shizofrenu psihozu (F20-29) prema kriterijima ICD-10. Instrumenti istraìvan-ja su bili psihijatrijski intervju, BPRS, skala neèljenih efekata, fiziolo{ki testovi, procjena op{teg klini~kog utiskai samoocjenske skale (skale apetita i potreba za uglji~nim hidratima). Evaluacija je bila klini~ko-psihijatrijska i sta-tisti~ka. Na osnovu procjene rezultata tretmana u sve tri grupe, evidentno je do{lo do pobolj{anja klini~ke slikeakutnog pogor{anja bolesti, ali i pojave neèljenih efekata koji su uglavnom bili umjereno izraèni i podno{ljiviza bolesnike. Niske stope incidencije i ne{to vi{e stope prevalencije uslovile su da se odbaci nulta, a potvrdi alter-nativna hipoteza, prema kojoj je incidencija i prevalencija akutnih neèljenih efekata u granicama o~ekivanja.Intenzitet neèljenih efekata bio je umjeren do blago izraèn, ~ime se prihvata osnovna teza ovog rada, a to je dase pravilnim odabirom klasi~nih ili tipi~nih antipsihotika, kao i njihovim pravilnim doziranjem, smanjuje ukupanskor akutnih neèljeni efekata. Na osnovu procjene fiziolo{kih testova moè se zaklju~iti da primijenjeni antipsi-hotici nisu u ve}oj mjeri uticali na psihofiziolo{ke funkcije pacijenata. Zna~i psihofiziolo{ke funkcije mjerene speci-fi~nim psihofiziolo{kim testovima su ostale u fiziolo{kim granicama, tj. ustanovljene promjene nisu imale bitan uti-caj na ukupni tretman. To potvr|uje i tezu ovog rada da se pravilnim izborom antipsihotika i pa`ljivim doziranjemantipsihoti~ne terapije zna~ajnije ne mijenjaju psihofiziolo{ke funkcije pacijenata. Ni u jednom slu~aju nije bilopotrebe da se prekine lije~enje antipsihoticima, bilo zbog izostanka terapeutske djelotvornosti, ili zbog izraènihneèljenih efekata antipsihoti~ne terapije, odnosno komplikacija u toku lije~enja.


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Abstract:

A prospective case study was conducted in theDepartment of Occupational Medicine, Tuzla. The pur-pose of this study was to indicate negative effects fromoccupational exposure to mercury on behavioural andmental health, memory and psychomotor function thatwas tested in 46 chloral-alkali plant workers (mean agewas 38. 8+/- 5. 7 years; mean age of occupational histo-ry 16. 5+/- 6. 0 years). Data on toxicological monitoringon atomic absorption spectrometer, and data on mentalhealth were collected, psychiatric and other subjectivesymptoms, and behavioural, psychomotor and memoryfunction tested. The data were compared to controlgroup, 32 healthy non exposed workers. The study wasdesigned to assess blood and urine mercury levels andlength of occupational exposure and investigate its rela-tionships to effects on the mental health. The mean airmercury levels were 0. 23 mg/m3, the mean blood mer-cury concentrations was 3. 6 µg/ dl and the mean urinemercury concentrations were 151.7 +/- 180.4 µg/l. In 25(53%) workers exposed to mercury vapour was identifiedDepression-Hypochondrias Syndrome (p trend < 0. 001)with higher scores for scales: Hysteria (p trend <0. 001),Schizoid and Psychoastenia (MMPI). All psychologicalparameters were in highly significantly correlations withmercury levels and length of occupational exposure.Pathological parameters were possible general identifiedif the concentration of blood mercury levels are >2. 9 µg/dl, or urine mercury levels > 87 µg/l workers exposed tomercury vapour knew that toxic effects in body result-ed in loosing some of intellectual abilities, and that peo-ple who handle chemicals had an increased health risk(ESW questionnaire). The occupational mercury exposedworkers had introvert behaviour (EPQ). Aggressivenesswas found in 71.7% workers. The cognitive disturbances:short-term memory loss, difficult to concentrate on taskswhich require attention and thinking, were significantlydiffered compared to those of controls (p trend < 0. 001).In 24 (52%) exposed to mercury workers we have deter-mined ego strength loss and regressive defensive mecha-nisms (LB). Handwriting disturbances-micrography wehave identified in 27 (58.7%) workers.

Keywords: poisoning to mercury vapour, chloral-alkaliplant workers, neuropsychological consequences

Introduction:

Chronic exposure to elemental mercury vapour is knownto cause major effects on the central nervous system.Behavioural effects of occupational mercury exposurehave been described for many years in numerous clinicalreports of exposure to metallic mercury, inorganic mer-cury compounds, and organic mercury. Recently, quanti-tative psychomotor testing of workers with chronic low-level exposure to inorganic mercury has expanded ourknowledge of the behavioural effects of mercury expo-sure. Mercury vapour poisoning is an uncommon butserious occupational disease causing a prominent neuro-toxic syndrome (1-4).

This study described chronic mercury poisoning in agroup of 46 men actually exposed to mercury vapourwhile performing routine maintenance work on the mer-cury cell of chloral-kali plant. Excessive exposure tomercury results in neurotoxic effects. There are still con-cerns that current occupational exposure levels maycause sub-clinical effects on the nervous system (3-6).Recent data have shown that mercury toxicity causeslong-term, probable irreversible neurological conse-quence (7). Central nervous system are known to accu-mulate mercury after vapour exposure and Buchet andcolleagues have reported that in a rat model brain con-centrations of mercury were not influenced by chelationtherapy (8). Additionally, Hargreaves and colleaguesreport a patient with mercury intoxication who had clini-cal improvement after chelation therapy, but 16 years let-ter at autopsy was found to have expansive elementalmercury deposits the brain (9). After long-term mercury vapour exposure, howeverbehavioural effects are less well recognized.

Methods:

A group of 46 men were exposed to elemental mercuryfor more than ten years while performing planned workon mercury cell lines at a chlorine manufacturing plantlocated in north-eastern Bosnia. The plant uses a mercu-ry cathode as a catalyst. The data were compared to con-trol group 32 non-exposed to chemical workers. Thegroups were identical in other common parametersincluding age, gender (all men), occupational history,

Assessment of chronic neuropsychologicaleffects of mercury vapour poisoning in chloral-alkaliplant workers

Nurka Pranji}, Osman Sinanovi}, Jasenko Karamehi}, Ru{id Jakubovi}, Clinical Hospital Tuzla, Department of Occupational Medicine, Department of Neuropsychiatry, Department ofPharmacology, University School of Medicine Tuzla, Bosnia and Herzegovina

alcohol consumption and smoking, place of living(urban-rural), and level of education (p< 0. 05). Workershad serial measure of blood mercury and urine mercury.The parameters of this monitoring were determined byatomic absorption spectrometer. Data on toxicologicalmonitoring, data of mental health were collected, psychi-atric and other subjective symptoms (by questionnaireself report scale), psychomotor and memory functiontested. For all workers (experimental and control groups)a battery of standard psychological tests was conducted.Testing on behavioural function was performed between5-6 months of age and included numerous measureinstruments: Environmental worry scale (EWS),Minnesota Modified Personal Inventory (MMPI), Purduetest of intelligence measuring intellectual function (test-ed on standard 25 minutes, and adapted for measureshort-term memory functiontested 10 minutes), visual-motor gestalt test Benders (tests of cognitive and visual-motor ability- LB), Eysenck Personality Inventory(EPQ) and Finger-tapping test.

Statistics: Statistical testing included Students t test andFishers exact test. The analysis of variance was carriedout comparing neurobehavioral tests. A probability factorof the p trend below < 0. 05 and < 0. 001 was consideredas significant.

Results and Discussion:

Table 1 has shown that the means and standard deviationair mercury concentration in all months of year in chlo-ral-alkali plant crossed over maximal permissible limit(MPL) 0. 05 mg/m3. The highest levels of the air con-centrations were found in summer-months (in the Augustwas 0.4 mg/m3, eight times higher than MPL) (3). Average air mercury level was 0. 23 mg/m3, averageblood mercury concentration was 3. 55 µgr/dl, and aver-age urine mercury concentration was 151.7+/- 180.4 µg/l(monthly retrospective urine samples over the past yearwere also available). Blood and urine mercury levels thecontrol subjects have been at the lower level of detectionin urine 2. 3+/-6. 2 µg/l. The results currently found animportant number of workers (in 43 of 46) with a urinemercury level greater than 50 µg/l, which is considered athreshold for health risk by several authors (1, 3, 7).

The most frequent the central nervous system symptomsin experimental groups (table 1) and were significantly inrelationship to control subjects were dizziness, have ofinjustice, to get angry, fatigue, suspicious, tremor andsleep changes (p trend <. 001). But all other evaluatedsymptoms were significantly in relationship to controlsubjects except have forgetfulness (p<. 05). This is anagreement with the results obtained by other authors (2-4).

The mean urine mercury concentrations dropped from 80µg/l to 700 µg/l (figure 2) in exposed workers (the meanurine mercury concentrations was 151. 7 +/- 180. 4 µg/l),while mean urine mercury concentrations in control sub-jects was 2. 37 +/- 6. 24 µg/l). The average blood mercu-ry monitor was 3. 55+/-2. 26 m g/dl (in control subjects5. 655x 10-1 +/- 0. 15).

Dizziness, fatigue, easy to get angry, suspicious, sleepchanges, tremor of hands, to have injustice, were mostprominent behavioural symptoms in exposed to mercuryworkers and were in highly significantly correlations inrelationships to symptoms of control subjects (p trend <0. 001). But all other tested behavioural symptoms:dispirited, to spend oneself, headache, panic, were in sig-nificantly correlations in relationships to symptoms ofcontrol subjects except to have forgetfulness (p trend<0.05; table 2). This is an agreement with the resultsobtained by other authors but not in all parameters (10,14). Our findings included neurobehavioural symptom abnor-malities in 25 of 46 workers from MMPI test (table III).The workers in experimental group (EG) had increasedvalues of scores from Depression (D), Hypochondria(Hs), Hysteria (Hy), Psychoastenia (Pt), andSchizophrenia (Sc) scales in comparison to the controlgroup. The EG results point to significantly increasedvalues of neurotic symptoms as well as characteristicMMPI profiles which are in direct connection to the levelof mercury exposure (figure 1). It means that is true thehypothesis the greater the mercury exposure, the greaterthe neurological impairment. The blood mercury dosewas generally associated with toxicity (7, 11, 13). EG distinguished the profile of symptoms Hy-D-Hs-Pt-Sc, which involve the expressive inclination tohypochondriac fixation for some symptoms and schizo-phrenia interpretation, has a tendency of psychotic devel-opment. Psychoastenia symptoms happen as a type ofassociation between emotional disturbances and somaticfunctioning. Workers exposed to mercury knew that toxic effectsresulted in loosing some of intellectual abilities (ESWquestionnaire).

Neuropsychological testing (Purdue, standard) showednormal levels of intellectual functioning in both groups.But, there was significant correlation between results oftesting modified Purdue. It means that mercury exposedworkers had difficulty in concentrating on various tasks.Although they showed no particular deficits in memory,psychomotor performance, learning ability, their moststriking deficit was one of impaired concentrations whichresulted in erratic performance on various tests. This dis-order was manifest primarily in emotional symptomswhich had secondary effects on performance on stan-dardized tasks of psychological performance (16).



In the first year of exposure to mercury the cognitiveabilities decreased in poisoned workers, and than after 10years of exposure to mercury. After 25 years there areever low scores of cognitive abilities. But this figureshowed that chronic exposure to mercury may results inshort-term memory disturbances in workers even whenthey had lower mercury doses. Memory dysfunction wasin relationship to duration time of exposure. Further evaluation in this group of mercury exposedworkers of the neuropsychological effects is underway,but long-term effects are evident (Depression-Hypochondria Symptom, short –term memory dysfunc-tion, tremor). Aggressiveness was found in 71.7%. The cognitive dis-turbances: short-term memory loss, difficult to concen-tration tasks which require attention and thinking weresignificantly deteriorated compared to those of controls(p trend < 0. 001). In 24 (52%) we have determined ego

strength loss and regressive defensive mechanisms (LB).Handwriting disturbances-micrography we have identi-fied in 27 (58.7%) exposed workers. We have identifiedfine tremor of the hands in 80 % workers. All psychological parameters were in strong correlationwith mercury levels and time-duration of exposure. Thenegative psychological effects we have detected of > 2. 9µgr/dl blood mercury levels and of > 87 µg/l urine mer-cury levels. This phenomenon has been described by oth-ers authors (7).

Conclusion

Exposure to mercury vapour toxicity causes long-term,probably irreversible psychological consequences. Inconclusion the results obtained showed a strong correla-tion between behavioural changes and blood and mercu-ry concentrations and duration time of exposure.

Mercury concentration Experimental group M +/-SD Control group M +/-SDBLOOD µg/dl 3. 55 +/- 2. 36 5. 655x 10-1 +/- 0. 15 URINE µg/l 151. 69 +/- 180. 41 2. 37 +/- 6. 24

AIR mg/m3 0. 23+/- 0. 19 -

Groups: Experimental ControlSYMPTOMS: p N % N %

DISPIRITED * 13 28.3 6 18. 8

DIZZINESS ** 29 63.0 2 6. 3

HAVE OF INJUSTICE ** 41 89.1 10 31.3

TO GET ANGRY ** 39 84.8 7 21.8

FATIGUE ** 39 84.8 6 18.8

SUSPICIOUS ** 15 32.6 3 9.4

TO SPEND ONESELF 33 71.7 7 21.8

HAVE FORGETFULNESS ** 27 58.7 13 40.6

HEADACHE * 25 54.3 12 37.5

PANIC * 27 58.7 10 31.3

TREMOR ** 27 58.7 6 18.8

SLEEP CHANGES ** 32 69.6 8 25.0

* p < .005 ** p < .001

Table 1: Blood, mercury and air concentration of mercury in experimental and control subjects

Table 2 Summary of central nervous symptoms reported by all evaluated workers


MEAN VALUES AND GROUPSPERSONAL INVENTORY Experimental Control Groups p

Hypochondria (Hs) 69. 6+/- 18. 1 51. 2+/- 10. 5 **

Depression (D) 63. 4+/- 17. 5 48. 0+/- 13. 2 **

Hysteria (Hy) 64. 4+/- 15. 6 51. 2+/- 10. 0 **

Psychopathy (Pd) 47. 8+/- 9. 4 45. 4+/- 8. 0

Paranoia (Pa) 52. 6+/- 14. 8 46. 4 +/- 12. 0

Psychoastenia (Pt) 57. 0+/- 9. 5 50. 7+/- 9. 0 *

Schizophrenia (Sc) 54. 1+/- 9. 7 48. 3+/- 8. 3 *

Hypomania (Ma) 52. 7+/- 11. 9 50. 1+/- 9. 7

Table 3. The mean scores of scale MMPI for groups

In 25 of 46 workers exposed to mercury vapour wasidentified Depression-Hypochondrias Symptom (p<0.001**) with higher scores for symptom scales:

Hysteria (p<0. 001), Schizoid and Psychoastenia(MMPI). This results is in accordance with the resultsobtained by other authors (3, 6,7).

Figure 1: Correlation found between blood mercury concentration and the T mean scores for scale Depression (D)from Minesota Multiphasic Personality Inventory (MMPI) in workers exposed to mercury vapour. This figure showedthat abnormal high T scores (>70) for scale Depression occurred when blood mercury concentration exceeded 3.6 µg/dl,but two workers had high scores when blood mercury concentration was < 1. 8 µg/dl.


Figure 2: Correlation found between blood mercury concentration and the T-scores of symptom scale introversion/extroversion (I/E) – Eysenck questionnaires (EPQ) in workers exposed to mercury vapour. This figure showed thatwhen blood mercury concentration exceeded 3.6 µg/dl workers had introvert behaviour. This phenomenon has beendescribed by others authors (4, 9, 13).

Figure 3: Correlation found between the scores of scale " Purdue 10" and age of duration of exposure to mercuryvapour in experimental group.


References:

1. Chang LW. Neurotoxic effects of mercury- a review. Environmental research 1977; 14: 329-373.2. Bunn WB, Mc Gill CM, Berber TE, Cromer JW, Goldwater Lj. Mercury exposure in Chlor-alkaly plants.

American Industrial Hygienist Association Journal 1986; 47: 249-254.3. Anonymous. Occupational mercury poisoning. Morbidity and mortality Weekly report 1980; 29: 393-395.4. Smith RG. Dose response relationship associated with known mercury absorption at dose levels of inorganic

mercury. In: Environmental Mercury contamination, eds: Harting R, Dinman B, Ann Arbor, Micigan: Arm ArborScience Publishers, 1972. pp 207-222.

