Rectal leiomyoma--report of two cases originating in muscularis mucosae

ROMANIAN JOURNAL OF

INTERNAL MEDICINE Volume 47 No. 1, 2009

CONTENTS

REVIEWS

BRÎNDUŞA DIACONU, Risk factors in chronic pancreatitis .................................................................................................... 3 I.R. DINU, SIMONA POPA, MIHAELA BÎCU, E. MOŢA, MARIA MOŢA, The implication of CNR1 gene’s

polymorphisms in the modulation of endocannabinoid system effects ............................................................................ 9

ORIGINAL ARTICLES

I. SPOREA, ALINA POPESCU, ROXANA ŞIRLI, MIRELA DĂNILĂ, CORINA VERNIC, Current situation of colonoscopy in Romania – 3 years of colonoscopy performance ..................................................................................... 19

VIOLETA MOLAGIC, VICTORIA ARAMĂ, A. STREINU CERCEL, NICOLETA IRIMESCU, ANA MARIA VLĂDĂREANU, M. OLARIU, S.S. ARAMĂ, A. RAFILA, CAMELIA DOBREA, S. COSTOIU, MARIA MÂRZA, D. OŢELEA, SIMONA PARASCHIV, DANA MAXIM, MĂDĂLINA POPA, H. BUMBEA, CRISTINA CIUFU, C. BĂICUŞ, RALUCA MIHĂILESCU, Preliminary data on the involvement of B,C and D hepatitis viruses in the etiopathogenesis of chronic lymphoproliferative syndromes in Romania ........................................................................ 25

D. ZDRENGHEA, DANA POP, MARIA ILEA, G. BODISZ, ADINA MĂLAI, M. ZDRENGHEA, The acute effect of Metoprolol upon NT-proBNP level in patients with congestive heart failure .................................................................. 35

D. ZDRENGHEA, DANA POP, ADELA SITAR-TĂUT, MIRELA CEBANU, V. ZDRENGHEA, Drug secondary prevention in post-menopausal women with ischemic heart disease ................................................................................ 41

A. FRANKE, M.V. SINGER, D.L. DUMITRAŞCU, How general practitioners manage patients with irritable bowel syndrome. Data from a German Urban Area .................................................................................................................... 47

F. CASOINIC, D. SÂMPELEAN, CĂTĂLINA BĂDĂU, LUCHIANA PRUNĂ, Nonalcoholic fatty liver disease – a risk factor for microalbuminuria in type 2 diabetic patients .................................................................................................... 55

INIMIOARA MIHAELA COJOCARU, M. COJOCARU, R. TĂNĂSESCU, IULIANA ILIESCU, LAURA DUMITRESCU, CECILIA BURCIN, CAMELIA VIDIŢIA GURBAN, FELICIA SFRIJAN, Phospholipase A2 in patients with noncardioembolic ischemic stroke and severe inflammatory reaction ............................................................................. 61

D. BODA, DIANA PĂUN, ADRIANA DIACONEASA, Evaluation of 5-α reductase activity on cultured fibroblast in patients with hyperandrogenemia ..................................................................................................................................... 67

POINTS OF VIEW

M. RIMBAŞ, MĂDĂLINA MARINESCU, M.R.VOIOSU, Bowel lesions in spondyloarthritides ............................................ 75 C. BĂICUŞ, SIMONA CARAIOLA, ANDA BĂICUŞ, R. TĂNĂSESCU, M. RIMBAŞ, Involuntary weight loss: case series,

etiology and diagnostic ..................................................................................................................................................... 87

ROM. J. INTERN. MED., 2009, 47, 1, 1–100

2

CASE REPORTS

ADRIANA HRISTEA, DANIELA NICOLAE, A.I. LUKA, RUXANDRA MOROTI CONSTANTINESCU, VICTORIA ARAMĂ, R. TĂNĂSESCU, Invasive pneumococcal infections: Austrian syndrome ...................................................................... 93

SABINA ZURAC, IRINA TUDOSE, GIANINA MICU, ALEXANDRA BASTIAN, ELIZA GAMADA, FLORICA STĂNICEANU, CRISTIANA POPP, D. SIMEANU, A. HAIDAR, Rectal leiomyoma – report of two cases originating in muscularis mucosae ................................................................................................................................... 97

REVIEWS

Risk Factors in Chronic Pancreatitis

BRÎNDUŞA DIACONU

“Iuliu Haţieganu” University of Medicine and Pharmacy, Third Medical Clinic, Cluj-Napoca, Romania

Chronic pancreatitis is an inflammatory disease followed by structural alterations – inflammation, fibrosis and acinar atrophy – pain emergence, exocrine and endocrine pancreatic insufficiency, severe alteration of quality of life. The pathogenetic mechanisms characteristic to this disease are not thoroughly known, but the identification of some genetic and autoimmune factors in certain entities has elucidated several pathogenetic links. The etiologic risk factors for chronic pancreatitis may associate each other and may cause different evolutions to the disease. By tracing out the risk factors and their typical working mechanisms, further pathogenetic treatments may occur, taking place precociously and preventing the evolution of the disease towards exocrine and endocrine pancreatic insufficiency.

Key words: chronic pancreatitis, risk factors, TIGAR-0 classification system.

Chronic pancreatitis is an inflammatory disease characterized by alteration of normal pancreatic architecture with irregular fibrosis, acinar and islet cell loss and inflammatory cell infiltrates, changes which are irreversible except for the obstructive chronic pancreatitis: in this case, removing the obstruction, the histological and functional changes may be reversible [1]. The histopathological examination of the pancreatic tissue remains the gold standard for diagnosing chronic pancreatitis [2]. But due to the fact that pancreatic tissue sampling is not easy to perform and implies some risks, the diagnosis is often established using a combination of clinical, functional and morphological criteria [2][3]. Chronic pancreatitis has a different evolution and prognosis according to the different risk factors. This is pointed out by the TIGAR-0 classification system from 2001 [4].

INCIDENCE AND PREVALENCE OF THE DISEASE

The incidence and prevalence of the disease vary throughout the world due to the different risk factors and diagnostic methods which are used. In Western countries, the estimated incidence is of 3.5–4/100000 inhabitants per year and the prevalence rates are of 10–15/ 100000 inhabitants [5]. In a recent study in Japan, the reported incidence and prevalence were much higher than those in the Western countries (12.4/100000 and

45.4/100000 inhabitants, respectively), probably due to the fact that diagnosis was based on very performant imaging investigations [6]. In some tropical regions, chronic pancreatitis is endemic, with a prevalence rate of 125/100000 inhabitants [7].

RISK FACTORS ASSOCIATED WITH CHRONIC PANCREATITIS

The etiologic risk factors associated with chronic pancreatitis are presented in Table I.

ALCOHOL

In the industrialized countries, 55–80% of the cases of chronic pancreatitis are due to alcohol consumption [8]. The risk of chronic pancreatitis is proportional to the duration and quantity of alcohol consumption. However, there is no apparent threshold of pancreatic toxicity [9]. Furthermore, only about 10% of the heavy drinkers develop alcoholic pancreatitis [10], the susceptibility to alcoholic chronic pancreatitis depending probably on other environmental (dietary factors, smoking, pattern of drinking, types of beverages consumed) and genetic factors. Despite many studies focusing on susceptibility factors for chronic alcoholic pancreatitis, there are no clear results, but one of the interesting research fields is that of the action of non-alcoholic compounds of alcoholic drinks on the pancreas [11].

ROM. J. INTERN. MED., 2009, 47, 1, 3–8

Brînduşa Diaconu 2 4

Table I

Etiologic risk factors associated with chronic pancreatitis: TIGAR-0 classification system [4]

Toxic-metabolic Alcoholic Tobacco smoking Hypercalcemia Hyperlipidemia Organotin compounds Chronic renal failure

Idiopathic Early onset Late onset Tropical

Genetic Autosomal dominant Autosomal recessive/ modifier genes

Autoimmune Isolated autoimmune chronic pancreatitis Syndromic autoimmune chronic pancreatitis

Recurrent and severe acute pancreatitis

Obstructive Pancreas divisum Duct obstruction (e.g., tumor) etc.

The mechanisms through which alcohol consumption determines acute and chronic pancreatitis are not well known because of the difficulty of inducing similar changes in animal models. Alcohol alone failed to produce chronic pancreatitis in animal models, but it seems that the currently used models are not the appropriate ones [12].

In experimental studies, alcohol has determined multiple effects on pancreatic level: on pancreatic duct permeability, on sphincter of Oddi function, on pancreatic secretion, pancreatic microcirculation, on the acinar and pancreatic stellate cells; it has also induced oxidative stress and had a role in pancreatic gene expression. Chronic alcohol consumption induces an increase in the production of trypsinogen, chymotrypsinogen, cathepsin B through cholinergic mechanisms [13], creating an imbalance of the trypsinogen/inhibitor of trypsin ratio which favours the premature activation of pancreatic enzymes [14]. Alcohol determines pancreatic ischemia which causes not only hypoxic lesions, but also trypsinogen activation [14]. In a study in which alcohol was given to rats for a short time, oxidative stress was generated in the liver, while longer administration produced oxidative stress in the liver and pancreas [15]. Norton et al. found that on rats, chronic ethanol administration determined an increase in pancreatic malondi-aldehyde without producing histopathologic damage, suggesting that oxidative stress can be a primary event [16].

Alcohol is metabolized in the pancreatic acinar cell through two pathways, the oxidative pathway, involving alcohol dehydrogenase and possibly cytochrome P4502E1, and the nonoxidative pathway, involving fatty acid ethyl ester (FAEE) synthases with the production of fatty acid esters [17]. Depending on the involved pathway, the effect of alcohol on transcription factors for inflammatory molecules is different: the production of FAEE activates NF-kB and activated protein (AP)-1 and the acetaldehyde inhibits the activation of NF-kB [18]. NF-kB is an activated factor in response to different injuries, which regulates the cytokine expression. Fatty acid esters with ethyl alcohol have also produced increased lysosomal fragility in vitro, they have altered the function and permeability of membranes and induced mito-chondrial dysfunction [19].

The pancreatic stellate cells, similarly to hepatic stellate cells, contain vitamin A-lipid droplets and stain positive for desmin in the quiescent state. In response to different stimuli they get activated, lose vitamin A and stain positive for actin [20]. Pancreatic stellate cells are responsible for the fibrogenesis in chronic pancreatitis. A study on cultured rat pancreatic stellate cells showed that alcohol can activate these cells via acetaldehyde and the production of intracellular oxidative stress [21]. Studies on pancreatic stellate cells also showed that alcohol can activate (AP)-1 and the MAP kinases [22].

SMOKING

Until now, there have been many studies which demonstrate the role of smoking as a risk factor for chronic pancreatitis [23–26]. In chronic alcoholic pancreatitis, alcohol and smoking are independent risk factors [23][26], and the risk of developing pancreatitis increases proportional to the cumulative amount of smoking [23]. Imoto and DiMagno showed that cigarette smoking increases the risk of pancreatic calcification in late-onset but not in early-onset idiopathic chronic pancreatitis [27], while Cavallini et al. showed that smoking has increased the risk of developing pancreatic calcification in patients with chronic pancreatitis [28].

Cigarette smoke contains many chemical compounds among which only a few are well characterized. Smoking decreases volume and bicarbonate output in healthy men [29] and increases the pancreatic enzyme secretion in

3 Risk factors in chronic pancreatitis 5

patients with pancreatitis [30]. These changes could theoretically lead to either ductal or acinar cell damage. In most experimental studies, nicotine failed to induce chronic inflammatory changes, but in a study by Wittel et al., the environmental tobacco smoke inhalation has induced a pancreatic inflammatory process with fibrosis [31]. More studies should be done in order to elucidate the mechanisms of smoking on the pancreas.

IDIOPATHIC PANCREATITIS

The percentage of patients with chronic idiopathic pancreatitis decreased to 3–9% in the last years due to the recognition of some entities such as hereditary pancreatitis, autoimmune and obstructive chronic pancreatitis. The age of onset is bimodal and the evolution of the two types is different. In early-onset idiopathic chronic pancreatitis the symptoms appear early (age < 35 years), pain is frequent and the progression to exocrine and endocrine insufficiency is slower. In late-onset idiopathic chronic pancreatitis pain is often absent and the exocrine and endocrine insufficiency develops fast. Moreover, patients with the early-onset type have frequent mutations in the inhibitor of trypsin gene [32], suggesting that the two types are etiologically distinct.

TROPICAL CHRONIC PANCREATITIS

Tropical pancreatitis is endemic in South India, Thailand, Bangladesh, Nigeria and is divided into tropical calcific pancreatitis and fibrocalculous pancreatic diabetes. Malnutrition and diet containing cassava had been excluded as etiologic factors; there are studies which suggest the role of mutations in the inhibitor of trypsin gene and the role of oxidative stress in the pathogenesis of the disease [33].