5. Triebig G, Buttner J. Neurotoxic occupational substances: Metal and their compounds. A literature review of theyears 1970-1982. Zentblatt fur Bakteriologie Microbiologie and Hygiene 1983; 177 : 11-36.

6. Goyer Ra. Environmentally related diseases of the urinary tract. Medical clinics of North America 1990; 74 :377-389.

7. Bluhm Renata, Bobbit GR, Larry WW, Alastair JJ, Wood J, Bonfiglio F,sarzen C, Andrew JH, Branch RA.Elemental Mercury Vapour Toxicity, Treatment and Prognosis After Acute Intensive Exposure in Chlor-alkalyPlant Workers. Neuropsychological Findings and Chelator 1992; 11 : 201-210.

8. Buchet JP, Lauwerys RR. Influence of 2,3 Dimercaptopropane -1-sulfonate and dimercaptosucinic acid on themobilization of mercury from tissues of rats pre-treated with mercuric chloride, phenilmercury acetate or mer-cury vapours. Toxicology 1989; 54: 323-333.

9. Hargreaves RJ, Evans JG, Janota I, Magos L, Cavanogh JB. Persistent mercury in nerve cells 16 years aftermetallic mercury poisoning. Neuropathology and Applied Neurobiology 1988; 14: 443-452.

10. Andersen A, Ellingsen DG, Morland T, Kjuus H. A neurological and Neuropsychological Study of Chlor-alkaliWorkers Previosly Exposed to Mercury Vapour. Acta Neurologica Skandinavica 1993; 88 (6): 427-433.

11. Bluhm R, Bobbit GR, Larry WW. Elemental Mercury Vapour Toxicity, Treatment and Prognosis After AcuteIntensive Exposure in Chlor-alkaly Plant Workers. Neuropsychological Findings and Chelator1992; 11: 201-210.

12. Fiedler NC, Maccia C, Kipen H. Evaluation of Chemically Sensitive Patients. Journal OccupationalMedicine1992; 34(5): 529-53.

13. Gothe CJ, Molin C, Nilsson CG. The Environmental somatization syndrome. Psychosomatics 1995; 36(1): 1-1.14. Mullerminy H, Erber D, Moller H, Mullerminy B, Bongartz G. Is there a hazard to health by mercury exposure

from amalgam due to MRI. Journal Magnetic Resonance Imaging , 1996; 6 (1): 258-260.15. Stewart De, Raskin J. Psychiatric assessment of patients with “ 20th century disease (“total allergy syndrome”).

Canadian Medical Association Journal 1985; 33: 1001-1100. 16. Yeates KO, Mortensen ME. Acute and chronic neuropsychological consequences of mercury vapour poisoning

in two early adolescents. Journal of Clinical Experimental Neuropsychology 1994; 16(2): 209-222.


Abstract:

The aim of this study was to made assessment of healtheffects in 37 workers exposed to gasoline, and its con-stituents at gasoline stations between 1985 and 1996.Thirty-seven persons who had been exposed to gasolinefor more than five years were examined. The evaluationincluded a medical / occupational history, haematologicaland biochemical examination, a physical exam, standard-ized psychological tests, and ultrasound examination ofkidneys and liver. The groups were identical in othercommon parameters including age, gender (all men), andlevel of education (P<0. 05). The data were compared totwo control groups: 61 healthy non-exposed controls and25 workers at gasoline stations exposed to organic leadfor only nine months. Peripheral smear revealedbasophilic stippling and reticulocytosis. We found inchronic exposed gasoline workers haematological disor-ders: mild leukocytosis (7 of 37), lymphocytosis (20 of37), mild lymhocytopenia (3 of 37), and decrease of redblood cells count (11 of 37). Results indicated that theyhave suffered from liver disorders: lipoid degeneration ofliver (14 of 37), chronic functional damages of liver (3 of37), cirrhosis (1 of 37). Ultrasound examination indicat-ed chronic kidney damages (8 of 37). These results sig-nificantly differed from those of controls (P< 0.05). In 13out of 37 workers at gasoline stations exposed to gasolinefor more than 5 years the symptom of depression anddecreased reaction time and motor abilities were identi-fied. The summary of diseases of workers exposed toorganic lead and gasoline are discussed.

Keywords: occupational exposure, long-term exposureeffects, tetraethyl lead, gasoline constituents, oxygenates,haematological effects, physiological effects.

Introduction

Gasoline is mixture of saturated and unsaturated hydro-carbons. Gasoline fuels contain approximately 62% alka-nes, 7% alkenes, and 31% aromatics and additives (1).Typically, gasoline contains more than 150 chemicals,including small amounts of benzene and tetraethyl lead.Many of these are toxic; some such as benzene are car-cinogenic. Gasoline presents a serious health hazard,which is rapidly being exacerbated by the increasingnumber of cars on the road. Tulsa, an industrial town of about 165 000 inhabitants, is

located in north- east Bosnia and Herzegovina. The num-ber of cars designated to be used with the leaded petrolhas doubled in our area in the last ten months from 35000 to 70 000 cars. Every day 59 workers employed atTulsa gasoline stations work directly with gasoline.Workers exposure occurring during the pouring of gaso-line result in chronic gasoline poisoning (2). Many the harmful effects seen after exposure to gasoline,is due to the individual chemicals in the gasoline mixture,such as benzene, lead and oxygenates. Breathing smallamounts of gasoline vapours can lead nose and throatirritation, headaches, dizziness, nausea, vomiting, confu-sion and breathing difficulties. Some effects of skin con-tact with gasoline include rashes, redness, and swelling.Allergic reactions (hypersensitivity) have been reportedbut these are rare occurrences (3-7).

Gasoline vapours can cause central nervous system(CNS) depression (8). Prolonged and repeated exposureto n-hexane can cause irreversible damage to the periph-eral nervous system. Two types of nervous system actionare seen: an acute narcotizing effect induced by high con-centrations and axonal neuropathy associated with repet-itive or continuous exposure to n-hexane (9).

Lead can produce adverse effects, not only on mentalfunctions, virtually on every system of the body. Ninetypercent or more of the lead in the body eventually accu-mulates in the bones and may stay there for a lifetime(10). In the United States, the Prevention Act of theEnvironmental Protection Agency (US EPA) passed reg-ulations in 1973 to rule out the use of lead in gasoline(11). Such act has not been passed in European countries.The excretion of inorganic and total lead was investigat-ed in the urine of workers who were exposed to tetraethyllead (11-12). Long-term exposure has been shown to leadmodified number of red blood cells (13). Lead, even atlow levels of exposure, is now recognized to be toxic,and it is difficult to discern a threshold for lead toxicityin a population exposed to lead at levels nearly 200 timesthose experienced by pre-industrial humans (14). Almostall of the blood lead is in erythrocytes, from which it istaken up by soft tissues and stored primarily in bone. Thissubstance has demonstrated half-lives for blood and softtissues, which can be less than a month and long-termstorage in bones with half-life of more than 20 years. Thetoxic effects of lead on the haemopoietic system, andkidneys are well known. Less clear, however, are the

Assessment of health effects in workers at gasoline station

Nurka Pranji}, Hamza Mujagi}, Mahmud Nurki}, Jasenko Karamehi}, Slobodan Pavlovi}University of Tuzla, TuzlaInstitute of Occupational Medicine, Tuzla, Bosnia and Herzegovina

toxic effects of this metal on the liver (15-16). As of now,there are still different views on the existence of negativeeffects of lead on the human and animal liver exposed tothis metal. Chronic, heavy exposure to volatile leadedgasoline results in encephalopathy, cerebellar and corti-cospinal symptoms and signs, dementia, mental statusalterations, and persistent organic psychosis (17).However, heavy metal lead may cause carcinogenicchanges (18-19). Some studies in gasoline- exposedworkers indicated an increased mortality risk from malig-nant melanoma. This was result of exposure to gasoline,benzene, or sunlight, or combination of these factors (20-23). Some laboratory animals that breathed high concen-trations of unleaded gasoline vapours continuously for 2years developed liver and kidney tumours. However,there is no evidence that exposure to gasoline causes can-cer in humans.

Benzene is an aromatic organic hydrocarbon present ingasoline. Benzene toxicity involves both marrow depres-sion and leukemogenesis caused by damage to multipleclasses of heematopoietic cells and variety ofhaematopoietic cell functions (24-26). In milder forms ofbenzene toxicity individual cytopenias may occur:anaemia, leucocytopenia and thrombocytopenia (26-28).The production of benzene metabolites, largely in theliver, is followed by their transport to the bone marrowand other organs. The covalent binding of hydroquinoneto spindle fibre protein could explain inhibition of cellreplication by benzene (29). Benzene and their metabo-lites were added impacts on oxidative stress and antioxi-dant factors (30).

The effects of lead are always the same regardless ofwhether it enters the body through breathing or swallow-ing. The no-effect level for lead is not known (31-35).The literature on the toxicology of lead provides littleevidence of the neurotoxicity of organic lead (36). It hasbeen well known for decades that organ-lead compoundsare potent neurotoxics on the central nervous system.Triethyl lead, the major metabolite of tetraethyl lead, wasshown to disrupt cytoskeletal elements, particularly neu-rofilaments, at very low levels (nanomolar concentra-tions) (37-45). Occupational exposure to gasoline hasbeen associated with numerous neurological signsincluding effects on intellectual capacity, modificationsof psychomotor and visual-motor functions and delayedmemory (45-50). After long-term gasoline lead expo-sure, however behavioural effects are less well recog-nized.

Oxygenates are used as antiknock agents in place of leadderivatives and as substitutes for high octane aromatics infuel (51-52). Oxygenates include substances such asethanol, methanol, methyl tertiary butyl ether (MTBE),ethyl tertiary butyl ether (ETBE), tertiary butyl alcohol

(TBA), and tertiary amyl methyl ether (TAME). Short-term effects symptoms of oxygenates such as nausea,headaches, and sensory irritation were reported. Cancereffects tumours have been observed at multiple sites inrats and mice after exposure to high levels of MTBE.After high MTBE exposure, female mice showedincreased incidence of hepatocellular adenoma and malerats showed increased incidence of renal tubular celltumours and interstitial –cell testicular tumours (53-54).Methanol is well absorbed in humans following inhala-tion. It produces a transient mild depression of CNS withheadache, vertigo, and vomiting. There is little evidencefrom available information of human health effects fromlow-level exposure which demonstrates that methanolvapours as a gasoline additive can cause acute adverseeffects to health (55-57). Few studies have been conduct-ed on the health effects of other oxygenates, such asETBE or TAME. They deserve substantial investigationof they are likely to be placed in widespread use (58-59).

Gasoline engines always produce carbon monoxide(CO). When CO is inhaled it bounds with haemoglobin,displacing oxygen and forming carboxyhaemoglobin in alack of oxygen to the body cells. Continued exposure canlead to vomiting, loss of consciousness, brain damage,heart irregularity, breathing difficulties, muscle weak-ness, and even death (60).

Exposure to gasoline compounds and lead in our countryhas not been controlled. We studied the relationshipbetween gasoline exposure and the effects of inhaledgasoline in a group of workers at gasoline stations, andevaluated a spectrum of potential exposure effects.

Subjects and methods:

A prospective cohort study was conducted in the Instituteof occupational medicine Tuzla. Persons who had beenexposed to gasoline for more than five years between1985 and 1996 entered the program. Exposure to gaso-line occurs by breathing vapours when filling cars fueltank.

During refuelling, total hydrocarbon concentration in theair fall within the range of 20- 200p p m by volume. InTuzla the lead content in the petrol additives is 0. 6 g/L.Benzene concentration in gasoline formulation is 3% byvolume in unleaded gasoline and 2.7% in leaded gasolinecurrently used at Tuzla stations. (2) The ratio between thedifferent gasoline mixture in 1997 was as following:leaded gasoline: unleaded gasoline: “diesel” gasoline(1:4:3). (2) About 110 million people are exposed togasoline constituents in the course of refuelling at self-service gasoline stations (61), an operation that requiresonly a few minutes per week, accruing to approximately


100 min/year. Well, the workers at gasoline stations areexposed to gasoline constituents many hours a week,approximately 8 086 800 min/year.

The study comprised 120 male participants divided intofour groups. All the participants worked under specialoccupational conditions. As such, they are obliged toundergo a systematic medical check-up every year.Clinical and laboratory evaluations included medical /occupational history, a thorough physical exam, tests ofcognitive and visual motor ability (Bonardel) and psy-chological symptom scale (Minnesota MultiphasicPersonality Inventory) and Purdue Pegboard- manualdexterity test) (62). Haematological and biochemicalexamination also were conducted, ultrasound examina-tion of kidneys and liver, complete blood cell counts,serum bilirubin concentrations (to 21µmol/L, normalrange), liver enzymes, and qualitative urine analyses.Complete blood cell counts included: red cell count(RBC, 4. 5- 5. 5 x 1012 /L, normal range), total white cellcount (5-10x 109 /L, normal range), absolute lymphocytecount (2500-3500, normal range), reticulocyte count (3-15 /103 RBC, normal range).

The long-term gasoline exposed group was composed of37 petrol-station workers of an average age 41. 5 +/- 6. 9years, who had a chronic exposure to petrol andtetraethyl lead for 18.3 +/- 8.4 years. The nitrous vapourexposed group was composed of 31 chemical workerswho worked outdoors and who had accidental nitrousvapour poisoning. They were of an average age of40.4+/-6.2 and length of occupational time 18.5 +/ - 5.9years. The non- exposed group consisted of 30 driversand keepers in the hospitals. They were of an average ageof 38.7 +/-9.0 and length of occupational time 16.3 +/-7.3 years. These persons served as a basic control group.The short-term gasoline exposed group consisted of 22young men, whose average time of occupational expo-sure was only 9 months. No effects of exposure to petroland organic lead were expected in this group and it wasintroduced as a control to the long-term gasoline exposedgroup. All subjects were men. The workers who leftTuzla during the war (1992-1996) were not included.These subjects were co-workers with similar sociologi-cal-economic and intellectual backgrounds. All groups(except for the young workers at the gasoline stations)were comparable in other common characteristics includ-ing: age, gender (all males), level of education, andoccupational history (ANOVA P<0. 05). The summary ofmorbidity of workers exposed gasoline was evaluated.Microscopic observations of peripheral smear were per-formed for search of basophilic stippling and neutrophilictoxic granulation. There was no activity, dietary or smok-ing restrictions during this time. At all the workplaces (allsubjects work out), the conditions were unfavourable:temperature was to low in winter and to high in summer.

All subjects brought stressful life with a lot of traumaticevents and psychological consequences for majority ofworkers who lived in the country during the war.Smoking status was classified as current smoker, formersmoker, or no smoker.

All workers were also given a comprehensive psycholog-ical symptom (self-report) scale Minnesota MultiphasicPersonality Inventory (MMPI). The test measures dis-tress in eight psychological areas (hypochondria, depres-sion, hysteria, psychoastenia, paranoia, psychoticism,hypomania, schizophrenia,) by having the patient rate.The test measures distress in 201 items ranging from thetrue to no true (63). Psychological analyses were doneconsulting the psychologist.

Statistical testing included Student t test and the Fisher’sprobability exact test. As multivariate technique a one-way analysis of variance (F-test) was carried out com-paring the red cell count, total white cell count, differen-tial blood cell count, reticulocyte counts, age, occupa-tional exposure, and neurobehavioral tests. Results werecompared Multiple range test (for the effects of these fac-tors), paired Student t test. A relationship was designatedas statistically significant when the P was < 0.05 and <0.001.

Results:

Peripheral smear revealed basophilic stippling and retic-ulocytosis in 25 of 37 in long-term gasoline exposedworkers, and 15 of 22 in short-term gasoline exposedworkers at gasoline stations group. The mean red bloodcell count (RBC) in both gasoline exposed groups waslower (4.55 x 1012/l +/- 0.21x 1012/L) comparing to thosein controls groups (4.75 x 1012/l +/-0.23 x 1012/L). Thelowest number of RBC were found in the workers inlong-term exposed group of workers 40 to 44 years ofage, and who had 19 and more years of occupational his-tory. These findings were statistically significant accord-ing to a multivariate analysis of variance (P < 0.05).Eleven of 37 long-term gasoline exposed workers suf-fered from decrease RBC, and 3 of 22 short-term gaso-line exposed workers. The analysis of mean count ofRBC and the mean numbers of age indicated that thelowest counts of erythrocytes had workers from 40-44years (duration of exposure from 14 -19 years). The datashowed a statistically significant relationship betweendecrease RBC count and mean gasoline exposure period(F= 8.75; P<0. 005). Haemoglobin was in its referencevalues (7.76-10.6mmol / L), and only one of gasolineexposed workers had lower value of haemoglobin.

The prevalence of leukocyte in long-term exposed work-ers is derived from the number of greater total white


blood cell (WBC) count - (the mean of WBC 8. 16 x 109

/L) 7 of 37. During the test years, statistically significantdifferences in values of leukocytes between long-termgasoline exposed and non-exposed group (from Studentt- test) (P< 0.05) occurred.

In twenty of 37 long-term gasoline exposed workerslymphocytosis (mean counts of lymphocytes 4100+/-900, table 1) was found. Statistically significant differ-ences between groups in absolute lymphocytes countwere found for all consecutive years (from ANOVA testF=7.41; P<0.05). There were no significant differences inabsolute numbers of lymphocytes between lead exposedgroups (t=0. 38). Lymphocytopenia as indicator of ben-zene toxicity was found in 3 of 37 workers of long-termexposed group and in 3 of 22 short-term gasolineexposed workers.

Total bilirubin has been found in the normal range (6,8-20,4 µmol / L), but there were statistically significant dif-ferences between long-term exposed group and non-exposed group (from paired samples t-test) (t=2.36; P <0.05).

By based ultrasound examination in 18 of 37 workers oflong-term lead exposed group had liver damages. In 14of 37 was found lipoid degeneration of liver, in 3 of 37chronic inflammation changes, and in 1 of 37 cirrhosis.Proteinuria was found in six of 37 of long-term leadexposed workers. From the study data it follows that 11of 37 long-term lead exposed workers suffered chronickidney damages.

The most frequent central nervous symptoms in long-term lead exposed workers was headache, fatigue, suspi-cious, sleep changes. The psychological testing analysesusing the “Purdue Pegboard test”- standard manual dex-terity test, showed that there were no significant differ-ences between the groups (from ANOVA). The scores of“Purdue Pegboard” test were in normal levels of intellec-tual functioning in all groups. But, the results of“Bonardel” standard motor control test (among thegroups from ANOVA) (P<0.05; table 2) there was signif-icant correlation between groups. It may mean thatchronic gasoline exposed workers had difficulty in con-centrating on various tasks because they needed signifi-cantly more time (172.0 +/- 69.16 sec) comparing toother groups. It means that gasoline exposed workers hadthe cognitive disturbances: decreased reaction time andmotor abilities.

Results of a Minnesota Multiphasic PersonalityInventory (MMPI) (which are presented in table 3), indi-cated that long-term gasoline exposed workers had statis-tically significantly different (higher) values than othergroups (ANOVA) on the following MMPI scales: scale

of odd answers, control scale, scale of hypochondria,scale of depression, and scale of hysteria (P <0.05).

The summary of morbidity, as shown in table 4, illus-trates that long-term gasoline exposed workers sufferedgastrointestinal and liver diseases (29 of 37), diseases ofgenitourinary system (19 of 37), cardiovascular diseases(18 of 37) and endocrine diseases and metabolism dam-ages in 17 of the cases.

Discussion:

Lead in gasoline remains a major problem although thelead content has decreased in many countries in the lastfew years (18-22). In Tuzla the amount of tetraethyl leadin polluted air in the past years from 1985 to 1996 hasbeen increasing fifteen times from 7501 kg to 115 584kg.

In the long-term gasoline exposed workers were foundmultiple haematological effects: the decreased red bloodcell counts (RBC), the red blood cells are microcytic(founded in 18 of 37 workers), and the increased numberof reticulocytes with basophilic stippling (in 22 of 37)during the entire period of testing. Also, decreased RBCcount was found in 11 of 37 of the long-term gasolineexposed workers, and in 3 of 22 short-term gasolineexposed workers working at gasoline stations. This is anagreement with the results obtained by other authors (33,35, 40).

Amongst long-term exposed workers, the presence ofneutrofilic toxic granulation was in 8 of 37. The researchon the workers exposed occupational to lead has shownsignificant chromatid and chromosomal aberrations inperipheral blood lymphocytes (31,43,49).

Limphocytosis was found in 20 of 37 long-term leadexposed workers. Limphocytosis was no usually found incase of lead exposure. Probable, lymphocytosis has beenassociated by variation by a wide variety of processesincluding minor viral infection or from multiple chemicalexposure in workers at gasoline stations exposed to dif-ferent gasoline compounds too.

The phenomenon is not a single event but a complexinterplay of gasoline constituent exposure (organic sol-vent, tetraethyl lead and other) as revealed by increasedlymphocyte count. It is possible that exposure to gasolineconstituents or tetraethyl lead may cause an impairmentin concomitant stimulation of humoral immunity (19, 21,42, 46,). The results have shown the presence of the lym-phocytopenia (minimal count 1200) in three of 37 long-term gasoline exposed workers (in every evaluated years)and in three out of 22 short-term gasoline exposed work-ers. Decreased count RBC and leukopenia were found in


the same workers, and the reason is due to exposure tobenzene (24,29,30,31). Haematological effects of expo-sure to gasoline are early sign of the gasoline constituenttoxicity (seen in short-term exposed group).

So far, there are still different views on the existence ofnegative effects of lead on the liver of people and animalsexposed to this metal (15-16). The results of the studyanalysis indicated liver damages in 18 of 37. Lead mayplay a role in the liver damages, but exposure to com-pounds of gasoline too.

Lead may produce a chronic interstitial nephropathy,most commonly with blood lead levels greater than 60mg /dl (44). Restek- Samarzija and colleagues suggestedthat lead poisoning in the past, overall duration of leadexposure, and age as a major confounding variable relat-ed to aging process of the kidney (45). Chronic, recurrentlead poisoning with a consequently increasing lead bodyburden can cause impairment in renal function and a con-comitant of humoral immunity (46). In long-term leadexposed workers was found chronic damages of kidneysin 11 of 37 and proteinuria in 6 of 37. This results is inaccordance with the results obtained by other authors(47).The somatic symptoms most commonly described wereheadache and fatigue in gasoline exposed workers. Fifty-eight workers were evaluated for potential health effectsrelated to organic and inorganic lead exposures. Findingsfor which no alternative medical explanations could befound included neurobehavioral abnormalities (18 of 39workers) and sensomotor polyneuropathies (11 of 31workers) (47).

The literature on the toxicology of lead provides littleevidence of the neurotoxicity of organic lead compounds,but n-hexane may produces polyneuropathies too (9).Tetraethyl lead induced changes in the concentration ofglial fibrillary acidic protein (31,43). Organic lead stimu-lates an increase of free arachidonic acid in HL-60 cells.Influence of metabolic inhibitors on metal inducedarachidonic acid liberation (31). Chronic, heavy expo-sure to leaded gasoline results in an encephalopathy,cerebellar and corticospinal symptoms and signs, demen-tia, mental status alternations, and persistent organic psy-chosis. Lead chelation therapy is not rational and has notbeen shown to benefit these patients (48).

Further evaluation in this group of long-term gasolineexposed workers of the neuropsychological effects isunderway, but long-term effects are evident in 11 out of37 (depression-hypochondrias symptoms). These symp-toms were quantified over time with the use of the MMPIas the relating scale, the gasoline exposed workers scoresmarkedly abnormal and remained so throughout a periodof long follow-up, indicating persisting subjective dis-

tress. Other measures of neuropsychological functionthat assased cognitive, visual and motor skills indicatedimpairment at the time of initial testing. Purdue Pegboardscores, which was not significantly different from popu-lation norms, but Bonardel scores worsened significantlyin long-term exposed group. This phenomenon is verycomplex interplay of gasoline constituents poisoning(tetraethyl lead, n-hexane, MTBE, CO, methanol andother), smoking and alcohol consumption, as risk factorsfor neurological and psychological disorders (52, 53, 57,58, 60). Cortical atrophy was more frequently seen in thegas station employees group.

These results suggest the contribution of leaded gasolineto its development (64). None of these workers visited adoctor for psychological abnormalities of any kind.Long-term lead exposed workers had difficulty in con-centrating on various tasks. Schwartz and the associateshad similar finding 1993 (50). Also, effects on behav-ioural changes, memory and psychomotor ability wereseen in long-term gasoline exposed group. The signifi-cant effects were seen in workers exposed for more than5 years.

Finally, the results of summary of diseases (table 4),showed that long-term lead exposed workers sufferedfrom diseases that are probably caused by leaded gaso-line exposure, such as gastrointestinal disorders, diseasesof liver, diseases of genitourinary system, cardiovasculardiseases and endocrine diseases. This phenomenon hasbeen described by others authors (6). High prevalence ofcardiovascular disease may be relatively signs of carbonmonoxyde toxicity. People with coronary artery diseaseare particularly sensitive to this effect because they haveimpaired ability to increase coronary blood flow. Whenblood flow through the heart is not sufficient to meet tooxygen demand, the heart becomes ischemic, resulting inchest pain or ECG changes. Even relatively low CO lev-els may bring on ischemia more quickly for some indi-viduals with coronary artery disease (60).

Conclusions:

The health effects of being exposed to gasoline are usu-ally exposed to many other things that also can causehealth effects. This study clearly demonstrates that gaso-line workers exposed workers suffer long term healthproblems including psychological disorders, mild haema-tological disorders, kidneys damage and liver damage.

Given the conclusive evidence on the negative effects ofleaded gasoline that have been collected worldwide andthe specific results of this study conducted in Tuzla, it isclear that action must be undertaken immediately to pro-tect public health. Preventive measures should be pro-posed and implemented to develop adequate procedures



for reduction of lead in petrol. Such measure would pro-tect both gasoline workers and larger public. As gasolinestation workers are particularly at risk, they should beencouraged through an information campaign to payscrupulous attention to personal hygiene especially handwashing to prevent the absorption of tetraethyl lead andother gasoline constituents through the skin. Thus, the

problem regarding the potential toxicity of gasoline isstill open, even though it is clear that modern unleadedgasoline present less risk to human health due to thelower quantities of benzene and lead. Today is the insuf-ficient data for determination of long-term effects inoccupationally exposed cohorts and inadequate informa-tion on general population exposure.

Long term gasoline exposed Nitrous vapour exposed Non exposed Parameters:(referent range) N=37 N=31 N=30

Leukocyte (5-10x 109 /L) 8. 12 +/- 3.54 * 6. 37 +/- 1. 28 6.43 +/- 1. 14Lymphocyte ( 2500-3500) 4100 +/- 900 * 3600 +/- 400 3600 +/- 200Total bilirubin (6. 8- 20. 4 µmol/l) 11. 97+/- 5. 81 9. 98+/- 3. 20 9.18 +/- 3. 22Aspartate aminotransferase(< 570 nkat/l)

534. 9+/- 374. 47 * 374. 5+/- 104. 9 346.3 +/- 102. 3

Alanine aminotransferase(< 750 nkat/l)

688. 4+/- 208. 8 * 542. 4+/- 283. 9 524.6 +/- 214. 1

ANOVA P< 0.05*Results are presented as mean +/-SD.

GROUPS "Bonardel"- score (sec) (M +/- SD)Long term lead exposed 172.0 +/- 69.16 F=3.45*Short term lead exposed 118.4 +/- 43.05 F=3.45*Nitrous vapour exposed 118.3 +/- 27.22Non exposed 117.7 +/- 42.4ANOVA *P< 0. 05

Table 1 Summary of peripheral blood parameter counts detected by all evaluated workers (exceptyounger workers at gasoline stations)

Table 2 The mean scores (sec) of “Bonardel” for all groups

Chronic occupational exposure to gasoline may cause bone marrow injury and haematopoietic toxicity including leuko-cytose, and lymphocytose. Results were compared to control groups from ANOVA (P<0.05).

A difference in “Bonardel” scores between groups (from ANOVA) indicated that long-term lead exposed workers need-ed significantly more time for realization of test. It means that lead poisoning workers had difficulty in concentratingon various tasks.


PERSONAL INVENTORYLong term

gasoline exposedShort term

gasoline exposedNitrous vapour

exposedNon exposed

(N=37) (N=22) (N=31) (N=30)

Scale of sincerity 60.2+/-10.5 65.1+/-9.5 59.2+/-9.7 59.4+/-9.8Scale of capriciousness 48.5+/-9.4 * 42.9+/-6.3 44.7+/-5.8 45.1+/-5.8Scale of strength 52.3+/-7.9 59.3+/-8.0 54.5+/-7.6 54.6+/-7.5Hypochondria 57.6+/-14.1 * 49.3+/-11.3 46.9+/-7.9 46.9+/-8.1Depression 56.8+/-11.3* 44.0+/-11.2 49.4+/-7.6 49.7+/-7.4 Hysteria 57.6+/-10.6 * 45.9+/-8.4 47.8+/-7.3 48.0+/-7.5 Psychopathy 46.4+/- 9.5 47..3 +/-9.4 45.5+/-7.9 45.3+/-7.6Paranoia 50.0+/-13.7 * 47.1+/-16.2 48.0+/-10.4 47.9+/-10.0 Psychoastenia 52.3+/- 11.3 * 49.2+/-9.9 49.1+/-9.8 49.2+/-9.9Schizophrenia 50.8+/-11.1 * 48.6.9+/-8.3 47.3+/-9.7 47.4+/-10.0Hypomania 54.8+/-9.2 54.9+/-8.6 52.4+/-8.9 52..3+/-9.0

ANOVA *P< 0.05

Table 3 The mean Minnesota Multiphasic Personality Inventory scores for all groups(except younger workers at gasoline stations)

In 13 of 37 long-term gasoline exposed workers was identified high scores for following MMPI scales: depression,hypochondria, hysteria, paranoia, psychoastenia, and schizophrenia (P< 0. 005) compared to other control groups.Results are presented as mean +/-SD.

Long term gasoline exposed Nitrous vapour exposed Non exposed Diseases: N=37 N=31 N=30Infective and viral diseases 2 1 1Tumours 1 - -Anaemia 11* 1 *Endocrine diseases 17* 3 3Mental diseases 2 1 1Neurological diseases 3 1 1

Eye diseases 10* 3 8

Ears diseases - 7 1

Cardio- vascular diseases 18* 7 5

Respiratory diseases 6 7 4

Gastro-intestinal diseases 29* 8 4

Skin diseases 3 8 1

Bone diseases 9 7 5

Genitourinary diseases 19* 4 1

Injuries 2 3 1

ANOVA P< 0.05* - Results are presented as mean +/-SD.

Table 4 Diseases of all evaluated workers (except younger workers at gasoline stations)


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Saètak

Profesionalna izloènost benzinu i njegovim komponentama ne nadzire se u na{oj zemlji. Cilj rada bilo je dugoro~no

otkrivanje zdravstvenih poreme}aja u 37 radnika koji rade na benzinskim stanicama i koji su kroni~no izloèni ben-

zinu vi{e od pet godina. Prospektivna studija tipa kohorte provedena je u Zavodu za Medicinu rada Tuzla od 1985-

1996 godine. Pra}eni su slijede}i parametri: godine starosti, laboratorijske analize krvi i urina, istorija bolesti,

fizikalni pregled, radni sta` (istovjetan periodu izloènosti), ultrazvu~ni pregledi bubrega i jetre, rezultati psi-

hologijskog testiranja. Svaki parametar je bio uspore?ivan sa istim u dvije kontrolne grupe: 61 benzinu neizloènih

radnika. Da bi uo~ili rane neèljene zdravstvene u~inke izloènosti benzinu pratili smo 22 radnika zaposlena na ben-

zinskim stanicama devet mjeseci. U Tuzlanskom podru~ju aerozaga|enje olovom je porasla petnaest puta od 1985

do 1996. godine. Sniène vrijednosti eritrocita na?ene su u 11 od 37 benzinu izloènih radnika i u 3 od 22 mlada

radnika. One poti~u stvaranje novih mladih stanica, pa se u razmazu periferne krvi nalaze bazofilne punktacije i

retikulocitoza. U radnika, dugoro~no izloènih benzinu, otkrivena je: blaga leukocitozu, sniènje vrijednosti

eritrocita, limfocitoza, limfocitopenia (3 od 37), kroni~na o{te}enja bubrega (8 od 37), masna promjenu jetre (14 od

37), kroni~na funkcionalna o{te}enje jetre (3 od 37), i ciroza jetre (1 od 37). Izloènost benzinu ne izaziva karak-

teristi~ne lezije na organima. Rezultati studije bili su signifikantno razli~iti u kroni~no izloènih benzinu i u uspored-

nim skupinama (P<0. 05). U 13 od 37 radnika identificirani su simptomi somatskih poreme}aja li~nosti. Zdravstveni

poreme}aji u radnika kratko izloènih benzinu (devet mjeseci) bili su: limfocitoza i blaga odstupanja na

psiholo{kom planu (2 od 22) u odnosu na kontrolne skupine. (Diskutiran je morbiditet radnika).