GENETIC FACTORS

AUTOSOMAL DOMINANT DISORDERS

The first evidence for the role of mutations in chronic pancreatitis came in 1996 from a genetic study in a family with chronic pancreatitis; the responsible gene was mapped on the long arm of

chromosome 7 and proved to be the cationic tryp-sinogen gene [34]. Cationic trypsinogen is secreted by acinar cells and plays the key role in activating all other digestive proenzymes. Trypsinogen is activated in the duodenum to trypsin by enterokinase. The premature activation of trypsinogen in the pancreas leads to acute pancreatitis.

Hereditary chronic pancreatitis is an autosomal dominant disorder with 80% penetrance and high risk of developing pancreatic cancer. The most frequent mutations in hereditary pancreatitis are those in the cationic trypsinogen gene: R122H, N29I but also N29T, R122C, R116C [35]. Studies using re-combinant trypsinogen observed an increased auto-activation of cationic trypsinogen in these variants, leading to recurrent attacks of acute pancreatitis and eventually to chronic pancreatitis [35].

A recent study found mutations in exon 1 of SPINK1 gene in patients with autosomal dominant hereditary pancreatitis which affected the secretory signal peptide of this protein: the resulting inhibitor of trypsin was quickly degraded intracellulary leading to an abolished SPINK secretion [36].

AUTOSOMAL RECESSIVE/MODIFIER GENES

MUTATIONS IN CATIONIC TRYPSINOGEN

A16V, D22G and K23R amino acid substitutions in the cationic trypsinogen were found in non-hereditary chronic pancreatitis.

MUTATIONS IN THE PANCREATIC SECRETORY TRYPSIN INHIBITOR (SPINK1) GENE

SPINK1 is synthesized by pancreatic acinar cells and inhibits about 20 of trypsin, acting as the first line of defense against prematurely activated trypsinogen [1]. The most frequent mutation in SPINK1 gene found in chronic pancreatitis is the N34S mutation in exon 3, especially associated with early-onset pancreatitis, tropical pancreatitis and to a lesser degree with alcoholic chronic pancreatitis [32][37][38]. The underlying mechanism of this mutation was supposed to be a loss of function of trypsin inhibitor, but in vitro studies failed to find differences in the trypsin inhibitory capacity of the wild type and mutant sequences of SPINK1 [39]. This mutation has been found in about 2% of the healthy people where it is against a dominant inheritance pattern and age of onset and the severity


of disease is similar for SPINK1 N34S homozygous and heterozygous patients with chronic pancreatitis, which is against a recessive inheritance pattern, suggesting a role as disease modifier [40].

MUTATIONS IN CFTR GENE

CFTR gene encodes a transmembrane protein functioning as a cAMP responsive chloride channel. The role of CFTR at pancreatic level is to promote the cAMP mediated fluid and bicarbonate secretion. Some patients with chronic idiopathic and alcoholic chronic pancreatitis are compound heterozygous for mild CFTR mutations [41]. An association of CFTR mutations and SPINK1 mutations further increases the risk for chronic pancreatitis [41]. Some possible mechanisms of these mutations in inducing pancreatitis are related to alkalinisation of pancreatic secretion with formation of protein plugs in pancreatic ducts and the alteration of the endocytosis processes in centroacinar cells [42].

AUTOIMMUNE PANCREATITIS

Autoimmune pancreatitis is characterized by hypergammaglobulinemia – especially IgG4, lympho-plasmocytic infiltration of the pancreatic ducts and the possible association with other autoimmune diseases. The autoantibodies found in autoimmune pancreatitis are the following: antinuclear antibodies, antilactoferrin antibodies, anti-carbonic anhydrase II antibodies. The profile of CD8- and CD4-cells in

pancreatic tissue and blood suggests a Th1-type immune response [43]. The particularity of this form of chronic pancreatitis is the response to corticoid therapy.

OBSTRUCTIVE CHRONIC PANCREATITIS

The obstruction of the pancreatic duct due to tumours, pancreas divisum, preampullary duodenal wall cysts, posttraumatic pancreatic duct scars, can determine lesions of chronic pancreatitis, which are sometimes reversible if the obstruction is treated in time. In patients with chronic pancreatitis secondary to pancreas divisum, the sphincterotomy of the minor papilla is a treatment option.

OTHER RISK FACTORS FOR CHRONIC PANCREATITIS

There are some rare risk factors for chronic pancreatitis such as hypercalcemia (in patients with hyperparathyroidism), hypertriglyceridemia, chronic renal failure, recurrent and severe acute pancreatitis, and organotin compounds.

Patients with chronic pancreatitis can associate more etiologic risk factors, which explains the differences in the evolution and prognosis of the disease. A better understanding and identification of these risk factors and of their pathogenic mechanisms will lead to better treatments of the disease with a significantly impaired quality of life.

Pancreatita cronică reprezintă o afecţiune inflamatorie care are drept consecinţă a alterărilor structurale – inflamaţie, fibroză şi atrofie acinară – apariţia durerii, insuficienţei pancreatice exocrine şi endocrine, cu alterarea severă a calităţii vieţii. Mecanismele patogenetice ale acestei boli nu sunt pe deplin cunoscute, dar identificarea unor cauze genetice şi a factorilor autoimuni în anumite entităţi au elucidat anumite verigi patogenetice. Factorii etiologici de risc pentru pancreatita cronică se pot asocia şi imprima diferite evoluţii bolii. Identificarea factorilor de risc şi a mecanismelor prin care acţionează vor putea conduce în viitor la un tratament patogenetic care să acţioneze cât mai precoce, împiedicând evoluţia bolii spre insuficienţa pancreatică exocrină şi endocrină.

Corresponding author: Brînduşa Diaconu

IIIrd Medical Clinic 19–21 Croitorilor, 400162 Cluj-Napoca, Romania E-mail: [email protected]

5 Risk factors in chronic pancreatitis 7

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Received June 23, 2008

The Implication of CNR1 Gene’s Polymorphisms in the Modulation of Endocannabinoid System Effects

I.R. DINU1, SIMONA POPA2, MIHAELA BÎCU1, E. MOŢA3, MARIA MOŢA2

1Emergency Clinical County Hospital Craiova, Diabetes, Nutrition, Metabolic Diseases Clinic 2University of Medicine and Pharmacy Craiova, Diabetes, Nutrition, Metabolic Diseases Department

3University of Medicine and Pharmacy Craiova, Internal Medicine - Nephrology Department

The endocannabinoid system (ECS) represents one of the most important physiologic systems involved in organism homeostasis, having various implications upon individual behavior and metabolic phenotype. It is composed of cannabinoid receptors CB1 and CB2, and their genes (CNR1 and CNR2), their endogenous ligands and the enzymes which mediate endogenous ligands’ biosynthesis and degradation. Anandamide and 2-arachidonoylglycerol are two endogenous agonists of the cannabinoid receptors. It is considered that ECS connects physical and emotional response to stress with appetite and energy balance, functioning like an after stress recovery system which remains inactive in repose physiologic conditions. It is involved in several physiologic processes like nociception, motor control, memory, learning, appetite, food intake and energy balance. This review analyzes the implication of 11 polymorphisms of CNR1 gene in the modulation of the ECS metabolic and central effects. A lot of studies show that rs12720071, rs1049353, rs806381, rs10485170, rs6454674, rs2023239 polymorphisms are associated with metabolic effects. From them rs12720071, rs104935, rs6454674, rs2023239 polymorphisms are also associated with central effects of ECS (substance addiction, impulsivity, resistance to antidepressive treatment). Other studies indicate that rs806368, rs1535255, (AAT)9,(AAT)12 and (AAT)n are correlated only with central effects (schizophrenia, substance addiction, impulsivity, Parkinson syndrome). The discovery of ECS and its signaling pathways opens a door towards the understanding of several important physiologic processes regarding appetite, food intake, metabolism, weight gain, motor control, memory, learning, drug addiction and nociception. The detailed analysis and validation of the ECS functioning can bring us very close to the discovery of new diagnosis and treatment methods for obesity, drugs abuse and numerous psychic diseases.

Key words: endocannabinoid system, CNR1 gene, CNR1 polymorphism, CB1 receptors, metabolic phenotype, central effects.

The endocannabinoid system (ECS) represents one of the most important physiologic systems involved in organism homeostasis, having various implications upon individual behavior and metabolic phenotype.

This system has several structural and physiologic characteristics. The history of the ECS discovery is very special. Although the psychoactive effects of Cannabis sativa plant were known also in antiquity, the basic active Cannabis component – ∆9-tetrahydrocannabinol (THC) – was identified only in 1964 [1]. In 1988 Howlett et al. discovered the receptors with high affinity for cannabinoids. Cannabinoid receptor 1 and 2 (CB1 and CB2) receptors were cloned later. Taking into consideration the presence of cannabinoid receptors, one has tried to determine the endogenous ligands of these receptors. Anandamide (N-arachidonylethanolamine)

and 2-arachidonoylglycerol (2-AG) are the first endogenous cannabinoids discovered.

THE STRUCTURE OF ECS

ECS is a complex system with multiple roles in organism. It is composed of cannabinoid receptors CB1 and CB2, their endogenous ligands and the enzymes which mediate endogenous ligands’ biosynthesis and degradation [2].

CB receptors belong to a big receptor family and are G protein-coupled receptors [3]. CNR1 and CNR2 are the genes which codify the cannabinoid receptors CB1 and CB2, being localized at 6q14-q15 level for CNR1 and 1p35-p36.1 level for CNR2. Endocannabinoid receptors are expressed mainly in the brain, but also in other organs involved in energetic homeostasis: adipose tissue,

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liver, gastrointestinal tract, pancreas and skeletal muscle [4][5][6]. CB1 receptor is the most frequent G protein-coupled receptor in the brain [4]. In the central nervous system there have been observed increased CB1 receptor densities in basal ganglia (globus pallidus, putamen, black substance) and in the cerebellum; these positions justify the ECS implication in locomotory activity. The increased level of CB1 expression in neocortical areas and hippocampus can be correlated with cannabinoids’ effects on memory and cognitive function. The low expression of CB1 in spinal cord and brainstem explains the low effects of cannabinoids upon cardiovascular and respiratory functions [7–9].

In the brain the receptor is predominantly expressed in the hypothalamus and at the level of pituitary gland and its activity modulates hypo-thalamic-pituitary-adrenal axis [4]. In the hypo-thalamus, CB1 receptor is an important component of neuronal circuits involved in appetite and caloric intake control. It is considered that CB1 receptors’ stimulation at the level of appetite modulator centers determines preferential ingestion of palatable food items.

CB2 cannabinoid receptor is expressed at the level of immune cells (B lymphocytes, T lymphocytes and monocytes), spleen and tonsils, indicating their implication in immune functions [5]. In case of an inflammatory status, CB2 receptors can be expressed also in microglial cells of the brain [11].

Recent studies on mice with CB1 and CB2 deletion indicated the existence of supplementary endocannabinoid receptors non-CB1 and non-CB2 in the brain [12][13].

ENDOGENOUS AGONISTS OF CANNABINOID RECEPTORS

Anandamide (N-arachidonylethanolamine) and 2-arachidonoylglycerol (2-AG) are two endo-genous agonists of the cannabinoid receptors. Several other endocannabinoid compounds were later isolated from the nervous system: virodhamine (O-arachidonoyl ethanolamine) [14] and noladin (2-arachidonoyl glycerol ether) [15]. These molecules seem to be generated by the same enzymatic mechanism as anandamide, but in much smaller quantities. The synthesis and inactivation of these compounds and their physiologic significance represent the subject of many studies in progress [16].

ENDOCANNABINOID BIOSYNTHESIS

Anandamide is synthesized at the level of nervous tissue through a condensation reaction ATP-independent of arahidonic acid and ethano-lamine [17][18]. A lot of studies have shown that this reaction is catalyzed by the reverse action of fatty acid amide hydrolase (FAAH), an enzyme whose direct action is to hydrolyze anandamide [19]. But this enzyme needs concentrations of anandamide precursors superior to those existing in physiologic conditions at the cellular level. That is why it is less probable for this enzyme to have a role in anandamide synthesis in physiologic conditions [20]. Another model of anandamide biosynthesis is represented by phospholipid precursor hydrolysis, N-arachidonoyl phosphatidyl-ethanolamine (PE), catalyzed by phospholipase D [21–23]. At the neuronal level there are two possibilities for 2-AG biosynthesis to occur: phospho-lipase C mediated hydrolysis of membrane phosphor-lipids resulting diacylglycerol, which is converted to 2-AG by diacylglycerol lipase (DAGL); alternatively phospholipase A1 can generate lisophospholipid consequently hydrolyzed by lisophospholipase C [24].

Both anandamide and 2-AG are generated and released at neuronal level through a mechanism which does not imply vesicular secretion [24]. Having different synthesis pathways, anandamide and 2-AG seem to have also different synthesis and releasing stimuli. It has been observed that activation of dopaminergic D2 receptors in striate increases anandamide and not 2-AG eliberation [25], while the activation of N-methyl-D-aspartate receptors (NMDA) by glutamine in cortical neurons increases 2-AG level, but it does not have any effect on anandamide [26].

ENDOCANNABINOID DEGRADATION

Anandamide hydrolysis is mediated by FAAH resulting ethanolamine and free fatty acid [27]. FAAH is a hydrolytic enzyme for both anandamide and 2-AG, but in high concentration [28–30]. Recent studies have shown that FAAH can also have a reverse action, mediating anandamide synthesis from arachidonic acid and ethanolamine [17][18]. 2-AG hydrolysis in fatty acid and glycerol is mediated by monoacylglycerol lipase [31].

3 CNR1 gene’s polymorphism

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PHYSIOLOGIC EFFECTS OF ECS (Fig. 1)

ECS seems to be present in all vertebrates and in some nonvertebrate species, but with some small differences in receptors’ structure and activity, which indicate their implication in vital biologic processes [4][32][33].

It is considered that ECS connects physical and emotional response to stress with appetite and energy balance, functioning like an after stress recovery system which remains inactive in repose physiologic conditions [3]. It is involved in several physiologic processes like nociception, motor control, memory, learning, appetite, food intake and energy balance [4][34][35].

Fig. 1. – CB1 receptors localization and the physiologic effects of their stimulation.

At neuronal level, the depolarization of

postsynaptic membranes leads to the de novo synthesis of 2-AG and anandamide through phospholipid dependent pathways [32][36]. Endo-cannabinoid synthesis as response to membrane depolarization depends on intracellular calcium increment [35][37][38]. Endocannabinoids are released in the synaptic gap and bind to CB1 receptor; this binding has a role in blocking neuromediator release which led to their synthesis and release (dopamine, GABA, glutamate) [32][36]. Endocannabinoids appear to be synthesized “on demand” where and when they are needed [36]. At peripheral level endocannabinoids are also

synthesized “on demand” and act in a paracrine or autocrine manner [36].

Cannabinoid receptors’ activation stimulates hunger and increases appetite, especially for sweets and palatable food items [4]. In mice, endo-cannabinoids levels from the brain rise shortly after food deprivation [39]. Also CB1 receptors are expressed at the level of mesolimbic dopaminergic reward circuits where the perceptions associated with pleasure and appetite stimuli are being processed [4]. Cannabinoid receptors’ agonists have antiemesis effect. Both high and low endocannabinoid levels were associated with mood disorders. The majority of preclinical studies have

ADIPOSE TISSUE

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shown that CB1 receptors blockade at central level is associated with anxious and depressive states [40–42]. Through CB1 receptors blockade (and probably also CB2) analgesic effects have been observed [43–45]. ECS participates in the reward process, but also in substance addiction effects (alcohol, opioids, nicotine) [46][47]. Endocannabinoids seem to contribute also to neuroprotection, high endocannabinoid levels are found in cerebral vascular accident, cerebral traumatisms, but also in some degenerative diseases such as Parkinson disease, Alzheimer syndrome and multiple sclerosis [46][48][49].

ECS seems to have a role in stimulating relaxation and rest, inducing, after a stress episode, forget of unpleasant memories and food ingestion stimulation. CB1 receptors’ activation seems to have an anxiolytic effect [50][51]. ECS is involved in balance regulation at synaptic level both on long and short term, suppressing both excitatory and inhibitory neurotransmission [4]. ECS induced hunger is considered to be the need to refresh the energy supplies after the stress episode.

ECS regulates organism’s energy balance and peripheral metabolism. Comparing mice with CB1 receptors deletion to the wild-type (with these receptors intact), one could observe that, under the same diet, mice without CB1 tend to be leaner and less hungry than wild-type mice [3]. This suggests the implication of CB1 receptors – endogenous factor – in weight control.

Hepatic CB1 receptors blockade is associated with decreased hepatic expression of the transcription factor SREBP-1c (sterol regulatory element-binding protein) and of its target lipogenic enzymes (acetyl coenzyme-A carboxylase-1 and fatty acid synthase) at mice and attenuates synthesis de novo of hepatic fatty acids in mice with hyperlipidic diet [52]. Hepatic CB1 receptors activation inhibits β-fatty acid oxidation [81][82].

CB1 receptor blockade increased adiponectin secretion from adipose tissue in obese or non-obese mice [52–54]. Matias et al. showed that stimulation of adipocytes CB1 receptors stimulated adipocyte differentiation and lipogenesis [58]. Preclinical data indicate that CB1 receptor blockade may produce increased glucose uptake at the adipocyte level [85]. Cota et al. showed that stimulation of mice adipocytes CB1 receptor increased lipoprotein lipase activity [3].

At metabolic level, CB1 receptors activation increases hepatic lipogenic enzymes expression in mice, while their suppression reduces these enzymes and attenuates synthesis de novo of hepatic fatty acids in mice with hyperlipidic diet. CB1 receptors suppression increases adiponectin level from adipose tissue in obese or non-obese mice [52–54]. The studies show that at the level of glucidic metabolism, CB1 receptors suppression ameliorates glycemic alteration in mice with diet induced obesity, muscle glucose uptake and in humans it reduces HbA1c in patients with type 2 Diabetes Mellitus who present high circulating endocannabinoids levels [55–59].

At the pancreatic β cells level, in vitro pharmacological activation of the CB1 receptors stimulated insulin secretion [10][58][86].

CB1 receptors activation at the gastro-intestinal tract level decreased intestinal motility and gastric emptying [83] and increased orexigenic effect of ghrelin [84].

CNR1 GENE’S POLYMORPHISMS AND WEIGHT GAIN

Considering the physiologic effects of ECS and the great differences between individuals, many scientists consider that the variations of CB1 receptor’s gene lead to obesity, adipose tissue distribution, metabolic alteration [60], but also various psychic disorders such as schizophrenia, depression, anxiety and drug addiction. More and more studies aim to discover and analyze the various polymorphisms of this gene, in order to anticipate and prevent several diseases based on ECS through CB1 variants.

In a study carried out on a group of European men, Paola Russo analyzed two variants of exon 4 of CNR1, scanning the gene for polymorphisms rs12720071 (3813A/G) and rs806368 (4895A/G). One could observe that allele 3813G was associated with the growth of abdominal circumference (AC), subscapular cutaneous skinfold and body mass index (BMI). Concerning rs806368 polymorphism, there have been observed no associations between these genotypes and the determined variables. The haplotype’s analysis consisted in the studying of 3 frequent haplotypes: A3813A4895 (AA), A3813G4895 (AG), and G3813G4895 (GG), haplotype GG was associated with the increase of the abdominal circum-ference and subscapular cutaneous skinfold [60].


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In another study, the polymorphism rs1049353 (1422A/G) was associated in men with a significant increase of the abdominal circum-ference, waist to hip ratio and of adipose mass after the adjustment for age and BMI [61]. Adipose mass percent presented a significant association which disappeared after the adjustment for age and BMI [61].

Another study carried out on Swedish and Danish subjects indicates the fact that poly-morphisms rs806381 and rs2023239 from the introns level associate with an increased BMI. Continuing the analysis of polymorphisms, the same group of researchers shows two poly-morphisms that associate better with an increased BMI: rs6454674 and rs10485170 [62].

Muller et al. study 8 polymorphisms in German children and adolescents: in region 5' (rs9353527, rs754387, rs6454676), in intron 2 (rs806379, rs1535255), exon 3 (rs2023239), intron 3 (rs806370) and in coding region (rs1049353), but they could not find any link to obesity [63].

THE IMPLICATION OF CNR1 GENE POLYMORPHISMS IN PSYCHIC DISEASES

The connection between ECS and affective state is very well known. The predominance of CB1 receptors in the brain areas responsible with affective state and psychic processes represents a proof for ECS participation in these processes. Numerous studies show the connection between several CNR1 gene’s polymorphisms and psychic diseases, especially hebephrenic schizophrenia. Hebephrenic schizophrenia appears at a very young age, most frequently at puberty or adolescence and has several polymorphic symptoms: oscillatory and childish behavior, with tendency towards antisocial and bizarre acts. The affective disorders are characterized by several aspects: the patient can switch quickly from a usually unmotivated good-humored state to a bad mood, irritability and even lamentation [64].

In a study carried out on Japanese population, Ujike et al. show that the repetition of AAT triplet in region 3’ can be associated with hebephrenic schizophrenia. Rs1049353 (1359G/A) polymorphism in codon 453 cannot be connected to this disease [64][65], but it is associated with resistance to antidepressive treatment [79]. Subjects who present a nine fold repetition of AAT triplet have a 2,3 times higher risk to develop schizophrenia [64].

The same (AAT)n repetition can be observed also in Spanish population [66]. The same poly-morphism seems to be present at different peoples in persons with schizophrenia [67][68], Parkinson disease [80].

Hamdani et al. analyzed rs1049353 poly-morphism and could not find any connection with the risk to develop schizophrenia, but only with treatment responsiveness, allele G being frequent in patients who do not respond to treatment [69]. In a study made on German subjects, Seifert et al. could not find any connection between the polymorphisms rs6454674, rs1049353, rs136096 and schizophrenia [70].

ALCOHOL AND DRUG ADDICTION RISK

CNR1 gene’s polymorphisms seem to control alcohol and drug addiction. Zuo et al. show that the risk increases in a direct ratio with the number of G alleles at the level of polymorphisms rs6454674 and rs806368. These two polymorphisms frequently associate with drug abuse, when they appear separately, but more intensively when they appear simultaneously [71]. Other studies show the connection between rs1049353 polymorphism and Alcohol withdrawal delirium [72]. Hutchison et al. indicated the existence of an association between the C allele of the rs2023239 polymorphism and alcohol abuse [77].

Ballon et al. show that the frequency of (AAT)12 and (AAT)n polymorphisms is increased in cocaine addicted patients [73]. Nicotine addiction was associated with rs12720071, rs806368, rs2023239 polymorphisms [78].

CNR1 VARIANTS AND AFFECTIVE DISORDERS

Chakrabarti et al. analyze 4 polymorphisms which associate with different striatal responses to happiness but not with disgust [74]. This shows a relationship between CNR1 gene’s variations and social behavior modulation. A study shows that the repetition of (AAT) triplet in CNR1 promotor region cannot be involved in the pathogenesis or in the psychotic symptoms of affective disorders [75].

A study carried out by Ehlers et al. shows the significantly statistical association of impulsivity with several polymorphisms: (AAT)12; (AAT)n/A6; rs1535255, rs2023239, rs1049353 and rs806368 [76]. (Table I).

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Table I

CNR1 gene polymorphism and ECS effects

Effects on CNS Polymorphism Metabolic Effects

Nicotine addiction rs12720071 ↑ Abdominal circumference ↑ Subscapular cutaneous skinfold ↑ Body mass index

Resistance to antidepressive treatment Alcohol withdrawal delirium Impulsivity

rs1049353 ↑ Abdominal circumference ↑ Waist to hip ratio ↑ Body mass index

We found no studies rs806381 ↑ Body mass index

We found no studies rs10485170 ↑ Body mass index

Substance addiction (alcohol, drugs) rs6454674 ↑ Body mass index

Substance addiction (alcohol, drugs) Impulsivity Nicotine addiction

rs806368 We found no studies

Impulsivity rs1535255 Studies infirmed such correlations

Alcohol abuse Impulsivity Nicotine addiction

rs2023239 ↑ Body mass index

Hebephrenic schizophrenia (AAT)9 We found no studies

Cocaine addiction Impulsivity (AAT)12 We found no studies

Impulsivity Parkinson syndrome Cocaine addiction Schizophrenia

(AAT)n We found no studies

CONCLUSION

The discovery of ECS and its signaling pathways opens a door towards the understanding of several important physiologic processes regarding appetite, food intake, metabolism, weight gain, motor

control, memory, learning, drug addiction and nociception. The detailed analysis and validation of the ECS functioning can bring us very close to the discovery of new diagnosis and treatment methods for obesity, drugs abuse and numerous psychic diseases.