Klju~ne rije~i: profesionalna izloènost, efekat dugoro~ne izloènosti, tetraetilno olovo, sastojci benzina, oksi-

genati, hematolo{ki efekti, psiholo{ki efekti.


Abstract

Over the past 13 years mitochondrial defects have beeninvolved in wide variety of degenerative diseases –Parkinson disease, Alzheimer dementia, arteriosclerosis,ageing and cancer. Mitochondria are believed to controlapoptosis or programmed cell death. Disturbance inmitochondrial metabolism has also been implicated inmany common diseases such as congestive hart failure,diabetes and migraine. Scientific investigations haveshowed complexities in mitochondrial genetics, but at thesame time, pathophysiology of mitochondrial diseases isstill enigma. Mitochondria and their DNAs are openingthe era of “mitochondrial medicine”. What we today call“a mitochondrial medicine” is only a part of the wholepanorama of diseases based on disordered mitochondrialfunction.

Keywords: mitochondria, mtDNA, mitochondrial dis-eases Abbreviations: mtDNA – mitochondrial DNA, ATP -adenosine triphosphate, OXPHOS - oxidative phospho-rylation

Introduction

It is interesting to note that the first cellorganelle ever to be linked to a humandisease was a mitochondrion. In 1962,Luft and his co-workers presented evi-dence of mitochondrial dysfunction inone patient - a 27-years old woman

with the highest oxygen consumption ever recorded. Shehad a hypermetabolic state, structurally abnormal mito-chondria and abnormalities in oxidative phosphorylation.Involvement of the thyroid gland was excluded. This raredisorder is known as Luft’s diseases.Almost eight years had passed before a few other patientswere reported with aberrations of mitochondrial function,although with quite different symptoms. In the meantime,a specific mitochondrial DNA was discovered in 1964.Nothing happened until 1988 when a new revolution inmitochondrial pathophysiology came onto scene, accord-ing to the report of an association of different sporadicencephalomyopathies with large deletions of mtDNA.This group of diseases - genetic mitochondrial disordersdue to mutations in mtDNA and dysfunction in electrontransport – have expanded dramatically during previous

13 years. This small mtDNA has become a Pandora’s boxof pathogenic mutation (1). Now, there are more than 100different pathogenic mtDNA pointed mutations and 200mtDNA deletions and insertions linked to the differenthuman diseases (2). In addition, several lines of evidencehave suggested that mitochondrial dysfunctions may playa role in the ageing process, as well as in some of themost common age-related disorders e.g. heart failure,some neurodegenerative diseases and diabetes mellitus(3).

Mitochondrial genomeand mtDNA genetics

Mitochondria are cytoplasmic organelles with double-membrane and they are responsible for cellular energyproduction. Adenosine triphosphate (ATP) is produced inmitochondria in metabolic process known as oxidativephosphorylation (OXPHOS). OXPHOS carries out byfive respiratory chain complexes located within the mito-chondrial inner membrane. This chain is a line of proteincomplexes that combines electron with oxygen to gener-ate potential energy in the form of ATP. A numerous pro-teins of respiratory chain are encoded by two genetic sys-tems - nuclear DNA and mitochondrial DNA. Mitochondrial DNA (mtDNA) is 16.569-nucleotide pair,double stranded circular chromosome located in mito-chondrial matrix (4).

Genetic characteristics of mtDNA are:• Maternal inheritance - mother transmits her

oocyte mtDNA to the offspring. Male gametedoes not contribute the inheritance process.

• Polyploidy - each human cell has a hundredsof mitochondria each containing severalmtDNAs.

• Heteroplasmy - if mutated mtDNA coexist inthe same tissue with normal-wild typemtDNA that is heteroplasmy.

• High mutation rate - 10-20 times highermutation rate in mtDNA than in the nuclearDNA.

For the further information highly recommended webpages are:Mitomap http://www.gen.emory.edu/mitomap.html/GOBASE http://megasun.bch.umontreal.ca/gobase/

Mitochondrial medicine - a key to solvepathophysiology of XXI century diseases

Emina Kiseljakovi}, Radivoj Jadri}, Sabaheta Hasi}, Lorenka Ljuboja, Jovo Radovanovi},Husein Kulenovi} and Mira Winterhalter-Jadri}Institute for Physiology and Biochemistry, Department of Biochemistry, Faculty of Medicine, Sarajevo


Mitochondrial diseases

Mitochondria are essential for the food we eat turninginto energy in the form of ATP. Mitochondrial diseasesresult from the failure of mitochondria to create energy(mitochondria are responsible for the creating of morethen 90% of energy needed by the cell). When they fail,less energy is generated, the whole system is beginning tosuffer and life is severely compromised. Mitochondrialdisease primarily affects children while in adults itbecomes more common.

Commonly Affected Systems inMitochondrial Disorders

The main problems associated with mitochondrial dis-eases - low energy, free radical production and lactic aci-dosis - can result in the variety of symptoms of many dif-ferent organs of the body. This diagram shows commonsymptoms of mitochondrial diseases, of which, mostpeople have a specific spectrum (5).

Table 1 presents some of disorders that can be caused bythe mutations of mitochondrial DNA. Nuclear mutationsor other processes that disrupt mitochondrial function canalso cause these medical conditions. (6)All of these problems start when something goes wrongin mitochondria. Some of them are direct result of theinterruption in energy supply, while others may developas a consequence to secondary build-up of toxic by-prod-ucts. Mitochondria play a central role in regulation of the pro-grammed cell death -apoptosis. These organelles can trig-ger cell death in many ways - by disruption of the elec-tron transport and energy metabolism or by the activationof proteins that mediate process. This mechanism may bean explanation of how mitochondria and their functionscontribute to the pathogenesis of human diseases. During the last 13 years we have witnessed the enormousgrowth of mitochondrial medicine. New investigativetechniques have continuously been adopted.Mitochondrial DNA mutations have been successfullylinked to diseases and disorders affecting virtually everysystem of the human body. Many linkages have been dis-covered recently, suggesting that in the future even more

Alzheimer’s disease Progressive loss of cognitive capacityCPEO (chronic progressive externalophthalmoplegia)

Paralysis of eye muscles and mitochondrial myopathy

Diabetes mellitus High blood glucose levels and various complications

DystoniaAbnormal movements, muscular rigidity; frequently accompanied bydegeneration of the basal ganglia of the brain

KSS (Kearns-Sayre syndrome)CEO combined with retinal deterioration, heart disease, hearing loss,diabetes and kidney failure

Leigh’s syndromeProgressive loss of motor and verbal skills and degeneration of thebasal ganglia; a potentially lethal childhood disease

LHON (Leber’s hereditary optic neuropathy)Permanent or temporary blindness stemming from damage to the opticnerve

MELAS (mitochondrial encephalomyopathy,lactic acidosis and stroke like episodes)

Dysfunction of brain tissue (causing seizures, transient regional paral-ysis and dementia) combined with mitochondrial myopathy and a toxiclevel of acid lactic and acidosis in the blood

MERRF (myoclonic epilepsy and ragged redfibres)

Seizures with mitochondrial myopathy, may involve hearing loss anddementia

Mitochondrial MyopathyDeterioration of muscle, manifested by weakness and intolerance forexercise; muscle often displays ragged red fibres, which are filled withabnormal mitochondria that turn red when exposed to a particular stain

NARP (neurogenic muscle weakness andretinitis pigmentosa)

Loss of muscle strength and coordination, accompanied by regionalbrain degeneration, ataxia and deterioration of the retina

Pearson’s syndromeChildhood bone marrow dysfunction (leading to loss of blood cells)and pancreatic failure


diseases may be linked to the mitochondria. If mitochon-dria are the root for even more diseases, further research-

es of mtDNA could hold the answers to the origin ofmany diseases that plague XX and XXI centuries.

References

1. DiMauro S and Andreu AL. Mutations in mtDNA: Are We Scraping the Bottom of the Barrel? Brain Pathology2000; 10:431-331

2. Howell N. Human mitochondrial diseases: answering questions and questioning answers. Int Rev Cyto l999;186:49 –116

3. Wallace DC. Mitochondrial defects in cardiomyopathy and neuromuscular disease. Am Heart J 2000; 139:S70-S85

4. DiMauro S and Moreas CT. Mitochondrial Encephalomyophaties. Arch Neurol 1993; vol 50:1197-1208

5. Picture taken from web page: http://www.mdausa.org/publications/Quest/q65mito.html

6. Kleiner IM, Bari} I, Borani} M. Vrijednost analize mitohondrijske DNA u klini~koj praksi. Paediatr Croat2000;44:145-150


Abstract

In recent years, drug release/dissolution from soliddosage forms has been the subject of intense and prof-itable scientific developments. Whenever a new soliddosage form is developed or produced, it is necessary toensure that drug dissolution occurs in an appropriatemanner. The pharmaceutical industry and the registrationauthorities do focus, nowadays, on drug dissolution stud-ies. The quantitative analysis of the values obtained indissolution/release tests is easier when mathematical for-mulas that express the dissolution results as a function ofsome of the dosage forms characteristics are used. This work discusses the analysis of data obtained for dis-solution profiles under different media pH conditionsusing mathematical methods of analysis described byMoore and Flanner. These authors have described differ-ence factor (f1) and similarity factor (f2), which can be

used to characterise drug dissolution/release profiles.In this work we have used these formulas for evaluationof dissolution profiles of the conventional tablets in dif-ferent pH of dissolution medium (range of physiologicalvariations).

Key words: dissolution testing, difference factor, simi-larity factor, quantification, comparison, solid dosageforms

Introduction

Over the years, dissolution testing has been employed asa quality control procedure in pharmaceutical production,in product development to assist in selection of a candi-date formulation. It was also used in research to detectthe influence of critical manufacturing variables, such as:binder effect (1), mixing effect (2), granulation procedure(3), coating parameters (4) and/or in comparative studiesof different formulations (5), in in vitro-in vivo correla-tions (6).

It becomes apparent that sensitive and reproducible dis-solution data derived from physicochemically and hydro-dynamically defined conditions are necessary in order tocompare various in vitro dissolution data. Such resultscan be used: as a surrogate for possible in vivo bioavail-ability, bioequivalence testing, and in vitro-in vivo corre-lation (ivivc). However, the influence of technologicaldifferences and process variables involved during manu-

facturing often complicates the decision making processin selection of the appropriate dissolution method andsubsequent data interpretation technique. Skoug and co-workers (7) stressed that this consequence is the reasonfor dissolution studies. The defined specifications sooften generate strong interest during regulatory review ofsolid oral dosage forms.

The currently available USP-25 has been one of the mostvaluable references to pharmaceutical scientists involvedin the area of dissolution studies. The available dissolu-tion methods within individual drug monographs havebeen divided, into immediate and controlled or extendedrelease products. Except the prominence given to differ-ences in specifications such as tolerance (Q) valuesbetween immediate and controlled release products, thereare no substantial differences in the methodologies usedto test these products.

When regulatory activities are concerned Emea-cpmpNote for Guidance (8) suggests below mentioned purposeof dissolution testing:

• to get information on the best test batchesused in bioavailability/bioequivalence stud-ies and pivotal clinical studies to supportspecification for routine quality control

• to be used as a tool in quality control todemonstrate consistency in manufacture

• to get information on the reference productused in bioavailability/bioequivalence stud-ies and pivotal clinical studies

• to compare reference products from differentMember States

• to help to ascertain similarity between differ-ent formulations of a drug substance (varia-tions and new, essentially similar productsincluded) and the reference medicinal product

Expected differences in dissolution test methods mayinclude variation in the pH of the dissolution mediumdepending on where the drug dissolves or depending onthe drug release rate, drug solubility and absorption pro-file. In most cases, the monographs are not up to date.The necessary refinement reflecting the recent advancesin research finding with the respect to changes in mediaand methods are not included.

Mathematical methods for quantification andcomparison of dissolution testing data

Edina Vrani}1, Aida Mehmedagi}2, Sabira Had`ovi}1

1Department of Pharmaceutical Technology, 2Department of Pharmacokinetics, University of Sarajevo, Faculty of Pharmacy, ^ekalu{a 90, Bosnia and Herzegovina

In the past decade many approaches have been proposedfor the comparison of dissolution profiles. In spite of thedevelopment of complicated approaches, the main prob-lem persists in the comparison process to define an exactmeasure of quantification.

The recent guidelines by the CDER at the FDA ( 9 )describes the necessary criteria for granting bio-waiversfor specific changes in drug product manufacturing suchus formulation changes or even changes in manufactur-ing site. To this end, the guidelines and specific publishedwork (10,11) on extended release solid oral dosage formsdescribe the mathematical treatment of dissolution dataderived from the pre- and post-approval changes by com-paring their release profiles using the “similarity factor,f2” which may be defined as follows:

where n is the number of dissolution time points, ωt is an

optional weight factor, Rt is the reference assay at time

point t. The “reference” and “test” products may be iden-tical formulations. Optimisation of release profiles maybe achieved by the appropriate adoption of standard oralternative dissolution methods.

In addition, moore and Flanner in their work alsodescribe a f1 fit factor or “difference factor” as follows:

where f1 describes the relative error between two disso-

lution profiles. “It approximates the percent errorbetween two curves. The percent error is zero when thetest and reference profiles are identical and increases pro-portionally with the dissimilarity between the two pro-files”.

In these work we use mathematical approach for evalua-tion of dissolution profile of diazepam conventionaltablets in pH of dissolution medium. In the period of timeneeded for dissolution (thirty minutes), the sampling timepoints were set-up to obtain profile which was supposedto have different characteristics for the dissolution mediaof different acidity (range of physiological variations).

Material and Methods

Commercially available conventional tablets of

diazepam (Bosaurin®, 5 mg tablets, Bosnalijek,Sarajevo, Bosnia and Herzegovina) were used as a testsample. Dissolution tests were performed using the uspApparatus 1 (ERWEKA DT, Heusenstamm, Germany).

Experiments were conducted to study the effect of vary-ing the pH value of the dissolution medium within thephysiological relevant range. Two different pH valuesbetween 1.0 and 2.0 were chosen. Media used for theseexperiments were 0.1mol/l hydrochloric acid and0.01mol/l hydrochloric acid.

Fixed volumes of the dissolution medium (5 ml) werewithdrawn at 5, 10, 15, 20, 25 and 30 minutes for UVassay at 242 nm. The amounts of diazepam released indissolution medium were calculated from calibrationcurves (Figure 1 and Figure 2). The data were analysedusing f1 and f2 equations proposed by moore and Flanner.

Figure 1 Calibration curve of diazepam in 0.1 mol / l HCl solution

Figure 2 Calibration curve of diazepam in 0.01 mol / l HCl solution

Results

The results of dissolution studies are summarised inTable I and Table II and Figure 3 which show the per-centage of drug dissolved as a function of time.


Table I: Percentage of dissolved diazepam fromthe tablets as a function of time in 0.1 mol / l HCl.

Table II: Percentage of dissolved diazepam fromthe tablets as a function of time in 0.01 mol / l HCl.

Figure 3 Comparative display of dissolution profilefor diazepam tablets in 0.1 mol / l HCl ( pH=1.0 )and in in 0.01 mol / l HCl ( pH=2.0 ).

The f2 value between 50 and 100 suggests that the disso-

lution profiles are similar. The f2 value of 100 suggests

that the test and reference release profiles are identical.As the value becomes smaller, the dissimilarity betweenrelease profiles increases. f2 equation is a logarithmictransformation of the sum of squared error. It takes theaverage sums of squares of the difference between testand reference profiles and fits the result between 0 and100. It is important to note that f2 equation is for thecomparison of dissolution curves in which the averagedifference between Rt and Tt is <100. The use of theweight factor allows some values to be more importantthan other values, where wt will be >1. If all values aretreated equally, then ωt =1.0.

Interpolating these data in f2 and f1 equation weobtained vales f2 = 23.16 and f1 = 34.15. These valuesindicate that there are great differences in drug releasefrom investigated diazepam tablets. Although pH valueof the dissolution medium was varied in relatively smallinterval (1.0 – 2.0), there was a great difference in drugrelease in different pH.

Conclusions

Application of f1 and f2 equations show expected dif-ferences in time-course of diazepam dissolution in arange of physiological conditions. Obtained differencesin the dissolution profile of diazepam conventionaltablets in different pH values indicate that dissolutionstudy should be followed through different pH values ofthe medium. With the advent of international harmonisa-tion of scientific protocols and implementation of Supacguidelines including site-to site manufacturing condi-tions, such process comparisons have important regulato-ry implications. Although not infallible, the most oftenused statistical approach at this stage appears to be theuse of f2 similarity factor and f1 difference factor. These

two model-independent measures surpass all other tech-niques for the profile comparison in their unique abilityto complete profile characterisation. However, more dataon their utility in conjunction with similarity of in vivodrug absorption profiles will provide the ultimate meas-ure of their discerning potential.