Sistemul endocanabinoid (SEC) este unul din cele mai importante sisteme fiziologice implicate în homeostazia organismului, cu diverse implicaţii asupra comportamentului individual şi fenotipului metabolic. SEC este alcătuit din receptorii canabinoizi CB1 şi CB2 şi genele lor (CNR1 şi CNR2), liganzii lor endogeni şi enzimele care mediază biosinteza şi degradarea liganzilor endogeni. Anandamida şi 2-arahidonoilglicerolul sunt doi agonişti endogeni ai receptorilor canabinoizi. Se consideră că SEC leagă răspunsul fizic şi emoţional la stres, cu


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apetitul şi balanţa energetică, funcţionând ca un sistem de recuperare după stres, care rămâne inactiv în condiţii fiziologice de repaus. Este implicat în diverse procese fiziologice ca nocicepţia, controlul motor, memoria, învăţarea, apetitul, ingestia de alimente şi balanţa energetică. În acest articol se analizează implicarea a 11 polimorfisme ale genei CNR1 în modularea efectelor metabolice şi centrale ale SEC. Numeroase studii au arătat că polimorfismele rs12720071, rs1049353, rs806381, rs10485170, rs6454674, rs2023239 sunt asociate cu efectele metabolice. Din acestea, polimorfismele rs12720071, rs104935, rs6454674, rs2023239 se asociază şi cu efecte centrale ale SEC (dependenţa de substanţe, impulsivitatea, rezistenţa la tratamentul antidepresiv). Alte studii au arătat că rs806368, rs1535255, (AAT)9, (AAT)12 şi (AAT)n sunt corelate numai cu efectele centrale (schizofrenia, dependenţa de substanţe, impulsivitatea, sindromul Parkinson). Descoperirea SEC şi a căilor sale de semnalizare deschide drumul spre înţelegerea mai multor procese fiziologice legate de apetit, aport alimentar, metabolism, creştere ponderală, control motor, memorie, învăţare, dependenţă de substanţe şi nocicepţia. Analiza detaliată şi validarea funcţionării SEC ne poate aduce mai aproape de descoperirea unor noi metode de diagnostic şi tratament pentru obezitate, abuz de substanţe şi numeroase afecţiuni psihice.

Corresponding author: Maria Moţa, Professor

Emergency Clinical County Hospital Craiova E-mail: [email protected]

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61. PEETERS A., BECKERS S., MERTENS I., VAN HUL W., VAN GAAL L., The G1422A variant of the cannabinoid receptor gene (CNR1) is associated with abdominal adiposity in obese men, Endocrine, 2007; 31(2):138–41.

62. BENZINOU M., CHÈVRE J.C., WARD K.J. et al., Endocannabinoid receptor 1 gene variations increase risk for obesity and modulate body mass index in European populations. Hum. Mol. Genet., 2008; 17(13):1916–21.

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64. UJIKE H., TAKAKI M., NAKATA K., TANAKA Y. et al., CNR1, central cannabinoid receptor gene, associated with susceptibility to hebephrenic schizophrenia. Mol. Psychiatry, 2002; 7(5):515–8.

65. UJIKE H., MORITA Y., New perspectives in the studies on endocannabinoid and cannabis: cannabinoid receptors and schizophrenia. J. Pharmacol. Sci., 2004; 96(4):376–81.

66. MARTÍNEZ-GRAS I., HOENICKA J., PONCE G., RODRÍGUEZ-JIMÉNEZ R. et al., (AAT)n repeat in the cannabinoid receptor gene, CNR1: association with schizophrenia in a Spanish population. Eur. Arch. Psychiatry Clin. Neurosci., 2006 Oct. 256(7):437–41.

67. CHAVARRÍA-SILES I., CONTRERAS-ROJAS J., HARE E. et al., Cannabinoid receptor 1 gene (CNR1) and susceptibility to a quantitative phenotype for hebephrenic schizophrenia. Am. J. Med. Genet. B. Neuropsychiatr. Genet., 2008; 147(3):279–84.

68. TSAI S.J., WANG Y.C., HONG C.J., Association study of a cannabinoid receptor gene (CNR1) polymorphism and schizophrenia. Psychiatr. Genet., 2000; 10(3):149–51.

69. HAMDANI N., TABEZE J.P., RAMOZ N., The CNR1 gene as a pharmacogenetic factor for antipsychotics rather than a susceptibility gene for schizophrenia. Eur. Neuropsychopharmacol., 2008; 18(1):34–40.

70. SEIFERT J., OSSEGE S., EMRICH H.M., SCHNEIDER U., STUHRMANN M., No association of CNR1 gene variations with susceptibility to schizophrenia. Neurosci. Lett., 2007; 426(1):29–33.

71. ZUO L., KRANZLER H.R., LUO X., COVAULT J., GELERNTER J., CNR1 variation modulates risk for drug and alcohol dependence. Biol. Psychiatry, 2007; 62(6):616–26.

72. SCHMIDT L.G., SAMOCHOWIEC J., FINCKH U., FISZER-PIOSIK E., HORODNICKI J. et al., Association of a CB1 cannabinoid receptor gene (CNR1) polymorphism with severe alcohol dependence. Drug. Alcohol Depend., 2002; 65(3):221–4.

73. BALLON N., LEROY S., ROY C., BOURDEL M.C., CHARLES-NICOLAS A. et al., (AAT)n repeat in the cannabinoid receptor gene (CNR1): association with cocaine addiction in an African-Caribbean population. Pharmacogenomics J., 2006; 6(2):126–30.

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74. CHAKRABARTI B., KENT L., SUCKLING J., BULLMORE E., BARON-COHEN S., Variations in the human cannabinoid receptor (CNR1) gene modulate striatal responses to happy faces. Eur. J. Neurosci., 2006; 23(7):1944–8.

75. TSAI S.J., WANG Y.C., HONG C.J., Association study between cannabinoid receptor gene (CNR1) and pathogenesis and psychotic symptoms of mood disorders. Am. J. Med. Genet., 2001; 105(3):219–21.

76. EHLERS C.L., SLUTSKE W.S., LIND P.A., WILHELMSEN K.C., Association between single nucleotide polymorphisms in the cannabinoid receptor gene (CNR1) and impulsivity in southwest California Indians. Twin. Res. Hum. Genet., 2007; 10(6):805–11.

77. HUTCHISON K.E., HAUGHEY H., NICULESCU M., SCHACHT J., KAISER A. et al., The incentive salience of alcohol: translating the effects of genetic variant in CNR1. Arch. Gen. Psychiatry, 2008 Jul., 65(7):841–50.

78. CHEN X., WILLIAMSON V.S., AN S.S., HETTEMA J.M., AGGEN S.H. et al., Cannabinoid receptor 1 gene association with nicotine dependence. Arch. Gen. Psychiatry, 2008 Jul., 65(7):816–24.

79. DOMSCHKE K., DANNLOWSKI U., OHRMANN P., LAWFORD B., BAUER J. et al., Cannabinoid receptor 1 (CNR1) gene: Impact on antidepressant treatment response and emotion processing in Major Depression. Eur. Neuropsychopharmacol., 2008 [Epub ahead of print]

80. BARRERO F.J., AMPUERO I., MORALES B., VIVES F., DE DIOS LUNA DEL CASTILLO J. et al., Depression in Parkinson’s disease is related to a genetic polymorphism of the cannabinoid receptor gene (CNR1). Pharmacogenomics J., 2005; 5(2):135–41.

81. MUOIO D.M., SEEFELD K., WITTERS L.A., COLEMAN R.A., AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target. Biochem J. Mar. 15, 1999; 338 (Pt 3):783–791.

82. KOLA B., HUBINA E., TUCCI S.A. et al., Cannabinoids and ghrelin have both central and peripheral metabolic and cardiac effects via AMP-activated protein kinase. J. Biol. Chem. July 1, 2005; 280(26):25196–25201.

83. MASSA F., STORR M., LUTZ B., The endocannabinoid system in the physiology and pathophysiology of the gastrointestinal tract. J. Mol. Med., 2005; 83(12):944–954.

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86. LIU B., HOBBS C., DOHERTY P., JONES P., PERSAUD S., Diacylglycerol lipase and cannabinoid receptor expression and function in pancreatic beta-cells (abstract 443). Paper presented at: 41st European Association for the Study of Diabetes (EASD) Annual Meeting; September 10–15, 2005, 2005; Athens, Greece.

Received September 26, 2008

ORIGINAL ARTICLES

Current Situation of Colonoscopy in Romania – 3 Years of Colonoscopy Performance

I. SPOREA1, ALINA POPESCU1, ROXANA ŞIRLI1, MIRELA DĂNILĂ1, CORINA VERNIC2

1Department of Gastroenterology, University of Medicine and Pharmacy Timişoara, Romania 2Department of Biophysics and Medical Informatics, University of Medicine and Pharmacy Timişoara, Romania

The aim of this paper is to evaluate the situation of colonoscopy in Romania in 2007, as compared to a study we performed in 2004.

Material and method. We performed a multicentric prospective study, by means of a questionnaire distributed to 36 centers of endoscopy from Romania, requesting the total number of colonoscopies performed in March 2007, the number of complete and incomplete colonoscopies, the number of colonoscopies performed with or without sedation.

A number of 28 centers responded to our questionnaire, 15 university and 13 non-university departments.

Results. According to the responses we received, 2,684 colonoscopies were performed in March 2007, with a mean number of 96 colonoscopies/month/center, 76.9% of them total.

In the university departments 1,776 colonoscopies were performed, 78.9% total, while in the non university departments 908 colonoscopies were performed, 73.1% total (p=0.0021).

46.7% of the colonoscopies were performed without sedation, 53.2% with sedation and 0.1% under general anesthesia (intra surgery colonoscopy).

In conclusion, this multicentric prospective study has shown a slight increase in the number of colonoscopies as compared to 2004 (though insignificant), but with an improvement in quality (total colonoscopies 76.9%-2007 vs. 70.5%-2004, p<0.001). There still exists a difference regarding the performance in university vs. non university departments, and the percentage of cases in which sedation was used during colonoscopy is approximately the same (46.7%), still very small.

Key words: colonoscopy, caecal intubation rate, national audit.

Colonoscopy is still the “gold standard” method for colonic evaluation, as it has the highest sensibility and specificity for the diagnosis of every kind of colonic pathology. Moreover, even if it is more expensive, it is probably the best method for the screening for colorectal cancer. Unfortunately, the investigation of patients with suspicion of colorectal pathology, as well as the screening for colorectal cancer in patients over 50 years old, require a very large number of colonoscopies, too many for any medical system.

Since in Romania, according to the 2002 Population Census, 6,659,784 subjects were over 50 years old, over the next 5 years 6,659,784 colonoscopies will be needed only for screening for colorectal cancer (considering that it should start at the age of 50 and that colonoscopy should be repeated every 5 years). In addition, we must not forget the number of colonoscopies that must be performed in symptomatic patients.

Beside quantity (the number of colonoscopies), another important aspect is the quality of the colonoscopies performed (caecal intubation being mandatory for a complete exploration, a target to reach in as many cases as possible, preferably in all the cases).

The aim of this paper was to evaluate the situation of colonoscopy in Romania in 2007, as compared to the data obtained from the study we performed in 2004, by means of a national multicentric prospective study.

MATERIAL AND METHOD

We performed a multicentric prospective study, by means of a questionnaire distributed to 36 centers of endoscopy from Romania, in which they were required to mention the total number of colonoscopies performed in March 2007 (from the

ROM. J. INTERN. MED., 2009, 47, 1, 19–24

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1st to the 31st), the number of complete colonoscopies, as well as the incomplete ones, and also the number of colonoscopies performed with or without sedation. For the incomplete colonoscopies, we enquired about the reason for which they were not complete (un-surpassable malignant or benign stenosis, insufficient preparation of the colon, or technical impossibility of total colonoscopy). We divided the endoscopic departments into university and non university ones, in order to compare the performances in colonoscopy for that matter.

From the 36 centers, only 28 responded to our questionnaire, out of which 15 were university departments and 13 non university ones. The remaining 8 centers did not respond to our

questionnaire, in spite of repeated requests, even if at first they agreed to participate in this multicentric national study.

RESULTS

According to the responses we received, 2,684 colonoscopies were performed in March 2007, with a mean number of 96 colonoscopies/ month/center, 76.9% of them total. The number of colonoscopies performed in the 28 centers which responded to our questionnaire, as well as the type of centre (university or non university) and the percentage of total colonoscopies are presented in Table 1.

Table I

Quantity and quality of colonoscopies performed during a month time, according to the type of department

Center University (U)/Non University (NU)

Number of colonoscopies

Percentage of total colonoscopies

1 U 78 73 2 U 37 91.9 3 NU 20 45 4 NU 67 79.1 5 NU 305 88.8 6 NU 105 71.4 7 U 221 85.9 8 U 143 93.7 9 U 65 80 10 U 70 85.7 11 U 356 78.9 12 U 81 67.9 13 U 43 81.4 14 U 31 70.9 15 NU 32 75 16 NU 31 32.2 17 U 246 78.8 18 U 52 88.4 19 NU 134 58.9 20 NU 5 20 21 NU 52 30.7 22 NU 68 91.1 23 NU 31 64.5 24 NU 22 54.5 25 U 48 66.6 26 U 183 61.2 27 U 122 79.5 28 NU 36 88.8

Total 2684 76.9 If we correct the percentage of total

colonoscopies by eliminating those that were stopped due to unsurpassable malignant stenosis, the percentage of total colonoscopies reaches 83.5%. In

the university departments 1,776 colonoscopies were performed, out of which 78.9% were total, while in the non university departments 908 colonoscopies were performed, 73.1% of them total (p=0.0021, ES).

3 Current situation of colonoscopy in Romania

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46.753.2

0.10

102030405060

withoutsedation

with sedoanalgesia

with generalanestesia

percentage of colonoscopies Fig. 1. – Sedation during colonoscopy in March 2007 in Romania.

Regarding the problem of sedation during

colonoscopy, we found out that 46.7% of the colonoscopies were performed without sedation, 53.2% with sedation and 0.1% under general anesthesia (intra surgery colonoscopy) (Fig. 1).

DISCUSSION

We performed a similar study in November 2004 (1st to 30th of November) (1, 2), which revealed the following data: 30 centers out of 37, from 18 cities, responded to our questionnaire, among which 16 were university departments and 14 non university ones.

The total number of colonoscopies performed in the 30 centers in November 2004 was 2,559, with a mean number of approximately 85 colonoscopies/ month/center, 1,804 of them total (70.5%).

Comparing the university centers to the non university ones, the situation was the following: 1,582 colonoscopies were performed in university centers, 74.1% (1,173) of them total, while in the non university centers 977 colonoscopies were performed, out of which 64.4% (631) were total (p=0.000027, ES).

Regarding sedation during colonoscopy, 46% of cases were performed without sedation; while in 54% of them sedation was used.

By comparing the data from those 2 studies we can observe that the number of colorectal procedures increased with approximately 4.8% as compared to 2004. More important yet is the obvious increase of the percentage of total colonoscopies, from 70.5% in 2004, to 76.9% in 2007 (p<0.001, ES).

We would like to discuss each of those two aspects separately. The first one is the total number of colonoscopies that were performed, which, sadly, increased very little (only with 4.8%), even

though the screening program for colorectal cancer should already have been well implemented in Romania, just like in other European countries (Italy, Germany or Poland). In the previous study we demonstrated that the number of colonoscopies performed in Romania was totally inadequate.

Starting from the 2,684 colonoscopies performed during 1 month in 2007 and applying a correction of 20% (unaccounted centers) the number of colonoscopies will increase to 3,220/month, meaning approximately 38,640 colonoscopies/year, still insufficient for the needs of our country. In the introduction of our paper we specified that according to the Romanian population census from 2002, approximately 1,330,000 colonoscopies/year are needed only for an adequate screening program (since there are 6,659,784 subjects over 50 years old, in need of one colonoscopy once every 5 years). Obviously, this number will be smaller due to a lack of addressability by all eligible subjects, but the symptomatic patients will add up. In these conditions, the number of colonoscopies that should be performed each year in Romania should increase tenfold.

On the other hand, even if in our previous study we emphasized the insufficient number of centers performing high quality colonoscopy [1][2] and also the insufficient number of colonoscopy specialists [3][4], nothing changed; the increase with only 4.8% of the number of colonoscopies was probably just an accident, and not the result of a national medical policy.

Regarding the mean number of examinations/ month/center, we found out that it increased from 85 in 2004 to 96 in 2007, a rise of approximately 13%, thus, a positive trend, so that the mean number of colonoscopies/center/year reaches approximately 1,200 colonoscopies/year, similar to data already published [5][6]. Despite this progress, the total

anesthesia

I. Sporea et al. 4

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number of colonoscopies performed in Romania each year remains very small, totally insufficient. A healthy medical policy must be applied in order to achieve a significant increase in the number of colonic explorations [4]. The likely solution is the development of other endoscopy centers, all over the country.

Thirdly, we would like to discuss about the performance at colonoscopy, meaning the rate of caecal intubation. If in 2004 it was 70.5%, in 2007 it reached 76.9% (thus an improvement of performance of approximately 7%). If we correct this percentage by eliminating the colonoscopies that had to be stopped due to unsurpassable malignant stenosis, the percentage of total colonoscopies would be 83.5%.

If we divide the colonoscopies according to the type of institution in which they were performed, 1,776 colonoscopies were performed in university centers, with a percentage of total colonoscopies of 78.9%, while 908 colonoscopies were performed in non university centers with a percentage of total colonoscopies of 73.1% (p=0.0021 FS). The data are similar to those obtained in 2004, when the performance in colonoscopy was significantly superior in university centers. Interesting, though, is the fact that the increase was uneven, in the university centers from 74.1% to 78.9% (approximately 4.8%), while in the non university centers from 64.6% to 73.1% (almost double – 8.5%).

In spite of these increases, the percentage of total colonoscopies is lower than the average of performant centers, below an average of minimum 90% [7–9]. Considering the fact that the study is a national one, including centers with different experience, the results are similar to those obtained by Bowles [5] in a study performed in 2004 in 3 areas from The United Kingdom, in which the mean rate of caecal intubation was 76.9% (also with differences between hospitals: 74.5% in general hospitals, 76.6% in teaching hospitals and 89.7% in private hospitals). Probably, in the real life, the overall rate of total colonoscopies is smaller than the ideal one, but, in university departments, it gets closer to the ideal of at least 90% [7][10].

Regarding sedation during colonoscopy in Romania, there were no significant changes between 2004 and 2007; 46% of the colonoscopies were performed without sedation in 2004 and 46.7% in 2007. Even if, 3 years ago [1][2], we emphasized that this practice is an incorrect one, that does not meet the international standards

[5][6][8], nothing changed. In a previous study [10], we proved that sedation during colonoscopy can improve the performance of lower endoscopy, and we maintain our position regarding the role of sedation during colonoscopy.

The national guidelines of the Romanian Society of Endoscopy should strongly recommend sedation during colonoscopy. An increased number of colonoscopies performed with sedation will probably improve the rate of caecal intubation, since some of the colonoscopies had to be interrupted due to the pain of the patient. Rex [11] related that sedation practice is related to technical performance of colonoscopy and unsedated procedures are associated with higher rates of failed caecal intubation.

Regardless who performs the analgesia, either the anesthesiologist present in the endoscopy room [12–14], or another gastroenterologist (different from the one who performs the endoscopy) [15] or even by a registered nurse [16–20], it will reduce the patient’s suffering, as well as the unpleasant memory of the exploration. In most of the countries, the percentage of colonoscopies performed under sedation is higher than 90% and there are virtually no cases of colonoscopy without sedation [5][8]. In France, in most of the cases the examination is performed under general anesthesia with intubation [7].

In addition to generalizing the practice of sedation during colonoscopy, we propose a new teaching program during residency [4][21–24], so that the fellow of gastroenterology should perform a sufficient number of colonoscopies under supervision [21], allowing a greater percentage of total colonoscopies and a smaller rate of complications [25–28].

In conclusion, our multicentric prospective study performed in Romania, in 2007, during a one month period, has shown a slight increase in the number of colonoscopies as compared to 2004 (yet insignificant), and a better quality (total colonoscopies in 76.9% of the cases in 2007, as compared to 70.5% in 2004 p<0.001). There are still differences between university and non university centers and the percentage of colonoscopies performed under sedation remained approximately the same (46.7%) totally unsatisfactory.

The most important conclusion is that the number of colonoscopies performed in Romania at this time is totally insufficient as compared to the need of colonic evaluation, and that is why a national strategy should be implemented.

5 Current situation of colonoscopy in Romania

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Scopul acestei lucrări este de a vedea care este situaţia colonoscopiei în România în 2007, comparativ cu un studiu efectuat in 2004.

Material şi metodă. Am efectuat un studiu prospectiv, prin care am solicitat pe bază de chestionar, la 36 centre de endoscopie din România înregistrarea numărului total de colonoscopii efectuate în luna martie 2007, numărul de colonoscopii totale, respectiv incomplete şi numărul de colonoscopii efectuate cu sau fără sedare.

Un număr de 28 centre au răspuns la chestionar, 15 centre universitare, iar 13 nonuniversitare.

Rezultate. Din datele obţinute reiese că în luna martie 2007 s-a efectuat un număr de 2684 colonoscopii, cu o medie/ centru de 96 explorari colonice/luna, procentul de colonoscopii totale fiind de 76,9%.

În centrele universitare s-au efectuat 1776 colonoscopii cu un procent de colonoscopii totale de 78,9%, iar în centrele nonuniversitare 908 colonoscopii, cu un procent de colonoscopii totale de 73,1% (p=0,0021).

46,7% colonoscopii au fost efectuate fără sedare, 53,2% folosind sedo-analgezia şi 0,1% folosind anestezia generală (colonoscopie intraoperatorie).

În concluzie, studiul prospectiv multicentric a demonstrat o uşoară creştere a numărului de colonoscopii faţă de 2004 (dar nesemnificativ), dar cu o îmbunătăţire a calităţii acesteia (colonoscopie totală la 76,9%–2007 faţă de 70,5%–p<0,001). Se menţine încă diferenţa în performanţe între centrele universitare şi nonuniversitare, iar procentul de cazuri la care colonoscopia s-a efectuat sub sedo-analgezie este aproximativ acelaşi (46,7%) şi total nesatisfăcător.

Acknowledgements. The authors would like to thank all the Centers of Endoscopy that participated in this study, especially the contact persons who answered the questions that made this multicentric study possible.

Arad: E. Miuţescu, T. Duşe Baia Mare: I. Măilătescu Bucureşti: M. Diculescu, C. Gheorghe, C. Bogdan, I. Dina, C. Chira, T. Bădescu, R. Voiosu, G. Constantinescu, M. Jinga, T. Nicolae Constanţa: E. Dumitru, F. Voinea Cluj: O. Pascu, S. Vălean, D. Sâmpelean, A. Drăghici Craiova: A. Săftoiu, V. Sbârcea, C. Şarpe Deva: D. Blendea Drobeta Tr. Severin: O. Lungulescu, M. Pătraşcu

Iaşi: C. Stanciu, A. Trifan, C. Cijevschi Oradea: A. Lenghel, O. Frăţilă Ploieşti: M. Vintilă Râmnicu-Vâlcea: N. Ignat Reşiţa: E. Başa, R. Dumitrescu Satu Mare: I. Brândeu, C. Ursu Sibiu: A. Frăticiu Suceava: L. Croitoru Târgu-Mureş: S. Băţagă, D. Dobru, L. Bancu Timişoara: V. Dănilă, A. Goldiş

Corresponding author: I. SPOREA 13, Snagov St., 300482 Timişoara, Romania Tel: +40-256-309455 Fax: +40-256-488003 E-mail: [email protected]

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21. FAIGEL O.D., BARON T.H., LEWIS B., PETERSEN B., PETRINI J., POPP J.E. et al., Ensuring competence in endoscopy. Available on line at: http://www.asge.org/uploadedFiles/Publications_and_Products/Practice_Guidelines/competence.pdf. Accessed 10.11.2008.

22. BOND J.H., FRAKES J.T., Who should perform colonoscopy? How much training is needed? Gastrointest. Endosc., 1999; 49: 657–9.

23. WEXNER S.D., GARBUS J.E., SINGH J.J., SAGES COLONOSCOPY STUDY OUTCOMES GROUP, A prospective analysis of 13.580 colonoscopies. Reevaluation of credentialing guidelines. Surg. Endosc., 2001; 15(3): 251–61.

24. AMERICAN SOCIETY FOR GASTROINTESTINAL ENDOSCOPY, Quality and outcome assessment in gastrointestinal endoscopy. Gastrointest. Endosc., 2000; 52: 827–30.

25. REX D.K., Speeding up cecal intubation: its role in the efficiency of colonoscopy delivery. Am. J. Gastroenterol., 2002; 97: 6–8. 26. NELSON D.B., MCQUAID K.R., BOND J.H., LIEBERMAN D.A., WEISS D.G., JOHNSTON T.K., Procedural success and

complications of large scale screening colonoscopy. Gastrointest. Endosc., 2002; 55: 307–14. 27. SHAH H.A., PASZAT L.F., SASKIN R., STUKEL T.A., RABENECK L., Factors associated with incomplete colonoscopy:

A population-based study. Gastroenterology, 2007; 132: 2297–303. 28. KO C.W., RIFFLE S., SHAPIRO J.A., SAUNDERS M.D., LEE S.D., TUNG B.Y. et al., Incidence of minor complications and

time lost from normal activities after screening or surveillance colonoscopy. Gastrointest. Endosc., 2007; 65(4): 648–56.