References

1. Omelczuk, M.O., McGinity, J.W. The Influence of Thermal Treatment on the Physical-Mechanical andDissolution Properties of Tablets Containing Poly (DL-lactic acid), Pharm. Res. 1993, 10, 542-548

2. De Villiers, M.M., Van der Watt, J.G. The Measurement of Mixture Homogeneity and Dissolution to Predict theDegree of Drug Agglomerate Breakdown Achieved through Powder Mixing. Pharm. Res. 1994, 11, 1557-1561

3. Gordon, M.S., Rudraraju, V.S., Dani, K., Chowan, Z.T. Effect of the Mode of Super Disintegrant Incorporationon Dissolution in Wet Granulated Tablets, J. Pharm. Sci. 1993, 82, 220-226

4. Rekhi, G.S., Jambhekar, S.S. Bioavailability and in vitro-in vivo correlation for Propranolol HydrochlorideExtended Release Bead Products Prepared Using Aqueous Polymeric Dispersions. J. Pharm. Pharmacol. 1996,48, 1276-1284

5. Naylor, L.J., Bakatselou, V., Dressman, J.B. Comparison of the Mechanism of Dissolution of Hydrocortisone inSimple and Mixed Micelle Systems. Pharm. Res. 1993, 10, 865-870

6. Fassihi, R.A., Ritschel, W.A. Multiple-Layer, Direct Compression, Controlled Release System: In vitro and Invivo evaluation. J.Pharm.Sci. 1993, 82,750-754

7. Skoug, J.W., Halstead, G.W., Theis, D.L., Freeman, J.E., Fagan, D.T., Rohrs, B.R. Strategy for the developmentand Validation of Dissolution Tests for Solid Oral Dosage Forms. Pharm. Tech. 1996, 20, 59-72

8. EMEA-CPMP Note for Guidance on the Investigation of Bioavailability and Bioequivalence,CPMP/EWP/QWP/1401/98

9. Pillay, V., Fassihi, R. Unconventional Dissolution Technologies. J.Pharm.Sci. 1999, 88, 843-851

10. Moore, J.W., and Flanner, H.H. Mathematical Comparison of Dissolution Profiles. Pharm. Tech. 1996, 20, 64-74.

11. O'Hara, T., Butler, J., Devane, J. A Review of Methods Used to Compare Dissolution Profile Data, PSTT, 1998, 1,214-223


Abstract

Lethal and sublethal genetical factors, including Rh fac-tor, represent endogenous risk factors of the pregnancyoutcome. These factors are most frequently inherited inrecessive way and they often lead to the negative out-come of pregnancies. They represent pregnancy (aprirori) risk of various degrees. Inheritance of Rh systemblood groups is linked to chromosome 1 and it could beexplained by two alternative theories; molecular Rh sys-tem genetics has not yet been completely explained. Thefirst formal-genetic theory postulates three closely linkedgene sites (loci C, D and E) while the second theory hasa monogenic character (one locus with several allelegenes).Data on 755 pregnancies, which were (for various rea-sons) estimated as increased risk pregnancies, were reg-istered at Gynaecology Clinic, Clinical Centre ofUniversity of Sarajevo, during the period from 1989 to1992. These data were collected from pregnant womenwho, according to the certain indications from theirfamiliar and personal anamnesis, demanded genetic con-sultations.The result of investigation of the basic Rh system pheno-type distribution shows no statistically significant differ-ence between monitored pregnant women. This result isassumed as valid for both pregnant women and their part-ners. The same result is suggesting that the observedincreased risk pregnancy samples do not significantlydiffer from the previously studied population samples.Therefore, it has been concluded that Rh factor is notclosely related to the increased risk of individual preg-nancy outcomes, that is, it does not have relevant influ-ence on the observed reproduction parameters. Thisresult is very interesting and deserves particular medicalattention and further evaluation in the future, particularlyconsidering known immunological phenomena resultingfrom relations between reproduction partners’ belongingto the basic Rh system phenotypes.Key words: Rh system, chromosome 1, prenatal diag-nostics, human reproduction, Sarajevo, pregnancy risks.

Introduction

Lethal and sublethal genetical factors, including Rh fac-tor, represent endogenous risk factors of the pregnancyoutcome. These factors are most frequently inherited in

recessive way and they often lead to the negative preg-nancy outcomes. They represent pregnancy (a prirori)risk of various degrees. Inheritance of Rh system bloodgroups is linked to chromosome 1 (Thomson et al. 1991)and it could be explained by two alternative theories;molecular Rh system genetics has not yet been complete-ly explained (Mouro et al. 1993). The first formal-genet-ic theory postulates three closely linked gene sites (lociC, D and E) while the second theory has a monogeniccharacter (one locus with several allele genes (Vogel etMotulsky 1986). Both theories equally satisfy well known heredo-graphi-cal facts regarding the Rh phenotype inheritance. Rh sys-tem phenotypes are usually divided to only two differentphenotypes - phenotype Rh+ and phenotype Rh- what hasbeen particularly investigated in many population-genet-ic studies. The four most common alleles, that is, allelecombinations have their total frequency percentageabove 96% in different populations (Giblett 1983). Haemolytic disease in newborns and transfusion incom-patibility are of particular medical importance and theyare both related to the Rh blood factors. Recently, moreattention has been paid to the other possible immune rela-tions beside those seen in the biological reproductionprocesses in humans. For these reasons, the frequency ofbasic Rh system phenotypes in pregnancy samples hasbeen recently more often investigated, not only consider-ing eventual haemolytic disease appearance, but alsoregarding the pregnancy outcome and possible influenceon the assessment of pregnancy risks.

Investigations of the frequency of Rh system phenotypesin population of Bosnia and Herzegovina are mostly spo-radically carried out. This is also pertaining to the preg-nancy samples classified considering their recorded out-comes (newborns, spontaneous abortions, artificial abor-tions). Comparative available data on pregnancy samplesin Sarajevo region have also been considered (Babi}1983 and 1988, Redì} 1996).

The objective of this paper-work was to stress the impor-tance and specificity of the frequency of Rh system phe-notypes in pregnancies with various risk degrees.Specific group genetic features of risky and unsuccessfulpregnancies (grouped in accordance with applied med-ical treatments) were investigated. Individual belongings

Frequency of Rh phenotypes in relation to theoutcome of pregnancy in the two groups of pregnant women

Amira Redì}*, Fatima Begi}***Department of Biology with Human Genetics, Faculty of Medicine, University of Sarajevo, êkalu{a 90,Bosnia and Herzegovina; E-mail: [email protected]**Cantonal Hospital “Irfan Ljubijanki}” Biha}, Bosnia and Herzegovina


of pregnant women and their partners to the basic Rh sys-tem phenotypes were monitored.

Material and methods

Data on 755 pregnancies, which were (for various rea-sons) estimated as increased risk pregnancies, were reg-istered at Gynaecology Clinic, Clinical Centre ofUniversity of Sarajevo, during the period from 1989 to1992 (Red`i} 1996). These data were collected frompregnant women who, according to the certain indica-tions from their familiar and personal anamnesis,demanded genetic consultation.

Amniocentesis (sub-sample A1) was performed in 409women while in other 346 women there were no indica-tions for amniocentesis performance (sub-sample A2).The other observed sample included 480 cases of sponta-neous abortions (sample E) registered at GynaecologyClinic and in Sarajevo Health Centres during the periodfrom 1992 to 1994 (Table 1).

Frequency of the basic Rh system phenotypes was deter-mined in all mentioned samples. At the same tame, dataon appropriate partners of the monitored pregnantwomen - potential fathers (if they were available) werealso analysed. Previously published data on Rh bloodtype frequency among mothers of newborns (Babi}

1983) and among abortions (Babi} 1988), in the samepopulation, were simultaneously presented and processed(Table 2). Statistically significant difference among studied (sub)samples of the examined material was estimated by the «ttest» method (Garret, 1959), and the results were pre-sented in Table 3. The same techniques were used for theeventual testing difference in relation to the other com-pared samples (Babi} 1983 and 1988).


The result of investigation of the basic Rh system pheno-type distribution shows no statistically significant differ-ence between monitored pregnant women. This result isassumed as valid for both pregnant women and their part-ners. The same result is suggesting that the observedincreased risk pregnancy samples do not significantlydiffer from the previously studied population samples.Therefore, it has been concluded that Rh factor is notclosely related to the increased risk of individual preg-nancy outcomes, that is, it does not have relevant influ-ence on the observed reproduction indicates. This resultis very interesting and deserves particular medical atten-tion and further evaluation in the future, particularly con-sidering known immunological phenomena resultingfrom relations between reproduction partners’ belongingto the basic Rh system phenotypes.

Samples No.Pregnant women %

Rh+Partners % Rh+

RiskyPregnancies

A1 409 89.5 75.1A2 346 82.9 73.7

Spontaneous abortions E 480 76.7 80.0Babi}, 1988 120 80.0 80.0

Newborns Babi}, 1983 7223 78.7 -

* Appropriate data on the abortion sample (Babi} 1983) and pregnant women sample during 1980-1981 (Babi} 1988) are also presented

A1 – Amniocentesis-performed (AC)A2 – Amniocentesis- not performed

Table 2 Frequency of Rh+ phenotypes in observed samples (pregnant women and their partners)*

Samples Samples Size

Pregnancies with indications for Amniocentesis- performed A1 409

Antenatal diagnostic(risky pregnancies)

AAmniocentesis-not per-formed

A2 346

Non-successful pregnancies E SPONTANEOUS ABORTIONS (1992-1994) 480

Antenatal diagnostics KMEDICAL ABORTIONS DURING THEPERIOD JUNE-SEPTEMBER 1996

532

T O T A L 1767

Table 1. General features and size of the observed samples


Frequencies of Rh+ phenotypes considering differencesamong the compared samples in relation to the variouspregnancy outcomes were studied in the same way (Table4). It was observed that our investigation samples did notsignificantly differ (according to statistics) from the ana-logue samples of the same or even broader population(Babi} 1983 and 1988).

Haemolytic disease in newborns is not the only aspect ofthe Rh immunological relations between parents thatinfluences on the parameter of significance for the repro-duction investigations. It was found that various forms ofRh incompatibilities between partners were related to the

frequency of (early) abortions what could be the subjectof our future investigations.

Conclusion

On the account of the Rh blood factor distributionprocess in pregnancy samples of various risk degrees andvarious outcomes it is concluded that there are no statis-tically significant differences between pregnant womenand their partners in all observed samples, as well as incomparison with the similar samples from a broader pop-ulation. The conclusion is that Rh factor does not expressrelevant influence on the observed bio-reproductionparameters.

EP ET A2P A2T A1PA1T 0.550 0.705 0.816 0.374 0.788A1P 0.251 0.099 0.047 0.392A2T 0.153 0.302 0.430A2P 0.293 0.145ET 0.155T - Pregnant women P - Partners* - For all determined results t% - p >0.95

Table 3 Significance of the differences of Rh+ phenotype percentage frequency(t% test results)* in observed (sub) samples

PREGNANCYOUTCOME

SAMPLE SAMPLE SIZE PERCENTAGE OF FREQUENCIES OF Rh+PHENOTYPES

PREGNANT WOMEN PARTNERS

SPONTANEOUSABORTION

(A) + E BABI}1988

553120

77.280.0

78.180.0

NEWBORNS (A) BABI}1983

6677223

87.078.8

75.7-

(A) - A part of pregnant women’ (sub)samples A1 and A2 with negative pregnancy outcomes

Table 4 Frequency of Rh+ phenotypes in relation to thevarious pregnancy outcomes in observed samples (pregnant women and their partners)


Saètak

U endogene uzroke rizi~nosti ishoda trudno}e ubrajaju se letalni kao i subletalni geneti~ki faktori me|u koje spadai Rh faktor. Ovi se faktori naj~e{}e naslje|uju recesivno i dovode do nepovoljnih ishoda trudno}e, odnosno uzroku-ju njihovu (apriornu) rizi~nost razli~itog stupnja. Naslje|ivanje krvnih grupa Rh sistema vezano je za hromosom 1i obja{njava se dvijema alternativnim teorijama; molekularna genetika Rh sistema jo{ uvijek nije sasvim razja{njen-ja. Prva formalno-geneti~ka terorija postulira tri tijesno vezana genska mjesta (lokusi C, D i E), a druga je mono-genskog karaktera (jedan lokus sa vi{e alelogena).U periodu 1989-1992 na Ginekolo{ko-aku{erskoj klinici KC Univerziteta u Sarajevu detaljno su registrovani podacio 755 trudno}a koje su iz razli~itih razloga unaprijed procijenjene kao povi{eno rizi~ne. Radi se o trudnicama kojesu na osnovu odgovaraju}ih indikacija iz porodi~ne i li~ne anamneze zatraìle geneti~ku konsultaciju. Ispitivanjem raspodjele osnovnih fenotipova Rh sistema konstatovano je da ne postoje statisti~ki zna~ajne razlikeme|u posmatranim uzorcima trudnica. Ovaj nalaz se odnosi kako na trudnice, tako i na njihove partnere. Isti ra~unje pokazao da posmatrani uzorci trudno}a povi{enog rizika nisu zna~ajno razli~iti ni od ranije prou~enih uzorakapopulacije. Istraìvanje, prema tome, ukazuje da Rh faktor nema vezu sa pove}anim rizikom neuspjeha individual-nih graviditeta, odnosno nema relevantan uticaj na posmatrane reprodukcijske pokazatelje.Posmatraju}i raspodjelu Rh krvnog faktora u uzorcima trudno}a razli~itog stupnja rizi~nosti i sa razli~itim ishodi-ma, konstatovano je da ne postoje statisti~ki zna~ajne razlike me|u trudnicama i njihovim partnerima u posmatran-im uzorcima kao i u pore|enju sa sli~nim uzorcima {ire populacije. To bi zna~ilo da Rh faktor nema relevantan uti-caj na pra}ene bioreprodukcijske pokazatelje.Klju~ne rije~i: Rh sistem, hromosom 1, prenatalna dijagnostika, humana reprodukcija, Sarajevo, rizici trudno}e.

References:

1. Babi}, N. V. (1983) Distribucija krvnih grupa ABO i Rh sistema i njen odnos prema nekim parametrima fertil-nosti u uzorku porodilja Sarajevskog regiona (1980 i 1981. godina). Magistarski rad. Univerzitet u Sarajevu.Medicinski fakultet.

2. Babi}, N.V. (1988) Neki imunogeneti~ki i kariolo{ki aspekti spontanog poba~aja. Doktorska disertacija.Univerzitet u Sarajevu. Medicinski fakultet.

3. Garret, H.E. (1959) Elementarna statistika, specijalno izdanje Psiholo{kog biltena, Beograd.

4. Giblett, E. R. (1983) Erythrocyte antigens and antibodies. IN: Williams, W. J., Beutter, E., Erslev, A. J. etLichtman M. A. (Editors): Haematology (Third Edition). Pp 1491-1505. McGraw-Hill. New York.

5. Mouro, I., Colin, Y., Cherif-Zahar, B., Cartron J. P., Le Van Kim, C. (1993) Molecular genetic basis of thehuman Rhesus blood group system. Nature Genetics, 5 (1): 62-5.

6. Redì}, A. (1996) Results and Risks of Amniocentesis (Follow up Study of 409 Cases of Genetic Counselling).Akademija Nauka i umjetnosti Bosne i Hercegovine. Radovi. Knjiga LXXXIX. Department of natural andMathematical Sciences 26: 119-125.

7. Thompson, M. V: McInnes, M. D. et Willard, H. F. (1991) Genetics in Medicine (Fifth Edition). B. SaundersCompany. Philadelphia.

8. Vogel, F. et Motulsky, A. G. (1986) Human Genetics - Problems and Approaches (Second Revised Editions).Springer Verlag. Berlin-Heidelberg-New York-Tokyo.


Abstract

Posttraumatic Stress Disorder (PTSD) very often occursaccompanied with other psychiatric disorders such as:Alcohol and Drug abuse, Personality Disorder, GeneralAnxiety Disorder, Obsessive Compulsive Disorder,Schizophrenia etc. Sometimes it might be a problem forclinicians to differ PTSD symptoms from symptoms ofcoexisting psychic disorders.The aim of this study was to present the most commonPTSD coexisting psycho-disorders.This research was conducted during the period fromApril 1998 to October 1999. Participants were divided in two groups each containing30 examinees. The first group consisted of 30 partici-pants with symptoms of PTSD only while the secondgroup included participants who suffered from bothPTSD and other psychic disorders (co-morbidity). Bothgroups were quite similar regarding participants’ genderand age. The scientific tools used in the research were: StandardPsychiatric Interview, Harvard Trauma Questionnaire(HTQ), Hamilton Anxiety Rating Scale, HamiltonDepression Rating Scale, and Drug and Alcohol AbuseChecklist.Our research results are indicating that PTSD symptomsare most common in middle-aged persons, regardless oftheir gender and age. We have found following coexist-ing psychic disorders: personality disorder 46.6% (fromwhich 13.3% is permanent personality disorder after thetraumatic experience); depression 29.9% (depressionwithout psychotic symptoms 23.3% and depression withcoexisting psychotic symptoms 6.6%); drug abuse 13.3;alcohol abuse 6.7% and dissociative (conversion) disor-der 3.3%. The results of our work are suggesting that co-morbidpsychic symptoms have significant regressive influenceon PTSD course and prognosis.

Key words: PTSD, co-morbidity, depression, personali-ty disorder

Introduction

Posttraumatic Stress Disorder (PTSD) represents adelayed response to the stress events or situations theseare threatening or catastrophic in their nature. PTSD is

likely to cause pervasive distress in almost every person.Typical (crucial) symptoms of PTSD are: episodes of thetraumatic experience recurrence induced by the memory(flashback), nightmares, lack of positive feelings andemotional emptiness, isolation from other people, anhe-donia and avoidance of activities and situations remind-ing of trauma. Anxiety and depression are commonlyassociated with above mentioned symptoms and signs, aswell as with not rare suicidal ideas.

Regardless of the trauma type, PTSD is frequently asso-ciated with other psychic disorders including differentsubstance abuse, depression and personality disorder (co-morbid symptomatology). Concerning the aetiology ofPTSD co-morbidity, we have to take in consideration twoaspects: type of trauma and type of personality.It is still unclear which disorder is primary one, andwhich is secondary according to their occurrence.Depressive, antisocial and substance abuse factors areconsidered as factors of high risk for the development ofPTSD during the exposure to trauma, but also, the samecharacteristics can appear as a function of PTSD symp-toms through the person’ try to overcome the stress. Thisbecomes the objective of many studies. There is a corre-lation between the exposure to war trauma and the occur-rence of drug and alcohol abuse (1). Possible explana-tions for such high percentage of PTSD and drug andalcohol abuse co-morbidity are neurobiological factors(disorder in adrenergic system and opiate system inPTSD). Adrenergic activity suppression, caused by theconsumption of alcohol, central depressors, marihuana orpsychoactive substances, should provide temporary reliefto those who suffer from intrusive and hyper arousalsymptoms. In this manner, deficit of opioids is regulatedthrough self-medication by heroin, methadone and otherpsychoactive substances (2, 3). Many authors have inves-tigated neurotransmitter alterations (serotonin and cate-cholamine) in CNS, as well as opioid deficit as a cause ofalcohol and drug abuse (4, 5, 6).High co-morbidity percentage makes the treatmentchoice more difficult because clinicians have to decidewhether to treat that associated disorder before or afterthe treatment of PTSD. There are certain scientific dilemmas according to thedifferentiation between pure and co-morbid PTSD formsfrom the prognostic and therapeutic point of view.Further researches have to be performed in order to

Posttraumatic stress disorder (ptsd) and co-morbidity

Ifeta Li~anin*, Amira Red`i}** * Psychiatric Clinic, Clinical Centre of University of Sarajevo, Bolni~ka 25, Bosnia and Herzegovina;

E-mail: [email protected]** Department of Biology with Human genetics, Faculty of Medicine, University of Sarajevo, ^ekalu{a 90,

Bosnia and Herzegovina; E-mail: [email protected]


obtain the clarification of these scientific dilemmasregarding the aetiology and personal constellation as fac-tors influencing the appearance of pure or co-morbidPTSD form.

Methodology

Type of research

The research was performed at the Psychiatric Clinic inSarajevo. According to its type it was epidemiological,retrospective-prospective and analytic-descriptive.

Sample

Subjects chosen for this research were patients ofPsychiatric Clinic - Department for Stress RelatedDisorders. All of them had expressed psychopathology ofPTSD and co-morbid disorders. Examinees were dividedin two homogenous groups according to their gender andage (majority was between 20 and 40 years of age). Thefirst group consisted of 30 examinees with PTSD symp-toms only while the second one had 30 examinees withboth PTSD and other psycho- disorders’ symptoms.This research was conducted during the period fromApril 1998 to October 1999.

Research tools

The tools of our research were: Standard PsychiatricInterview, Harvard Trauma Questionnaire (HTQ),Hamilton Anxiety Rating Scale, Hamilton DepressionRating Scale, and Drug and Alcohol Abuse Checklist.Working diagnosis was free from psychiatrists’ subjec-tivism and done according to the structured clinical inter-view that was based on DSM-IV criteria, ICD-10 criteriaand HTQ questionnaire. Final diagnosis was establishedafter both criteria were completely fulfilled (7, 8).

Results

Relevant research data were analysed and adequately sta-tistically processed.

Demographic characteristics of examined patients

The predominant age of patients ranged from 25 to 44years with 18 examinees (60%) in the first group and 15examinees (50%) in the second group of patients. On thesecond place was group aged 45-64 years, with 10 exam-inees (33.3%) from the first group and 20 (66.6%) fromsecond group of patients. Only 7 patients were youngerthan 24 years - 1 (6.6%) from the first group and 5(16.6%) from the second group of patients.X2-test value is suggesting that there is no significant dif-ference between the groups according to their gender andage.

Overview of co-morbid symptoms related to PTSD

All types of psychic disorders associated with PTSD arepresented in Tables 2 and 3.

As it is presented in Table 2 and Table 3 the most com-mon psychic disorder associated with PTSD is personal-ity disorder-from group F 60.3 (emotionally unstable per-sonality disorder, impulsive type) in 7 male patients(23.3%). The same disorder was not observed amongfemale patients.These two Tables are presenting PTSD co-morbid disor-ders as follows: the most common is personality disorder46.6% (14 male patients), while among female patients itwas not recorded (0%); on the second place is depres-sion-depressive episodes without psychotic symptoms (F32) in 8 patients (26.6%) from which 5 males (62.5%)and 3 females (37.5%); on the third place is drug abuse(F 10) in 4 male patients only (13.3%); on the fourthplace is alcohol abuse (F11) in 2 male patients only(6.7%) and the last one - dissociative disorder (F 44) inonly one female patient (3.3 %).

Discussion

PTSD rarely occurs in its pure form and it is usually man-ifested combined with other serious psychic disorders.Co-morbid PTSD form incidence is relatively high.

Age

GenderUp to 24 years 25-44 years 45-64 years Total

Group I Group II Group I Group II Group I Group II Group I Group IIMale 2 (100) 5 (100) 13 (72.2) 14 (93.3) 6 (60.0) 17 (85.0) 21 (70.0) 26 (86.6)Female 0 (0) 0 (0) 5 (27.8) 1 (6.7) 4 (40.0) 3 (15.0) 9 (30.0) 4 (13.4)

Total 2 (6.6) 5 (16.6) 18 (60.0) 15 (50.0) 10 (33.3) 20 (66.6) 30 (100) 30 (100)

X2 = 15.107 p = 0.0091X2m = 1.845 p = 0.397X2f = 1.034 p = 0.5963

Table 1 Age according to the patients’ gender


According to our research results and adequate literaturedata it may be noticed that PTSD is equally present inboth genders, mostly in persons aged between 25 and 45years (Table 1). Similar results considering demographiccharacteristics may be observed in previous researchesdealing with the same medical problem (9).Many publications, like those describing VietnamVeterans’ psychic disorders, are indicating a high psychicPTSD co-morbidity in this population. Mc Fall et al.studied the other diagnosis predictions (PTSD co-mor-bidity) in a broader Vietnam Veteran population (accord-ing to the different stress event) collected from the clini-cal and non-clinical resources. The results are indicatingthat PTSD combined with dissociative disorders may bebest explained in the war context and in relation to theother diagnostic psycho-combinations. It was noticedthat high risk special tasks, as well as exposure to thebizarre deaths were more notable than other stressfulexperiences regarding other diagnostic psycho-combina-tions (9).As it is presented in Tables 2 and 3 we have found the fol-lowing psycho disorders associated with PTSD: person-ality disorder in 46.6% patients from which (F 60.3)Borderline personality disorder 23.3%, (F 62.0) person-ality disorder after catastrophic event 13.3%, personalitydisorder- impulsive types (F 60.31) 6.7% and (F 60. 41)3.3%. It is important to emphasize that there was nowomen among patients with personality disorders. Onthe second place is depression in 29.9% of patients:depression without psychotic symptoms (F 32) wasfound in 26.6% of patients and depression with psychot-ic symptoms (F 33) in 3.3% of patients. In this group ofpatients there were two times more female than malepatients. On the third place is drug abuse (F 10) in 13.3% of patients - males only, following by the alcoholabuse (F 11) in 6.7% of patients - males only.Dissociative disorders (F 44) are on the last place,

recorded in 3.3% of patients - females only. Our research results are similar to those found in the rel-evant scientific references. Many authors have exploredPTSD with coexisting disorder. A group of authors hasfound PTSD associated with personality disorder -impulsive type in 10% of patients (10). Other authorshave recorded PTSD associated with Borderline person-ality disorder as a frequent medical problem, in up to69.5% of patients. They have also found PTSD associat-ed with other psycho-disorders: severe depression, obses-sive-compulsive disorder, social and specific phobias.These authors have concluded that patients sufferingfrom Borderline personality disorder are prone to devel-op PTSD and other psycho-disorders twice as much thanpatients without this type of personality disorder (10, 11).In our research we have found that, among all personali-ty disorders, Borderline type is the most frequent (foundin 23.3% of patients). Numerous literature data havepointed out co-morbidity of severe depression associatedwith PTSD - found in 50% of patients (12, 13, 14). Agroup of authors have concluded that the health recoveryis six months longer in cases of PTSD with co-morbidity(15). PTSD associated with depression (found in 50% ofpatients), as well as PTSD associated with other psycho-disorders (anxiety, affective, drug and alcohol abuse) hasbeen elaborated by the group of authors in their research(16). Mollica et al. have explored relations between trau-ma and PTSD symptoms in Cambodian refugees andhave discovered that 39% of patients has PTSD withdepression (among them 25% with invalidity). They havediscovered a greater risk for invalidity development inpatient with co-morbid PTSD, irrespective of age, traumaor previous health status. Same authors have found PTSDco-morbid with depression in 49% of patients emphasiz-ing pre and post-migrational risk (17). Many authors have explored functioning level in patientswith PTSD and major depression and have concluded

Diagnosis

Gender F10 F11 F32.0 F32.2 F32.3 F33.3 F44.7 F60.3 F60.31 F60.4 F62.0 TotalMale 4 (100) 2 (100) 3 (60.0) 1 (50.0) 1 (100) 1 (100) 0 (0) 7 (100) 2 (100) 1 (100) 4 (100) 26(86.6)Female 0 (0) 0 (0) 2 (40.0) 1 (50.0) 0 (0) 0 (0) 1 (100) 0 (0) 0 (0) 0 (0) 0 (0) 4 (13.4)Total 4 (13.3) 2 (6.7) 5 (16.6) 2 (6.7) 1 (3.3) 1 (3.3) 1 (3.3) 7 (23.3) 2 (6.7) 1 (3.3) 4 (13.3) 30 (100)

X2= 4.021 p=0.134

Table 2 Types of psychic disorders associated with PTSD

Diagnosis

Gender F10 +F11 F32 F33 F44 F60 F62 Total Male 4 (100) 2 (100) 5 (62.5) 1 (100) 0 (0) 10 (100) 4 (100) 26 (86.6)Female 0 (0) 0 (0) 3 (37.5) 0 (0) 1 (100) 0 (0) 0 (0) 4 (13.4)Total 4 (13.3) 2 (6.7) 8 (26.6) 1 (3.3) 1 (3.3) 10 (33.3) 4 (13.3) 30 (100)

X2= 4.021 p=0.134

Table 3 Types of psychic disorders associated with PTSD


that co-morbidity is characterized by more the severesymptoms than PTSD without co-morbidity (18). As it is listed in the results of our research, according tothe frequency, PTSD associated with drug abuse is on thethird place (13.3%) while alcohol co-morbid with PTSDis on the fourth place (6.7%). These co-morbidity typeswere observed among males only. A group of authorshave had similar findings during their study and theyhave emphasized the presence of high risk for the addic-tion development during the treatment of different psy-chic disorders what should be taken in permanent con-sideration (19). Some authors have found even 60-80%of drug and alcohol abuse among clinically treatedVietnam Veterans (20, 21). Drug abuse that has occurred after the attempts of self-medication, with heroin, methadone and cocaine, hasbeen reviewed by many authors (11).

Conclusion

Our research has revealed the following PTSD co-mor-bidity:

• Personality disorder 46.6% (1/3 permanentpersonality disorder),

• Depression 29.9%, (1/4 associated with psy-chotic symptoms),

• Drug abuse 13.3%, • Alcoholism 6.7%, • Dissociative disorders 3.3%.

Our research findings are indicating that co-morbidsymptoms have significant regressive influence on thePTSD course and prognosis.

Saètak

Postraumatski stresni poreme}aj (PTSP) i komporbiditet

Posttraumatski stresni poreme}aj (PTSP) je ~esto pra}en prisustvom drugih psihi~kih oboljenja kao: zloupotrebadroga i alkohola, depresija, poreme}aj li~nosti, generalizirani anksiozni poreme}aj, opsesivno-kompulzivniporeme}aj, shizofreni poreme}aj i sl. Ovo donekle predstavlja klini~ki problem u razlu~ivanju ~istih simptomaPTSP u odnosu na istovremeno postojanje i drugih psihi~kih poreme}aja.Cilj rada je prikazati naj~e{}e psihi~ke poreme}aje koji se javljaju uz PTSP.Od aprila 1998. do oktobra 1999. godine, analizirali smo dvije grupe ispitanika podijeljenih na po 30: prva grupa jeformirana od pacijenata sa simptomima PTSP, a druga od ispitanika sa PTSP i nekim drugim psihi~kimporeme}ajem (komorbiditetna). Podjednako su zastupljena oba spola unutar obje grupe bez bitnije prevalencijedekadnog ìvotnog doba. Od instrumenata istraìvanja kori{ten je standardni psihijatrijski intervju, HTQ,Hamiltonove skale: za depresiju i za anksioznost, Check lista na ovisnosti. Od simptoma komorbiditetne grupe evi-dentirali smo sljede}e poreme}aje: poreme}aj li~nosti sa 46,6 % od ~ega je najvi{e (13,3%) sa trajnim poreme}ajemli~nosti nakon traumatskog iskustva; depresija sa 29,9%; (depresija bez psihoti~nih simptoma 26.6%, i 3,3% depre-sija sa psihoti~nim simptomima; zloupotreba droga sa 13,3%; zloupotreba alkohola sa 6,7 % i disocijativni (kon-verzivni poreme}aj) sa 3,3%. Psihi~ke poreme}aje sa PTSP imaju ~e{}e osobe srednje ìvotne dobi, bez obzira naspolnu, dobnu, edukativnu, bra~nu situaciju. Komorbiditeti imaju signifikantno regresivni uticaj na tok i prognozu PTSP.

Klju~ne rije~i: PTSP, komorbiditet, depresija, poreme}aji li~nosti


References

1. Quimette, P. C. et al., (1998): Course and Treatment of Patients with both Substance Use and posttraumatic stressDisorders. Addict Behav. Nov-Dec; 23 (6): 785-95. Review

2. Yehuda, R. (1999): Linking the Neuroendocrinology of Post-Traumatic Stress Disorder with RecentNeuroanatomic Findings. Semin. Clin. Neuropsychiatry. Oct; 4 (4): 256-265

3. Bremner, J. D. (1999): Alterations in Brain Structure and Function Associated With Post-traumatic StressDisorder. Semin. Clin. Neuropsychiatry. Oct; 4 (4): 249-255

4. Stewart, S. H. et al., (1998): Functional Associations among Trauma, PTSD and Substance Related Disorders.Addict Behav. Nov-Dec; 23 (6): 797-812

5. Chilcoat, H. D., et al., (1998): Posttraumatic Stress Disorder and Drug Disorders; Testing Causal Pathways. Arch.Gen. Psychiatry. Oct; 55 (10): 913-7.

6. Pitman, R. K., et al., (1999): Psycho-physiological Alterations in Post-traumatic Stress Disorder. Semin. Clin.Neuropsychiatry. Oct; 4 (4):234-241

7. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV). Washington, D. C.: A. P. A., 1994.

8. International Classification of Diseases, X edition. Geneva: World Health Organisation, 1994.9. Mc Fall, M. et al., (1999): Analysis of Violent Behaviour in Vietnam Combat Veteran Psychiatric Inpatients with

Posttraumatic Stress Disorder. J. Trauma Stress. Jul; 12 (3): 501-1710. Brady, K. T. (1997): Posttraumatic Stress Disorder and Co-morbidity: Recognizing the Many Faces of PTSD. J.