Received December 3, 2008

Preliminary Data on the Involvement of B, C and D Hepatitis Viruses in the Etiopathogenesis of Chronic Lymphoproliferative Syndromes in Romania

VIOLETA MOLAGIC1, VICTORIA ARAMĂ1, A. STREINU CERCEL1, NICOLETA IRIMESCU1, ANA MARIA VLĂDĂREANU3, M. OLARIU1, S.S. ARAMĂ2, A. RAFILA1, CAMELIA DOBREA4, S. COSTOIU5, MARIA MÂRZA5, D. OŢELEA1, SIMONA PARASCHIV1, DANA MAXIM1, MĂDĂLINA POPA1, H. BUMBEA3,

CRISTINA CIUFU3, C. BĂICUŞ2, RALUCA MIHĂILESCU1 1“Professor Dr. Matei Balş” National Institute of Infectious Diseases, Bucharest

2“Carol Davila”University of Medicine and Pharmacy, Bucharest 3Hematology Department, Bucharest University Emergency Hospital

4“Victor Babeş” National Institute for Research and Development in Pathology and Bio-medical Sciences, Bucharest 5Electrotechnics Research and Designing Institute, Bucharest

The aims of the study. Evaluation of the prevalence of HBV, HCV, HDV infection in patients with chronic lymphoproliferative diseases (CL), identification of the most involved viral genotypes, correlation between viremia dynamics and CL evolution, detection of molecular mechanisms implicated in CL pathogenesis, identification of lymphocytic receptors for viral antigens and biologic markers for early diagnosis of CL.

Methods. We present preliminary results of the first year of our research grant. This is a prospective, analytic, observational study in patients diagnosed with CL and HBV, HCV, HDV chronic infection. We included the following forms of CL: non-Hodgkin malignant lymphoma (NHL), Hodgkin lymphoma (HL) and chronic lymphocytic leukemia (CLL). We used the following commercial test kits: HCV RNA Real time PCR on a COBAS TaqMan (Roche Diagnostics) analyzer with 28 to 140.000.000 UI/ml detection range for HCV viremia, HBV DNA Real time PCR on a COBAS TaqMan (Roche Diagnostics) analyzer with 6 to 110.000.000 UI/ml detection range for HBV and the Roboscreen-RoboGene AJ kit with 10-10.000.000 replica/ml detection range for HDV.

Results. We have included 20 patients with CL and chronic hepatitis infection so far. Median age of the patients was 61 years.

The identified CL forms were: B cell NHL (15 cases), T cell NHL (1 case), CLL (3 cases), Hodgkin lymphoma (1 case), equally distributed in aggressive and indolent forms of CL.

HCV infection was diagnosed in 10 patients with CL, HBV infection was found in 10 patients with CL, 3 of them having co-infection HBV+ HDV.

In 4 patients with HBV infection viremia was over 20.000 IU/ml and the pattern of the CL was the aggressive form of the disease. The feature of the co-infection HBV+HDV was the predominance of indolent forms of CL. Among patients with HCV infection, only 3 cases were detected with viremia over 600.000 IU/ml and CL was represented by aggressive forms of the disease.

We also have immunohistochemical data available in 19 cases, which seem to confirm the role of hepatitis viruses in lymphoproliferative disease etiopathogenesis.

Conclusions. We ascertained an almost equally represented prevalence of HCV and HBV infection in patients with CL. The levels of HBV, HCV and HDV viremia were low in most of the cases. The most frequent form of CL was B cell NHL. We found an equal distribution between indolent and aggressive forms of NHL associated to hepatitis virus infection.

Key words: HBV, HCV, HDV, chronic lymphoproliferative disease.

Hepatitis B, C and D viruses (HBV, HCV, HVD) infections are a major public health issue for the entire world, especially in developing countries. It has been estimated that 5% of the world’s population (400 million people) is affected by HBV and 3% (170 million people) by HCV, with wide prevalence variations between ethnic groups within the same geographical areas.

For our country the estimated HBV prevalence is 6% and HCV prevalence 5–6%. Romania is classified by W.H.O. as a medium endemic risk area for HBV infection. C.D.C. includes Romania in the highly endemic areas for HVD, but without sufficient data to estimate the prevalence of this type of infection in our country [1–6].

ROM. J. INTERN. MED., 2009, 47, 1, 25–34

Violeta Molagic et al. 2 26

Limited studies revealed that HCV genotype 1b is predominant in Romania, but there is insufficient data on the most common HBV and HDV genotypes in our country.

It is currently accepted that chronic HBV and HCV infection naturally evolve to cirrhosis of the liver (CL), sometimes hepatocellular carcinoma and ultimately death is currently unanimously accepted. But during the last few decades HBV and HCV were involved in the etiopathogenesis of a growing number of extra hepatic diseases of autoimmune and chronic lymphoproliferative (CL) nature [7].

Non-Hodgkin lymphomas (NHL) are malignant blood disorders that usually involve B type lymphocytes (BL). Their incidence has alarmingly increased during the past decade, by at least 5% per year. Currently NHL occupies the 5th place on a list of the most common types of neoplasic diseases in the world and the third place in the incidence growth rate list. Simultaneously, NHL mortality is increasing by 2% per year in the USA. In the industrialized countries NHL is currently the 7th most common cause of death. In most cases NHL starts in the lymph nodes, but an increase of extra nodular lymphoma was observed during the last few decades. While the involvement of EBV, HIV, HTLV and HHV8 viruses in the etiopathogenesis of NHL is already confirmed and well documented, the involvement of HBV and HCV in CL diseases is a recent discovery and the intimate mechanisms of lymphoproliferation are not known well enough yet.

The epidemiologic correlation between chronic HBV and HCV infection and a number of CL, especially B cell NHL, was proven by numerous studies in various geographical areas [1][8–18]. The results confirm that chronic HBV and HCV infection is an independent risk factor for B cell NHL, increasing the overall morbidity and mortality in these infections. For a long time HBV and HCV were considered to be liver specific, but in the last decades they were proven to be lymphotrope as well, a fact demonstrated by proof of viral replication inside peripheral mononuclear lymphocytes, lymph nodes and hematopoietic marrow [7].

For NHL patients worldwide, HBV infection rate ranges between 3 and 30% [7][10][11] and HCV infection rate between 8 and 32% [7][13–16][19–21].

The correlation between hepatitis viruses and NHL in Romania was rarely and insufficiently studied, little related data being currently available

in the local specialty literature. A study published in 1999 by Cucuianu A. [8] reported a significantly higher HBV and HCV infection rate in NHL patients compared to the general population. Out of 68 NHL patients 20 (29.5%) were positive for anti-HCV antibodies, 21 (30.8%) were positive for HBs antibodies and 6 cases were diagnosed with mixed HBV and HCV infection. A study published by Munteanu C [22] in 2002, who analyzed 50 NHL patients compared with a control group of 6087 blood donors, reported a higher HCV infection rate in NHL patients (22% vs. 1.44%; p<0.001).

OBJECTIVES

Regarding the facts described in the introduction, a new multidisciplinary study in Romania is needed to evaluate the involvement of hepatitis viruses in CL diseases. For this purpose “Professor Dr. Matei Balş” National Institute of Infectious Diseases – Bucharest (INBIMB) submitted and won a research grant in a competition organized by The National Authority for Science Research (ANCS) as part of the Priority Domains Partnership Program, named “Correlation of diagnostic methods to evaluate the hepatitis virus involvement in chronic lympho-proliferative diseases – infection rates and research on the molecular mechanisms for the oncogenesis. (Acronym: LIMFO-VIR)”

This is a complex multidisciplinary study that will span over three years (2007-2010). The grant brings together 4 prestigious medical institutions (INBIMB; “Carol Davila” University of Medicine and Pharmacy Bucharest – UMFCD, “Victor Babeş” National Institute for Research and Development in Pathology and Bio-medical Sciences Bucharest – INCDVB and the Hematology Department of the Bucharest University Emergency Hospital – SUUB) and 2 non-medical organizations (the Electro-technics Research and Designing Institute Bucharest – ICPE and the Bucharest Polytechnic University – UPB).

This project’s objectives are presented in Table I. The expected results for the LIMFO-VIR

grant are:

− An estimate of the HBV, HCV and HDV infection rates in patients suffering from CL diseases in Romania and the identification of the most common viral genotypes involved;

3 B, C and D hepatitis viruses and chronic lymphoproliferative syndromes

27

Table I

The research aims of LIMFOVIR grant

The research aims of LIMFOVIR grant

Evaluation of the prevalence of HBV, HCV, HDV in 150 consecutive patients with chronic

lymphoproliferative diseases (CL) Identification of molecular mechanisms of hepatitis viruses infections implicated in CL pathogenesis Detection of links between viral proteins and tumoral/lymphocytic receptors Study of the role of essential mixed cryoglobulinemia (EMC) associated with hepatitis viruses

chronic infection in CL Finding a possible correlation between viral genotype and histological form of CL Detection of links between viremia dynamics and CL evolution Study of a possible correlation between the hepatic disease stadium, the age of hepatitis virus chronic

infection and beginning of clinical manifestations of CL Comparative study of clinical and hematological evolution in infected/ noninfected patients under

standard chemotherapy Detection of biologic markers for early diagnosis of CL.

− To establish standard techniques for the

molecular diagnostic of hepatitis virus infection, not yet widely used in Romania

− To discover some of the molecular mechanisms of HBV and HCV involvement in CL diseases by identifying the lymphocyte receptors for viral antigens

− To define some therapeutic and prognostic implications.

MATERIAL AND METHODS

In this text we shall present the preliminary results of the first year of LIMFO-VIR grant study.

Research teams from SUUB and INCDVB defined the clinical, hematological, histological and immunohistochemical characteristics for the CL studied cases. The diseases included in the study were: Hodgkin lymphoma, non-Hodgkin malignant lymphoma (NHL) and chronic lymphatic leukemia (CLL).

The serological and virological characteristics of hepatitis virus infections in CL patients were analyzed by research teams from INBIMB and UMFCD.

For immunophenotype of the malignant cells and 2008 OMS Classification of the CL immuno-histochemicals tests were performed on 19 cases included in the study (INCDVB); in some cases, immunphenotyping by flow-cytometry was also performed (SUUB).

HBV, HCV and HDV viremia was determined by the Molecular Genetics Laboratory of INBIMB using commercial test kits: HCV RNA Real time

PCR on a COBAS TaqMan (Roche Diagnostics) analyzer with 28 to 140.000.000 UI/ml detection range for HCV, HBV DNA Real time PCR on a COBAS TaqMan (Roche Diagnostics) analyzer with 6 to 110.000.000 UI/ml detection range for HBV and the Roboscreen-RoboGene AJ kit with 10–10.000.000 copies/ml detection range for HDV.

Because of technical reasons, the genotype identification of the HBV, HCV and HDV has not been assessed yet; it will be done retroactively from the stocked serum samples.

The ICPE and UPB research teams have created and monitored the electronic database. Working in parallel, they defined the mathematical and statistical models to be used for the partial and definitive analysis of the complex multidisciplinary data resulted from the 5 research partners.

RESULTS AND DISCUSSION

Between September 2007 and September 2008, 24 patients diagnosed by SUUB and INCDVB with CL diseases were subjected to a complex set of tests to account for both serological (specific HBV, HCV and HDV antigens and antibodies) and virological (ARN-HCV, ADN-HBV and ARN-HDV molecular biology detection and quantification) characteristics of the hepatitis virus infections.

Out of the 24 patients diagnosed with CL diseases by the Hematology Department of SUUB only 20 were confirmed by INBIMB as positive for at least one hepatitis virus, thus being qualified to be included and monitored in the LIMFO-VIR project study.


The demographic characteristics of the monitored group showed a sex distribution of 12 females/8 males and an average age of 61 years old

(17 of the total of 20 were over 50 years old at the moment they were enlisted in the study group) (Fig. 1).

Fig. 1. – Age groups distribution.

Immunohistochemistry (IHC) is an indirect

method for assessing the cellular phenotype on paraffin embedded tissue. For lymphoid samples, the cellular antigens, grouped together in the CD (cluster of differentiation) system, were identified using specifically monoclonal antibodies. This method is useful for the immunophenotyping of the malignant cells (B or T/NK cells) and classification of the lymphoproliferation according to the 2008 WHO Classification; as well, IHC is able to assess some markers with prognostic implication, like markers for cell proliferation [26].

For this study, a large panel of monoclonal antibodies was performed: panB markers (antiCD20/ clone L26 and antiCD79a), for T cells antigens (for panT antigens CD3 and CD45RO/clone UCHL1, for CD4 positive helper T cells and CD8 positive suppressor T cells). The aberrant expression of some antigens was important for the diagnostic (such as

CD5, T cell marker, also expressed in B-CLL and mantle B-cell NHL; CD23, a dendritic cells marker, positive also in B-CLL). The study of cytoplasm expression of kappa and lambda light chains was useful for clonality investigation. AntiBCL2 (for the bcl2 oncogene protein) was performed, together with antiCD10 or antiBCL6 for the diagnosis of the B-cell follicular NHL. AntiCD15 and antiCD30 were used for the Reed-Sternberg malignant cells in Hodgkin Lymphoma. AntiKi67/clone MIB1 was the most suitable antibody for cellular proliferation rate.