Clin. Psychiatry. 58 Suppl. 9: 12-511. Zimmerman, M., Mattia, J. I. (1999): Axis I Diagnostic Co-morbidity and Borderline Personality Disorder.

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Course of Psychiatric Disorders Associated with Combat-Related Posttraumatic Stress Disorder. Am. J.Psychiatry. Nov; 149 (11):1568-74

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Personality Profiles of American World War II Prisoners of War. J. Nerv. Ment. Dis. Apr; 179 (4): 181-716. Dow, B., Kline, N., et al., (1997): Antidepressants Treatment of Posttraumatic Stress Disorder and Major

Depression in Veterans. Ann. Clin. Psychiatry. Mar; 9 (1): 1-517. Blanchard, E. B., Buckley, T.C., Hickling, E. J., Taylor, A. E., (1998): Posttraumatic Stress Disorder and Co-mor-

bid Major Depression: is the Correlation an Illusion? J. Anxiety Disord. Jan-Feb; 12 (1): 21-3718. Bleich, A., Koslowsky, M., Dolev, A., Lerer, B. (1997): Post-traumatic Stress Disorder and Depression. An

Analysis of Co-morbidity. Br. J. Psychiatry. May; 170: 479-8219. Mollica, R. F., Mc Innes, K., Poole, C., Tor, S., (1998): Dose-Effect Relationships of Trauma to Symptoms of

Depression and Posttraumatic Stress Disorder among Cambodian Survivors of Mass Violence. Br. J. Psychiatry.Dec; 173: 482-8

20. Shalev, A. Y., et al., (1998): Prospective Study of Posttraumatic Stress Disorder and Depression FollowingTrauma. Am. J. Psychiatry. May; 155 (5): 630-7

21. Brown, P. J., Stout, R. L., Gannon-Rowley, J. (1998): Substance Use Disorder-PTSD Co-morbidity. Patients'Perceptions of Symptom Interplay and Treatment Issues. J. Subst. Abuse Treat. Sept-Oct; 15 (5): 445-8

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26. Najavits, L. M., et al., (1998): "Seeking Safety": Outcome of New Cognitive-Behavioural Psychotherapy forWomen with Posttraumatic Stress Disorder and Substance Dependence. J. Trauma Stress. Jul; 11 (3): 437-56.

Abstract

Sustained-release theophylline pellets formulation foronce-daily evening administration significantlyimproved patients compliance and adjusted serum levelsprofile of the drug. The patients conversion from i.v. top.o. therapy is one of the most critical steps in the treat-ment of asthma according to its chronopathophysiologi-cal character.

In our study we have examined safety and efficiency ofthis conversion in twelve hospitalised asthmatic patientswho were given the new sustained-release theophyllinepellets formulation for once-daily evening administra-tion. The lung function parameters (FEV1, VC, RV, andRt) and serum theophylline concentrations were moni-tored. So, the values obtained for the last day of i.v. ther-apy and the fifth day of p.o. therapy were compared. Wefound that 75% of the patients had no change orimproved lung function on the conversion. Our results indicate that this conversion from i.v. to p.o.theophylline therapy is safe and could be efficacious.Also, the maximum theophylline serum levels couldsafely be predicted by measuring only one serum con-centration in p.o. therapy with sustained-release theo-phylline pellets formulation for once-daily eveningadministration.

Key words: theophylline, sustained-release, pellets, asth-ma, theophylline serum concentrations, lung functionparameters, once-daily evening administration

Introduction

Asthma is a chronic inflammatory disease of the airways,characterised by hyper-responsiveness to a variety ofstimuli (1). Clinically, these features are manifested byattacks of cough, wheezing, and dyspnoea. The nocturnalworsening of asthma is a common event in asthmapatients. It has been recognized since the 5th century A.D.by Aurelianus Caelius (2). Nocturnal asthma, defined asan exacerbation of asthma at night, is associated withincreases in symptoms and need for medication,

increased airway responsiveness and worsening of lungfunction (3).The mechanisms of nocturnal asthma are intimately relat-ed to circadian rhythms, which influence inflammatorycells and mediators, hormone levels and cholinergic tone.Patients with nocturnal asthma symptoms may havegreater night-time activation of inflammatory cells andmediators, lower levels of epinephrine and increasedvagal tone (4).In the long term treatment, especially in patients withnocturnal asthma, theophylline has a major impact onlung function parameters. This observation correspondswell to the hypothesis of a predominantly antinlammato-tory action of theophylline (5). Theophylline is not onlya bronchodilator but also has other effects (inhibition ofmediator release, enhancement of mucociliary clearance,enhancement of diaphragmatic contractility) that arepotentially beneficial to asthmatic patients. An importantfeature of oral controlled-release theophylline therapy istiming of drug administration. Because airway reactivityincreases and airway caliber decreases at night, it hasbeen suggested that the greatest benefit is derived fromtheophylline when peak serum concentration is reachedduring the night or the early morning.

It has been confirmed that once-daily theophylline (Th)regimens are as effective as twice daily regimens for con-trol of chronic asthma in adult patients (6,7,8). In the studies where the once-daily theophylline formula-tion was administered in the evening, there were signifi-cant benefits in spirometric values and symptom controlin favour to the once-daily regimen (8,9,10). This con-firmed the dynamic nature of the asthma process, in otherwords that the patients’ greatest physiological needs forbronchodilatation are between 2 and 8 a.m. In order toachieve effective theophylline serum concentrations(STCns) within the range of 8-20 mg/l once-daily dosingrequires single doses greater than 600 mg of theo-phylline, which presumes linear and pH independentdrug release from a dosage form.New sustained-release theophylline pellet formulationfor once-daily evening administration meets theserequirements, providing STCns profile adjusted to thechronopathophysiological character of asthma.


The new trends in theophylline therapy

Aida Mehmedagi}1, Edina Vrani}2, Sabira Had`ovi}2, Milena Pokrajac3, Jela Mili}4,Bakir Mehi}5, Hasan @uti}5, Abdulah Konjicija5

1Department of Pharmacokinetics, 2Department of Pharmaceutical Technology, University of Sarajevo, Faculty ofPharmacy, ^ekalu{a 90, Bosnia and Herzegovina3Department of Pharmacokinetics, 4Department of Pharmaceutical Technology, University of Belgrade, Faculty of Pharmacy, Vojvode Stepe 450, Yugoslavia5Clinic for lung diseases and tuberculosis “Podhrastovi” Clinical Center of the University of Sarajevo, Bardak~ije 90, Bosnia and Herzegovina

In order to individualize theophylline dosage, estimationof minimum and maximum STCns is essential. Whereasthe minimum (trough) concentration can readily be esti-mated from the pre-dose level for sustained-release theo-phylline pellets formulation, the nocturnal maximumconcentration can be predicted by taking one blood sam-ple between 7 and 9 a.m. The resultant STCns represent90% of the maximum STCns, with a range of 80 to 100%in individual subjects.The safe clinical conversion from i.v. amonophylline tothis sustained-release theophylline pellets formulationfor once-daily evening administration could have multi-ple advantages for the patients, providing most suitableconditions for the chronical treatment, and was examinedin this study.

Subjects and Methods

Twelve hospitalised asthmatic patients (reversibility ofthe obstruction was proven by a beta-agonist inhalation)participated in the study. Mean patient’s age 49,614,7years (range 24-73 years), mean body mass was 76,713,8kg (range 50-107 kg), eight were nonsmokers (sixfemale and two male), and four smokers (one female andthree male). Before the conversion, all the patientsreceived i.v. amonophylline (250 or 500 mg twice daily).Inhaled beta-agonists and inhalled or orally administrat-ed steroids (part of some patient’s regular therapy) werecontinued throughout the trial. The patients were asked tokeep their daily dosage of these medications constant,and it was supervised.Drugs known to affect theophylline metabolism were notallowed during the trial. The patients took their regular meals three times daily,additional food intake was not restricted (except xanthinecontaining foods and beverages), and was recorded; so

was the occurrence of possible side-effects.One the conversion, the patients were administrated (at 8p.m., one hour after a meal) one or two capsules (eachcontaining 375mg of anhydrous theophylline) of a sus-tained-release theophylline pellets formulation for once-daily evening administration (average dose of 8,642,17mg, range 4,66 to 11,36 mg). Fixed dose of the prepara-tion all the patients received for five subsequent days.Blood samples for the STCns determination were takenaccording to the following shedule:

• The last day of i.v. aminophylline at 8.p.m.(just before the first p.o. dose)

• Every day during five trial days at 8 a.m.• Just before the fifth p.o. dose at 8 p.m.• After fifth p.o. dose at 2 a.m.

Five mililiters of blood were taken, serum was separated,and frozen at –20oC until analysed.Theophylline in serum was determinated by the spectro-densitometric method (11)The function parameters (spiropletismography was used)were monitored for six days betwwen 8 and 9 a.m. Forthe presentation of the results and the conversion safetyassesment, we chose the lung function parameters(FEV1-forced expiratory volume in the first second, VC-vital capacity, RV-residual volume and RT-total resist-ance) for the last day of i.v. aminophylline, and the fifthday of p.o. sustained release theophylline pellets for once–daily evening administration.

Experimental RESULTS anddiscussion

The main results are shown in tables, I, II and III, and inFigure 1.Table 1 - minimum (trough) serum theophylline concen-


Table 1.

Patient Age (Yr) Sex Body mass(kg)

Th(mg/kg/day)

Smoking status(S-smoker, NS-

nonsmoker)

Cmin.i.v.

(mg/l)Cmin.p.o.

(mg/l)

M.A. 42 F 50 7.5 NS 3.8 7.1R.R. 26 M 80 4.6 S 12.5 6.5G.B. 24 M 73 5.1 S 6.2 4.5K.K. 73 M 76 9.8 NS 8.3 13.8K.N. 57 F 107 7.08 NS 7.0 7.1T.R. 65 F 78 9.6 NS 8.1 11.2G.A. 44 M 73 10.2 S 5.7 5.4P.J. 50 F 76 9.8 NS 5.8 23.5

B.U. 48 M 94 8.0 NS 6.0 6.1P.J. 63 F 71 10.5 NS 7.5 15.6

H.M. 46 F 76 9.8 NS 4.7 10.6S.S. 57 F 66 11.3 S 5.7 8.3

Mean ± S.D. 49.6 ± 14.7 76.7 ± 13.8 8.6 ± 2.1 6.8 ± 2.2 10.0 ± 5.4

trations (STCns) after i.v. aminophylline and p.o. sus-tained-release theophylline pellets formulation for once-daily evening administration, determined for twelve asth-matic patients

Table 2 - Lung function parameters for the last day of i.v.aminophylline ( i ), and the fifth day of p.o. sustained-release theophylline (Th) pellets formulation for once-daily evening administration ( II )

Table 3 - Steady state theophylline (Th) concentrations at8 a.m. and 2 a.m., and their ratio, in sera of twelve asth-matic patients after five p.o. doses of a sustained-releasetheophylline (Th) pellets formulation for once-dailyevening administration

Figure 1. Mean serum theophylline concentrations(STCns) at 8 a.m., in sera of 8 asthmatic nonsmokingpatients receiving p.o. sustained-release theophylline pel-

lets formulation for once-daily evening administration

Table I contains minimum STCns after i.v. amino-phylline, as well as after four p.o. doses of sustained-release theophylline pellets formulation. It can been seenthat in the case of non-smoking patients the trough con-centration rose, confirming the sustained-release proper-ties of the formulation, while in the case of smokingpatients indicated the need for the adjustment of thedosage and timing of the administration (increase thedose and administer one third in the morning)During the study all the conditions were constant, so theclinician, on the basis of the lung function comparison,could draw a conclusion about possible change duringthe conversion (Table II). It can be seen that 75% of thepatients had no change or improved lung function on theconversion.STCns in patients sera taken at 8 and 2 a.m., and theirratios are listed in Table III. As can be seen in eight non-


Patient C 8 a.m. (mg/l) C 2 a.m. (mg/l) C8 /C2 (%)M.A. 10.96 96% 12.80R.R.* 9.38 8.84 105.11G.B.* 6.03 8.51 70.86K.K. 19.55 21.40 91.35K.N. 7.67 7.93 96.72T.R. 19.33 22.00 87.86

G.A.* 6.63 11.57 57.30P.J. 20.80 25.40 81.89

B.U. 9.68 11.31 85.59P.J. 15.21 17.64 85.56

H.M. 13.65 17.81 76.64S.S.* 9.10 6.92 131.50

* - smokers

Table 3.

PatientI II

ConclusionFEV1 VC RV RT FEV1 VC RV RT

M.A. 81% 88% 113% 0.25 95% 94% 139% 0.29 no changeR.R. 71% 96% 185% 0.19 89% 91% 176% 0.18 no changeG.B. 89% 107% 179% 0.24 92% 111% 204% 0.18 improvementK.K. 26% 61% 141% 0.60 35% 68% 181% 0.35 improvementK.N. 55% 75% 181% 0.28 54% 65% 166% - no changeT.R. 57% 83% 190% 0.32 41% 75% 201% 0.34 worseningG.A. 36% 79% 315% 0.60 35% 78% 376% 0.56 worseningP.J. 60% 74% 332% 0.30 67% 94% 88% 0.20 improvement

B.U. 16% 45% 294% 1.54 55% 98% 160% 0.29 improvementP.J. 24% 64% 335% 0.57 39% 59% 217% 0.80 no change

H.M. 36% 78% 260% 0.31 31% 75% 277% 1.16 worseningS.S. 46% 85% 425% 0.45 80% 89% 258% 0.30 improvement

Table 2.

smoking patients, this ratio ranged between 76.64% and96.72 (mean 86.53 ± 5.97).In Figure 1., non-smoking patients’ 8 a.m. STCns aregiven for the five trial days. It is shown that all the con-centrations were within therapeutic range of theophylline(mean 14.83 ± 0.86).The results obtained indicate that this conversion fromi.v. to p.o. theophylline therapy is safe and could be effi-cacious, and that maximum STCns could be predicted bymeasuring 8 a.m. steady state concentration

Conslusion

However, comparing individual STCns with lung func-

tion parameters, one of the mayor conclusions in theo-

phylline therapy arose.

This is that each patient must be treated individually in a

context of SCTns, lung function parameters and his spe-

cific needs for theophylline in the therapy of asthma.


Figure 1.

References

(1) Horowitz RJ, Busse W W. Inflammation and asthma. Clin. Chest.Med. 1995, 16(4): 583– 602 (2) Smolensky MH, Martin RJ. Nocturnal asthma: Role of circadian rhythms in its mechanisms and therapy.

Chronobiol Int 1999, 16 ( 5 ) : vii – ix .(3) Martin R.J, Banks-Schlegel S. Chronobiology of asthma. Am JRespir Crit Care Med 1998, 158: 1002– 1007 (4) Martin R.J. Nocturnal asthma: An overview. In: Martin RJ, editor. Nocturnal asthma: Mechanisms and treat-

ment. Mount Kisco (NY): Futura; 1993. pp. 71–115 .(5) Kunkel G., Siebert B. Biochemical and cellular basis for circadian rhythms in obstructive lung disease and

implications for theophylline therapy. Arzneimittelforschung 1988; 38(II):1200-1202(6) Yoshida S., Tomioka H., Kagami M., Nakazawa T. (Chronotherapy of bronchial asthma: circadian rhythms in

peak expiratory flow. The report II: Circadian rhythms of peak expiratory flow in asthmatic patients andeffect of sustained-release theophylline on various types of the circadian rhythms), Arerugi, 2001; 50(8):679-688

(7) Martin R.J. Nocturnal Asthma and the Use of Theophylline. Clin Exp Allergy, 1998, 28 Suppl 3:64-70(8) Burioka N., Sasaki T. Chronopharmacology and Chronotherapy for Asthma by Using PEF. Nippon Rinsho,

1996, 54 (11):2956-2961(9) Welsh, P.W., Reed, C.E., Conrad, E. Timing of Once a Day Theophylline Dose to Match Peak Level With

Diurnal Variation in Severity of Asthma, Am. J. Med., 1986, 80, 1098-1102(10) Arkinstall, W.W., Atkins, M.E., Harrison, D. and Steward, J.H. Once-Daily Sustained Release Theophylline

Reduces Diurnal Variation in Spirometry and Symptomatology in Adult Asthmatics, Am. Rev. Respir. Dis., 1987, 135, 316-321

(11) Pokrajac, M., Varagi}, V.M. Spectrodensitometric Determination of Theophylline in Plasma. Acta Pharm. Jugosl. 1983, 33, 23-28

Abstract

Having in mind the fact that cerebrovascular disease(CVB) takes today in medicine, in spite of diagnostic andtherapeutic modernisation, the third place of mortalitycauses in the world (behind cordial and malignant dis-eases, but in front of depressions), and the second placeof invalidity cause (right after trauma) as well as the sec-ond place of dementia cause (after Alzheimer disease), iturges primary prophylaxis.Developing countries, but before all countries of East andMiddle Europe, where is our country, are highly riskedareas where CV disease has trend of incidence and totalfrequency increase. In the neighbouring Croatia todayCV disease is at the first place of mortality causes.In the world today 5 million people annually suffer CVdisease, in Europe about 700.000, but frequency of suf-fering on Balkan is about 5 prom.Age of CV disease effecting unfortunately movedtowards young age, and today 49% of effected by CVdisease are of 46 to 59 years of age. Early detection andtreatment of risk factors (before hypertension, smoking,diabetes mellitus, hyperlipidaemia, stress and physicalnon-activities) are the first aspect of CV disease prophy-laxis.Together with this aspect of primary prophylaxis is earlydetection of complications of mentioned risk factors onthe walls of blood vessels, before all changing in sense ofarteriosclerosis, with consequence of disorder of cerebralhaemodynamics. With that objective – verification of circulator and totalhaemodynamic disorders, there is obvious disclose ofnon-invasive diagnostic methods, and one of them isTranscranial Doppler Sonography (TCD).

TCD is method comfortable for patient, reliable andrather precise, dynamic, and can be repeated severaltimes, without side effects and in comparison with othersrather cheap.

Key words: cerebrovascular disease (CVB), TranscranialDoppler Sonography (TCD)Educational article

Transcranial Doppler Sonography is non-invasive diag-nostic method, which ensures insight into the status ofcirculation of intra cranial part of posterior – VB basin aswell as intra cranial, ending part of front – carotid basinand their anastomosis on the brain basis, which is Williscircle. Method is based on application of Doppler effect

or principle, which consists of application or shift ofreceived frequencies between two bodies in move. Whenthose two bodies are drawing closer received frequencyincrease and when drawing away received frequencydecrease. (This knowledge Doppler published in hiswork named “About coloured light of double stars andsome other sky’s bodies”).

As source of the waves serves piezo electric crystal,which oscillate and emit ultrasound wave of known fre-quency, which is directed through probe of apparatus toblood vessel, but before all to red corpuscles, which flowby certain velocity, reflects from them as well as fromblood vessel wall.

On the basis of change of frequency emitted and reflect-ed ultrasound wave is calculated velocity of blood flow,which serves as indicator of blood vessel status.

Frequency change (Doppler frequency change) is calcu-lated as per the following formula:

2fe x v x cos αDf = c

The above mentioned abbreviations show the followingvalues:

fe = frequency of emitted ultrasound wavev = middle velocity of blood flowa = angle between probe and blood vesselc = velocity of ultrasound through tissue

(1540 m/sec in soft tissues)

Blood velocity flow is proportional with Doppler shiftand it is calculated as per the following formula:

Df x c V = 2 fe x cos α

Frequency value is calculated in hertz (Hz), and bloodflow velocity in m or cm / sec.

Results are presented in measure units for velocity, asproposed Doppler Committee for Nomenclature ofAmerican Echocardiography Society 1986.

Velocity of ultrasound in blood depends of temperature,


Transcranial doppler sonography as diagnostic method

\elilovi}-Vrani} JasminkaNeurological Clinic of University Hospital in Sarajevo

content of proteins and haematocrit value. Flow can becalculated only by application of pulsating wave, whichprovides precise estimation of the spot at which theobservation is concentrated with responding anglebetween probe and blood vessel.

It is known that through middle part of blood vessel flow,red corpuscles, erythrocytes, and towards sides (i.e. wallof vessel) are moving thrombocytes and other cell’s ele-ments.

This is normal – laminar blood flow in blood vessel, butin case of stenosis of the vessel, since as vessel is morenarrow velocity is higher and considering physics princi-ples that through narrow space the same quantity ofblood has to pass within unit of time, that flow of bloodafter stenosis passing over into turbulent flow, which issign of pathologic state.

Picture 1 Schematic review of changes in stenosis– laminar flow passing over at narrow spot intohigh frequency flow, then comes turbulences andlater on flow is established again.

In 1955 Laksell found out, during echo encephalo-graphic X ray, that ultrasound can intrude throughscull and brain structure of different density andvelocity of conductivity, when one may establishlesion. However, problem was how and which ultra-sound frequency can achieve such.

Norway physiologist and cybernetic Rune Aaslidresolved that problem, which could not be bridged overat that time, by construction of probe of 2MHz. Until thattime investigation of intra cranial circulation by ultra-sound was possible with children up to one year of age,whose large fontanel is not closed or during intra neuro-logical intra surgery, because scull bonds were largeobstacles and cause weakening of ultrasound waves.When probe of 2MHz is placed on the thinnest part oftemporal bond it is achieved direct insight into state ofintra cranial circulation.

Kinds of ultrasound waves

Doppler diagnostic today applies two kinds of ultrasoundwaves: Doppler with continuing Doppler provides ultra-sound wave, which constantly emits from piezo – electriccrystal, placed in the same probe where is a receiver ofreflected waves (i.e. returned ultrasound waves), but pul-sated ultrasound wave provides crystal with purpose toemit and receive reflected waves, and emission is fromtime to time. Ultrasound is directed to the certain depth(different from continuing), goes through tissue (averagevelocity is 1540 m/sec), it returns, suffers weakening(attenuation), which is as rule 10 decibels per centimetreof tissue depth (always double because of two ways ofshift). It is considered that ultrasound travels 13 millisec-onds per centimetre of depth.

Brain requires permanent and proper blood supply,because of which circulation resistance is very weak. Incase of increase of resistance at first happens decrease ofdiastolic flow. In arteries responsible for brain supply,quantity of diastolic flow is function of circulation resist-ance of that area and can be presented through formula,which Pourcelot introduced 1971.

A - DR = A

The above abbreviations show the following values:

R = Resistance indexA = Maximum velocity of flow in systole D = Maximum velocity of flow in diastole

Vertebral arteries are difficult to access because of itsanatomic localisation, starting from palpation, ausculta-tion, as well as testing on Doppler principle. Apart fromthat they are in bone channel subject to often asymmetryand anomaly at the very beginning.

Transcranial Doppler Sonography – TCD, became veryimportant non-invasive method for testing of intra cranialcirculation; it is different from angiographies, which isinvasive method used only in some cases, mostly in pre-surgery state.

Apart from the above TCD method is very reliable andbecause of that it displaced angiographies. Following lit-erature TCD method is in correlation with angiographiesabout 75%, what understands high percent of accuracy ofa method itself.

TCD method gives the information on state of intra cra-nial circulation in real time, which provides following upof fast changes of brain bloodstream in intervals less than


a minute and testing can be repeated numberless times.This method is much cheaper in comparison with others,with special importance in economic sense.

Diagnostic areas of TCD are as follows:

• Detection of intra cranial stenosis and occlu-sions

• Haemodynamic evaluation of tendem’s(extra – intra cranial) lesion;

• Evaluation of collateral possibilities,• Estimation and monitoring of vasospasm • Diagnosis of artery – vein malformations

(AVM)• Intra surgery monitoring and monitoring in

units of intensive care• Estimation of brain death• Classification of cerebrovascular insult • Evaluation of manifested physiological

reactions• Early detection and prevention of cerebrovas-

cular insult

Considering VB basin, TCD method found the applica-tion in case of suspected stenosis, occlusion or asymme-try of vertebral arteries.Doppler signals can be registered intra cranial only at theplaces where bound of the scull are very thin and providepassing of UZ waves, called bound windows. “Lighting”of the scull provides disclosing of the windows on tem-poral bound, side part of front bound, on orbit, orbitalroof and in the area of occipital bound. Size of the win-dow varies from person to person. The most often, inyounger persons, is possible to achieve rather good sig-nal from relatively large area, but in older persons some-times is very difficult to achieve it because of hyper ossi-fication of bounds. That happens more often in females.(5, 6, 11)Temporal windows are used usually for testing of circu-lation of branches of Willis circle, orbital for search ofart. ophthalmia and sub-occipital for vertebral and basilararteries.

Picture 2 Bound windows on scull

Techniques of testing

Every blood vessel, when tested, has certain rules,such as: through which window is the easiest to verifyit, at which usual depth, and when defined ones, it isnecessary to recognise its spectrum, determine direc-tion of flow, whether towards probe or from the probeand at the end its mean, i.e. average velocity of bloodflow.

In the process of identification of blood vessels, in par-ticular in intra cranial part, it is important to determinesegment of individual blood vessel.

Having in mind that Doppler shift of frequency is pro-portional with cosine of angle between probe and bloodvessel, as angle is smaller the mistakes are less and irrel-evant.The smallest angle of insonation through temporal win-dow has medial cerebral artery, where is that angle about0 degrees, and achieved values are close to absolute val-ues of blood flow velocity, but through sub-occipitalwindow, the smallest angle of insonation has basilarartery, which is always below 30 degrees.

Through temporal window are disclosed the easiest arter-ies of Willis circle as follows: MCA, ACA, PCA, as wellas SC, AcoA and PcoA, but VA and BA are disclosedthrough sub-occipital or transforamental window.Testing can be performed in sitting position (most often)or position of lying on the stomach. Method is comfort-able for patient, without side effects, and it is enough ifpatient co-operates during testing, to be quite and do nottalk. In order to improve the contact with blood vessel isused cream, which is applied, in thin layer on the spot.

Picture 3 Techniques of testing

After finding and identification of responding bloodvessel, began determination of value of blood flowvelocity (SBSK).


Blood flow velocity in large brain arteries can beexpressed as systolic, diastolic or average blood flowvelocity. Average blood flow velocity has the largestphysiological importance, because it depends less fromcentral cardio-vascular factors like heart activity, con-tractions, total peripheral resistance and aortic compli-ance, than systolic and diastolic value.

Average value in respect of time (time – mean velocity)has better correlation with values of perfusion than max-imum and other values. Following literature, referencevalue for adults are blood flow velocity in cm/sec as perAaslid. (7, 11)

Table 1 shows: depth of insonation, direction of bloodflow and value of SBSK for arteries of Willis circle andVB basin.

In the most cases value of average blood flow velocityhas higher value in carotid than in VB basin.

During testing was observed the following: normal (lam-inar) flow – Picture 4a, weakened flow – picture 4b, andflow of high frequency – picture 4c.

Picture 4a

Picture 4b

Weakened flow is present when received ultrasoundwave is deafened by red corpuscles, because of some rea-sons. Spectrum is entirely of lower maximum velocitywith expressed peripheral resistance, but without neces-sarily disturbed flow. Typical weakened flow appearswith calcificated blood vessel (Picture 4-b)

High frequency is noticed when lesion or process, whichcaused it, reaches critical point and creates clear haemo-dynamic disturbance of circulation. Lesion is criticalwhen reduce inside diameter of blood vessel lumen andcreate increased pressure down the narrowed part (pic-ture 4-c) (11, 12, 13, 14).


Blood vessel Depth in mm Blood flow direction SBSK valuesACA 65-75 From probe 50+- 12 cm/secCS 60-80 54+- 23 cm/sec

MCA 40-60 Towards probe 62+- 12 cm/secPCA 55-70 Towards probe 42 +- 10 cm/secBA 65-100 From probe 42+- 10 cm/secVA 50-60 From probe 36+- 9 cm/sec

Table 1.


Saètak

Transkranijalna doppler sonografija kao dijagnosti~ka metodaImaju}i u vidu ~injenicu da je cerebrovaskularna bolest (CVB) danas u medicini, i pored dijagnosti~kih i terapeut-skih napredaka, na 3. mjestu uzroka smrtnosti u svijetu (iza kardijalnih i malignih bolesti, a ispred depresija), na 2.mjestu uzroka invaliditeta (odmah iza traume) i tako|e na 2. mjestu uzroka demencija (iza Alchajmerove bolesti) tose nalaè hitnost primarne prevencije. Zemlje u razvoju, prije svega zemlje Isto~ne i Srednje Evrope, a gdje spadai na{a zenlja, su visoko rizi~na podru~ja u kojima CV bolest ima tendenciju rasta incidence i ukupne prevalence. Unama susjednoj Hrvatskoj danas je CV bolest na 1. mjestu uzroka smrtnosti.U svijetu godi{nje oboli od CV bolesti oko 5 miliona ljudi, u Evropi je taj podatak oko 700.000, dok se prevalenci-ja na ovim na{im balkanskim prostorima kre}e oko 5 promila.Starosna dob oboljevanja od CV bolesti se naàlost pomjerila ka mla|oj dobi, tako da je danas 49% oboljelih od CVbolesti u ìvotnoj dobi 46-59 godina ìvota. Rano otkrivanje i lije~enje faktora rizika (prije svega hipertenzije,pu{enja, dijabetes melitusa, hiperlipidemije, stresa i fizi~ke neaktivnosti) su prvi vid prevencije CV bolesti.Uz to rano otkrivanje komplikacija navedenih faktora rizika na zidovima krvnih sudova, prije svega promjena usmislu arterioskleroze, {to ima za posljedicu poreme}aj cerebralne hemodinamike, je tako|e vid primarne preven-cije. U tom cilju – u verificiranju cirkulatornih i ukupnih hemodinamskih poreme}aja, danas se daje prednost grupineinvazivnih dijagnosti~kih metoda, a jedna od tih je Transkranijalna Doppler Sonografija (TCD).TCD je metoda komforna za pacijenta, pouzdana i prili~no ta~na, dinami~ka, moè se ponavljati vi{e puta, bez {tet-nih efekata i uz to, u odnosu na druge relativno jeftina.

Klju~ne rije~i: Cerebrovaskularna bolest (CVB), Transkranijalna dopler sonografija (TCD)Edukativni ~lanak

Bibliography

1. Aaslid R, Lindegaard KF, Sorteberg W, Vomes H, Cerebral Auto-regulation dynamics in humans, Stroke, 1989,20:45-52

2. Aaslid R, Lindegaard KF, Cerebral Haemodynamics, U. Waslid R, Transcranial Doppler Sonography, Wien-New York, Springer Verlag 1986.

3. Ackerstaff R G A, Ultrasonic duplex scanning in atherosclerotic diseases of the vertebrobasilar arterial system,Netherlands, University of Utreht, 1985, 195 str. Disertacija

4. Adams RJ, Nicholas FT, Hess DC: Normal Values and Physiological Variables U: Newell DW, Aaslid R,Transcranial Doppler, Raven Press, 1992, 41-48.

5. Berni A, Tromba L, Cavaiola S, Trombesi T, Castellani: Classification of the subclavian steal syndrome withtranscranial Doppler, Journal of Cardiovascular Surgery, 1997, 38(2), 141-145.

6. Bluth EI, Merritt CRB, Sullivan MA.A screening test for carotid stenosis: preliminary feasibility study.Radiology 1997:205(S):696-671.

7. Bray JM, Missoum A, Dubas F, Emile J, Lhoste P,: Detection of vertebrobasilar intracranial stenosis:Transcranial Doppler Sonography versus angiography, Journal of Ultrasound in Medicine, 1997, 16(3), 213-218.

8. Breyer B. Fizika ultrazvuka. U: Kurjak A. Ultrazvuk u klini~koj medicini, Medicinska naklada, Zagreb, 1997,1-13.

9. Breyer B. Medicinski dijagnosti~ki ultrazvuk, Školska knjiga, Zagreb, 1982, 37-103.10. Caplan LR, Migraine and vertebrobasilar ischemia, Neurology, 1991, 41: 55-61.11. Demerin V, Transkranijalni Doppler (TCD). U: Demerin V, Štitkovac M, Thaller N, Doppler – sonografija

krvnih ìla, Školska knjiga, Zagreb, 1990, 89-126.12. \elilovi} J., Vrijednost TCD u dijagnostici VB insuficiencije, Sarajevo, Medicinski fakultet 2001. 21-27,

Magistarski rad13. Gavranovi} M: Terapija cerebralnih komplikacija kod hipertenzije, Soc. Med. 1997, 2-91.14. Illuminati G, Calio FG, Bertagni A, Vietri F, Martinelli V: Transcranial Doppler validation of haemodynamic

vertebrobasilar insufficiency diagn., Revista EuropaPer Le Scienze Mediche Farmacologiche, 1996, 18(5-6):213-216.

15. Micieli G, Bosone D, Marcheselli S, Cavallini A, Rossi F, Nappi G, Cerebral haemodynamics in primaryheadaches: The transcranial Doppler experience Cephalalgia, Italy, 1998.18 Suppl. 117-22.

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