The CL type distribution showed a clear predominance of B-cell NHL (Fig. 2), with an equal distribution of aggressive (10 cases) and indolent (10 cases) types. These preliminary results do not agree with the current literature, which states that CL diseases associated to hepatitis virus infections are usually non-aggressive [7][15][23–26].

NHL – Non-Hodgkin Lymphoma; CLL – Chronic Lymphocytic Leukemia; HL – Hodgkin Lymphoma

Fig. 2. – Histological forms of chronic lymphoproliferations in the study group.

3

11

6

0

2

4

6

8

10

12

20-49 years 50-69 years 70-90 years

* there were no cases under 30 years

Included in LIMFOVIR study

Diffuse large B-cell lymphoma Diffuse small B-cell lymphoma

Lymphocytic lymphoma Marginal zone B-cell

B-cell NHL T-cell NHL CLL HL


29

The quantum of aggressivity degree was based on the classification proposed in 1995 by Saul Rosemberg, St. Pilieri and Lorenzo Leonini (Table II). The one we used takes into account not only the histological criteria, but also global

prognostic elements [26]. So, we used both the clinical aggression criteria (rapid progression of the disease, multi-organic lesions) and prognostic evaluation based on clinical, immune phenotype, genetic and molecular criteria.

Table II

Classification of lymphoproliferative diseases used in the study [26]

WHO classification of lymphoproliferative diseases based on malignity grade

INDOLENT LYMPHOPROLIFERATIVE DISEASES

B-cell lymphoproliferative diseases Chronic lymphocytic leukemia/small lymphocytic lymphoma. Lymphoplasmacytic lymphoma/immunocytoma Follicular lymphoma (1st and 2nd grade) Marginal zone B-cell lymphoma Hairy cell leukemia Plasmacytoma/plasma cell myeloma T-cell lymphoproliferative diseases T-cell chronic lymphocytic leukemia/prolymphocytic leukemia T-cell granular lymphocytic leukemia. Mycosis fungoides/Sézary syndrome

MILD AGGRESSIVE LYMPHOPROLIFERATIVE DISEASES

B-cell lymphoproliferative diseases B-cell prolymphocytic leukemia Mantle cell lymphoma Follicular lymphoma (3rd grade) T-cell lymphoproliferative diseases T-cell chronic lymphocytic leukemia/prolymphocytic leukemia Adult T-cell lymphoma/leukemia Angioimmunoblastic T-cell lymphoma

AGGRESSIVE LYMPHOPROLIFERATIVE DISEASES

B-cell lymphoproliferative diseases Diffuse large B-cell lymphoma T-cell lymphoproliferative diseases Peripheral T-cell lymphoma, not otherwise characterized Enteropathy-type intestinal T-cell lymphoma Anaplastic large cell lymphoma Adult acute T-cell lymphoma/leukemia

VERY AGGRESSIVE LYMPHOPROLIFERATIVE DISEASES

B-cell lymphoproliferative diseases Precursor B-cell lymphoblastic lymphoma/leukemia Burkitt lymphoma/Burkitt cell leukemia Burkitt-like lymphoma T-cell lymphoproliferative diseases Precursor T-cell lymphoblastic lymphoma/leukemia

HODGKIN LYMPHOMA

Nodular lymphocyte–predominant Hodgkin lymphoma Classical Hodgkin lymphoma Nodular sclerosis Hodgkin lymphoma. Lymphocyte-rich classical Hodgkin lymphoma. Mixed-cellularity Hodgkin lymphoma. Lymphocyte-depleted Hodgkin lymphoma


Of the 20 studied patients, 3 died during the first year of the study.

When diagnosed with CL diseases only 5 patients were known to suffer from hepatitis, the average interval between the diagnostic of hepatitis and the diagnostic of hematological disease being

3 years and 8 months. A simultaneous hemato-logical disease and hepatitis diagnostic was reached in 6 cases. The rest of 9 patients were diagnosed with the hematological disease before the discovery of hepatitis (Fig. 3).

Fig. 3. – Case distribution at the moment of diagnosis of the hematologic/hepatic disease.

It should be mentioned that at the time they

were diagnosed with a hematological disease and included in the study only 10 patients suffered from cirrhosis (ALT > 2x normal value) and 3 suffered from cholestasis. Until this moment the histological data about liver lesions, evaluated with the Fibromax technique, have not been analyzed yet.

Regarding the type of hepatitis virus detected (Fig. 4), HBV and HCV had a similar distribution: 10 patients were diagnosed with HBV, 3 of them suffering from simultaneous infection with HDV; the other 10 were diagnosed with HCV infection.

Fig. 4. – Cases distribution based on the hepatitis viruses

identified in the study groups.

DETAILS ABOUT THE GROUP OF 7 PATIENTS WITH CL DISEASE ASSOCIATED WITH

HBV INFECTION

In the group of 7 patients with CL disease associated with HBV infection we noticed the preponderance of negative antigen HBe with positive anti-HBe antibodies cases (6 out of the 7 patients). This pattern is characteristic for precore or BCP (core promoter basal) mutant HBV infection. It is estimated that up to 85–90% of

all HBV infections in Romania are with precore mutant HBV [27].

Because all the studied patients were immuno-suppressed from the hematological disease and its chemotherapy, we expected high values for viremia in all cases. Actually we detected HBV viremia in only 6 of the 7 cases and only 4 of them registered HBV-DNA values over 105 UI/ml.

The clinical and histological LC type distribution of the 7 patients (Fig. 5) was: 6 cases of NHL and one case of CLL. The 6 cases of NHL were further distributed as 4 cases of large B cell NHL and 2 cases of small B cell NHL.

Fig. 5. – Distribution of patients with HBV chronic infection

based on the type of lymphoproliferative disease.

Among the 7 patients with positive HBV serological markers we identified 3 rare serological and viral profiles: 1. One CL patient had anti-HBc and anti-HBe

antibodies without HBs antigen, anti-HBs antibodies or HBV-DNA. For an immuno-competent patient this serological profile would be interpreted as cured HBV infection.

CLL

small B-cell lymphoma

large B-cell lymphoma

associated with HBV infection

initial hematologic diseaseinitial hepatic disease

consecutively diagnosed simultaneously diagnosed

included in LIMFOVIR study


31

But for an immunosuppressed case due to hematological or HIV infection this kind of serological profile calls for extreme caution in both the clinical interpretation of the HBV infection and in its management. Immuno-suppression due to malignant hematological diseases can lower the anti-HBs levels below protection limits or even below the minimum detectable level, thus allowing a relapse of the HBV infection, proved by positive HBs antigen and HBV-DNA. Usually a HBV relapse occurs after aggressive chemotherapy [7][11].

2. Another CL patient had HBV-DNA in his blood without being positive for HBe antigen or anti-HBe antibodies, a very rare occurrence. Medical literature describes periods of HBe conversion and reversion during the evolution of a HBV infection (disappearance of HBe antigen with the appearance of anti-HBe antibodies or reappearance of HBe antigen without anti-HBe antibodies being present)[28].

3. Another CL case in our study had HBs antigen and low doses of anti-HBs antibodies, with a viremia value of 517 copies/ml. Detecting defective anti-HBs antibodies in a case of chronic HBV infection is interpreted as a contributing factor for a persistent infection [29].

All the 5 cases of this lot that we already have immune phenotype results for had bcl-2 present in the tumors.

UCLH1 was positive in small lymphocytes only in 2 cases and L26/CD20 was detected in the tumor cells of all 5 cases.

DETAILS ABOUT THE GROUP OF 10 PATIENTS WITH CL DISEASE ASSOCIATED WITH

HCV INFECTION

Among the 10 patients with chronic HCV infection only 3 had viremia over 600.000 UI/ml, the others registering lower values. Patients with viremia under 600.000 UI/ml are considered to be Low Viral Load (LVL) cases. It appears that LVL chronic HCV cases are more responsive to antiviral treatment. It is now estimated that cases with viremia under 400.000 UI/ml have the biggest chance for a sustained response to antiviral therapy [31][32].

The cases distribution based on the histological type of CL (Fig. 6) indicates NHL as preponderant (7 cases), with 6 B cell type and one T cell NHL. The

other 3 were: 2 cases of chronic lymphatic leukemia (CLL) and one with Hodgkin lymphoma (HL).

Fig. 6. – Distribution of patients with HCV chronic infection

based on the type of lymphoproliferative disease.

Immunohistochemically, BCL2 was expressed by the malignant cells in indolent B cells NHL; in aggressive NHL, only reactive small B and T lymphocytes were positive. In 2002 Zignego proved that HCV induces anti-apoptotic activity through t (14;18) translocation that activated the bcl-2 gene and took it near the antibodies heavy chain (IgH) gene. A high frequency of monoclonal B type lymphocytes was noticed that expressed the same rearrangement of the antibodies heavy chain. Bcl-2 gene rearrangement (present in 75% of CL plus HCV cases) disappeared under antiviral treatment [33].

In 5 cases UCHL-1 was present in the lymphocytes, but not in tumor cells. L26/CD20 was identified in all the studied cases.

In 5 of the 10 cases with CL associated with chronic HCV, at least one serological HBV marker was also identified, caused either by the disease or a previous vaccine. HBV serological profiles of these cases were:

2 patients with post-vaccine anti-HBV anti-bodies: one case with low protection dose of 34.02 UI/ml and the other with protector dose of 278.63 UI/ml;

3 cases with anti-HBV antibodies resulted from the disease: • 2 with anti-HBs antibodies (one case with

protective dose of 165.76 UI/ml and another with low protection dose of 48.52 UI/ml);

• One with anti-HBc antibodies and all the other serological markers (including viremia) negative.

associated with HCV infection

CLL

HL

B-cell NHL

T-cell NHL


This last situation can be explained by: cured HBV infection, naturally immunized patient from a past HBV infection, with anti-HBs antibodies in doses too low to be detected, a false positive result for anti-HBc antibodies or anti-HBs antibodies in doses too low to be detected even if the patient may suffer from a present HBV chronic infection.

All these situations in which a patient suffering from a malignant hematological disease has at least one positive HBV marker require continuous clinical, biochemical, serological and virological monitoring to diagnose a potential HBV relapse that can occur after chemotherapy. If it occurs, it will increase the immunosuppression caused by the hematological disease [7][23][34].

The viral reactivation mechanism consists in the restart of viral replication after immuno-suppression chemotherapy and is detected by: an over 10 times increase of the HBV-DNA dose in the serum, the presence of HBe antigen and the immediate T type lymphocytes response that will destroy the infected liver cells and subsequently increase AST and ALT doses in the serum [7][34].

The HBV relapse can occur in the following situations: chronic HBV infection, chronic inactive HBs positive case without detectable HBV-DNA in the serum, naturally HBV immunized patient (HBs negative, positive for anti-HBc antibodies, positive for anti-HBs antibodies) and silent HBV infection (HBs negative, HBV-DNA positive in serum or liver cells, positive for anti-HBc antibodies, positive or negative for anti-HBs antibodies)[34].

The most recent international guidelines recommend nucleoside analogues (Lamivudine) pro-phylaxis for all HBs positive patients undergoing chemotherapy during the entire chemotherapy treatment and 3 to 6 months after it [34.

DETAILS ABOUT THE GROUP OF 3 PATIENTS WITH CL DISEASE ASSOCIATED WITH HBV+HVD

SIMULTANEOUS INFECTION

The serological examination showed anti-HBe antibodies present in all 3 cases. Case distribution by the viral replication levels of the two viruses was: − One case of high HBV replication (HBV-DNA

over 2.140.000 UI/ml) with undetectable HDV viremia;

− One case of HDV replication (HDV-RNA = 143.08 copies/ml) with HBV viremia below 6 UI/ml;

− One case with replication of both viruses, with high values for HBV viremia (HBV-DNA = 17.300 UI/ml) and lower HDV viremia (HDV-RNA = 2.470 copies/ml).

All the 3 patients were diagnosed with B cells NHL. Immunohistochemically, CD20/L26 was positive in the malignant cells in all 3 cases.

CONCLUSIONS

We believe that the small number of cases currently analyzed in the multidisciplinary LIMFO-VIR research prevents us from formulating definitive conclusions, but the data acquired so far allows us to express a few ideas that may generate practical applications: 1. CL associated with B, C and D hepatitis

viruses appeared to be more frequent at ages over 50 years old.

2. HBV and HCV were equally involved in CL diseases

3. HBV, HCV and HDV viremia in CL cases was low. 4. In the chronic HBV infection group most

patients had positive anti-HBe antibodies with negative HBe antigen (precore mutant virus). There was no case of silent HBV infection in CL disease cases.

5. B cell NHL was preponderant. Aggressive and non-aggressive forms of NHL were equally distributed.

6. Immunohistochemical data (partly available only for 19 cases) appeared to support the hepatitis viruses’ involvement in chronic lymphatic proli-ferations, but at this stage of the study they lack statistic importance.

Acknowledgements. We bring our sincere thanks to all members of the research teams in all 5 institutes which have helped us along this project, to the entire medical team in the “Prof. Dr. Matei Balş” National Institute of Infectious Diseases – Bucharest and the Hematology Clinic of the Emergency University Hospital – Bucharest and to the patients and their families who agreed to participate in this project.

Funding. This paper contains the preliminary results from the first 2 stages of a research grant won through competition (Contract 41–012 / sept 2007), financed from the State Budget – National Authority for Science Research, in the 4th Program (Priority Domains Partnership Program).

The coordinating institute is “Prof. Dr. Matei Balş” National Institute of Infectious Diseases – Bucharest. The project manager is Associate Professor Victoria Arama, MD, PhD.


33

Obiective. 1) Evaluarea impactului HAART asupra viremiei CMV la pacienţii co-infectaţi CMV-HIV, în absenţa terapiei anti-CMV specifice; 2) compararea a două tehnici de biologie moleculară pentru detectarea şi cuantificarea ADN-CMV la aceşti pacienţi.

Metode. Prezentăm datele preliminare ale unui studiu prospectiv asupra pacienţilor nou diagnosticaţi cu infecţie HIV într-un spital de îngrijire terţiară, în perioada iunie 2006-iunie 2008. Monitorizarea clinică, virusologică (viremie CMV şi HIV) şi imunologică (CD4/mmc) a fost trimestrială. Încărcătura virală CMV a fost determinată cu RoboGene Human Cytomegalovirus (HCMV) Quantification kit (aj Roboscreen). Serurile pacienţilor cu CD4 <50/mmc şi viremie CMV nedetectabilă au fost retestate cu CMV PCR kit (Qiagen Diagnostics). Ambele PCR au fost efectuate cu ajutorul ABI Prism 7000 (Applied Biosystems).

Rezultate. Până în prezent, au fost incluşi în studiu 105 pacienţi cu infecţie HIV si anticorpi anti-CMV IgG pozitivi. Durata medie de urmărire a fost de 18 luni. Viremia CMV a fost detectabilă în 21 de cazuri la prima vizită şi alte 5 cazuri la a doua vizită. 22 de pacienţi au avut CD4<50/mmc, dintre care 14 cu viremie CMV nedetectabilă. Prin ambele tehnici moleculare s-au obţinut rezultate similare. S-a prescris HAART la 86% dintre pacienţi; toţi pacienţii cu viremie CMV detectabilă au primit HAART, dar nu şi terapie specifică anti-CMV. Sub HAART, toate viremiile CMV detectabile retestate au devenit nedetectabile după o perioadă mediană de 16,5 săptămâni.

Concluzii. Viremia CMV a fost utilă în diagnosticarea precoce a infecţiei CMV asimptomatice. Spre deosebire de diagnosticarea infecţiei CMV la pacienţii cu transplant, tehnicile de biologie moleculară pentru cuantificarea ADN-CMV la pacienţii cu infecţie HIV nu sunt standardizate. În studiul nostru, cele 2 kituri RoboGene Human Cytomegalovirus (HCMV) Quantification kit (aj Roboscreen) şi CMV PCR kit (Qiagen Diagnostics) au dat rezultate comparabile. Terapia antiretrovirală a negativat viremia CMV, în absenţa terapiei specifice anti-CMV. Ca în cazul altor infecţii oportuniste, evoluţia infecţiei CMV pare să se fi ameliorat prin controlul infecţiei HIV.

Corresponding author: Victoria Aramă, Associate Professor “Prof. Dr. Matei Balş” National Institute of Infectious Diseases Bucharest

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cell non-Hodgkin’s lymphoma in a hepatitis B endemic area: A case control study. Jpn. J. Cancer Res., 93; 471–477, May 2002.


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2006;375–380. 35. www.data.euro.who.int

Received January 25, 2009

The Acute Effect of Metoprolol upon NT-proBNP Level in Patients with Congestive Heart Failure

D. ZDRENGHEA, DANA POP, MARIA ILEA, G. BODISZ, ADINA MĂLAI, M. ZDRENGHEA

Deptartment of Cardiology, Rehabilitation Hospital, Cluj-Napoca, Romania

Brain natriuretic peptide (BNP) is a sensitive and specific marker of left ventricular (LV) function. The acute effect of β blockers upon plasma BNP levels in CHF patients has been less studied but it is important because of the initial possible depressing effect upon LV function.

Purpose. To investigate the acute effect of oral Metoprolol upon plasma proBNP levels in CHF patients.

Methods. There were included 56 patients with congestive heart failure, 38 with ischemic heart disease and 18 with idiopathic dilated cardiomyopathy, 40 males and 16 females, aged between 25 and 65 years, who were compared with 19 healthy individuals, 12 males and 7 females, of the same age. All patients were free of beta blockers treatment. Plasma Nt-proBNP was determined in fasting state using ELISA method (NV <250 fmol/mL). After this, every patient received 50 mg Metoprolol succinate and at three hours (considered as peak plasmatic concentration) venous blood samples were again obtained and Nt-proBNP determined.

Results. NT-pro BNP was increased (1400±130 fmol/mL) in heart failure patients and normal (187±17.2 fmol/mL) in healthy controls. After Metoprolol the plasmatic level of NT-proBNP was not significantly different in both healthy controls (162±13.3 fmol/mL) and heart failure patients (1419±133 fmol/ml) in comparison with baseline values. After Metoprolol NT-proBNP decreased (from 1266±121 to 1120±107, p>0.05) in III NYHA class patients and increased (from 1457±142 to 1530±150, p<0.05) in IV NYHA class patients. It remained unchanged in patients with LVEF >30% (1384±140 vs 1389±129 fmol/mL) and increased (from 1480±134 to 1690±161 fmol/mL, p<0.05) in patients with LVEF <30%; it was not significantly modified in patients with atrial fibrillation in comparison with those in sinus rhythm (1348±132 vs 1516±168 fmol/mL).

Conclusion. Beta blockers do not have a severe depressant effect on left ventricular performance in all patients with systolic heart failure. A LVEF>30% suggests, but the lack of modification of NT-proBNP levels after administration of 50 mg Metoprolol confirm, that the beta blocking treatment can be initiated with higher doses than those recommended until now.

Key words: brain natriuretic peptide, beta-blockers, congestive heart failure.

Brain natriuretic peptide (BNP) represents a quick and sensitive marker for the changes of left ventricular performance, increasing at rest and during exercise in patients with congestive heart failure [1][2]. BNP also increases in acute heart failure, representing a useful diagnostic tool and, at the same time, is used in the follow-up and proving the efficacy of the applied emergency treatment [1][3].

The effect of beta blockers upon plasmatic level of BNP was studied during short and long time treatment, the first one increasing cardiac peptides and the second one decreasing them in relationship with improvement of left ventricular performance [4][5]. Cardiac peptides increase during exercise in heart failure patients, but also in healthy people [1–3]. This seems to support the idea that in heart failure patients the treatment with

beta blockers has to be initiated with very small doses. On the other hand, in clinical practice, mainly in patients with atrial fibrillation, it is often necessary to administrate higher doses of beta blockers, doses as high as 100–150 mg Metoprolol/ day being recommended [6].

Thus, the aim of the present study is to investigate the acute effect of beta blockers upon BNP in heart failure patients, a subject that was less studied until now, even if it has important clinical implications, because of the initial possible depressant effect upon left ventricular function.

MATERIAL AND METHOD

In the present study there were included 56 patients with congestive heart failure, 38 with

ROM. J. INTERN. MED., 2009, 47, 1, 35–40

D. Zdrenghea et al. 2

36

CHF HC

baseline

after Metoprolol

1400±130 187±17.2

p>0.05

162±15.3

1419±133

ischemic heart disease and 18 with idiopathic dilated cardiomyopathy, 40 males and 16 females, aged between 25 and 65 years, who were compared with 19 healthy individuals, 12 males and 7 females of the same age.

The diagnosis was sustained on a clinical and echocardiographic (2D) basis, with LVEF <40%, which was also the inclusion criteria in the heart failure group.

All patients were free of beta blockers treatment during the last month before the study and they received no treatment on the examination day.

Plasma NT-proBNP was determined in fasting state using ELISA method (NV <250 fmol/mL). After this, every patient received 50 mg Metoprolol succinate and at three hours (considered as peak plasmatic concentration) venous blood samples were again obtained and NT-proBNP determined.

In all patients there were also considered NYHA class (33 patients in class III NYHA and 23 in class IV NYHA), LVEF (<30% in 38 patients and >30% in 18 patients) and atrial fibrillation (noted in 30 patients).

STATISTICAL ANALYSIS

The data were analyzed using SPSS 8.0 for Windows. We calculated mean and standard deviation for normal distributed quantitative variables. Differences between quantitative variables were examined using Student test (independent-sample T test), and for qualitative variables we used χ2 test. A

p value less than 0.05 was considered significant from the statistical point of view.

RESULTS

At the initial determination (Fig. 1) NT-proBNP was increased in heart failure patients (1400± 130 fmol/mL) and normal in healthy controls (187± 17.2 fmol/mL).To prove the effect of Metoprolol, blood pressure and heart rate were measured before and three hours after Metoprolol administration. Blood pressure decreased insignificantly from 111±10.2 to 105±10.1 mmHg in heart failure patients and increased insignificantly from 128±1.7 to 130±12.2 mmHg in healthy controls. In turn, in heart failure patients was registered an important decrease of the heart rate from 98±8.7 to 72±6.2 bpm (p<0.01), the decreasing being much less in healthy controls (74±6.5 to 64±6.9 bpm, p<0.05). The decrease in heart rate proves, in the absence of the determination of the plasmatic concentration, that Metoprolol was efficient and the modification of the plasmatic concentration of NT-proBNP can be attributed to it. At three hours after Metoprolol administration in both, healthy controls and heart failure patients, the plasmatic concentration of NT-proBNP was not significantly different (162±13.3 fmol/mL in healthy controls and 1419±133 fmol/mL in heart failure patients) in comparison with the baseline values (p>0.05).

Fig. 1. – NT-proBNP levels in healthy controls versus heart failure patients.

3 Effect of Metoprolol on congestive heart failure

37

-11.53

5

-4.4

6.56

0

14.19

-15

-10

-5

0

5

10

15

NYHA III NYHA IV FiA - FiA + FE>30% FE<30%

% variation of NT-pro BNP

The individual analysis showed that NT-proBNP values remained the same in three patients (3900±371 fmol/mL), increased in 32 patients (from 1236±112 fmol/mL to 1426±131 fmol/mL, p<0.03) and decreased in 21 patients (from 1418±120 fmol/mL to 858±79 fmol/mL, p<0.03).

When we took into consideration the NYHA class, NT-proBNP decreased in NYHA III patients (from 1266±121 to 1120±107 fmol/mL, p>0.05) and increased (from 1457±142 to 1530±150 fmol/mL, p>0.05) in NYHA IV class patients.

With respect to the relationship between NT-proBNP and LVEF, in patients with LVEF<30%, NT-proBNP increased from 1480±134 to 1690±

161 fmol/mL, p<0.05, but remained unchanged in patients with LVEF>30% (1384±140 vs 1389± 129 fmol/mL).

Finally, in atrial fibrillation patients NT-proBNP increased insignificantly from 1265±124 to 1348±132 fmol/mL and decreased insignificantly in patients in sinus rhythm (from 1587±149 to 1516±168 fmol/mL, p>0.05).

Also, after Metoprolol NT-proBNP values were significantly higher in patients with IV NYHA class, atrial fibrillation or LVEF <30% in comparison with III NYHA class patients, patients in sinus rhythm or LVEF >30% (Fig. 2).

* p<0.05

Fig. 2. – NT-pro BNP levels percent variation after administration of Metoprolol in relationship with some severity parameters.

DISCUSSION

The increased mean plasmatic level of NT-proBNP proves again its value for the diagnosis and evaluation of heart failure patients [7–9].

The plasmatic values of NT-proBNP, almost six times more than normal, can be attributed to the generally severe depression of the left ventricular

systolic function in patients with dilated cardio-myopathy and to the fact that all patients were included in NYHA class III or IV.

More important for the purpose of the present study is the acute effect of a mild dose of Metoprolol (50 mg) upon NT-proBNP plasmatic level. As we already showed, the highly significant decrease of heart rate in dilated cardiomyopathy patients proved, indirectly but strongly, that at three

*

*

%

*


38

hours after administration, plasmatic Metoprolol concentration was in the therapeutic range. In turn, NT-proBNP levels were not significantly modified in both, heart failure patients and healthy controls, even if they slightly increased in heart failure patients and slightly decreased in healthy controls.

The results support the idea that even such a high initial dose of Metoprolol as 50 mg does not impair the hemodynamic disturbances, intracardiac pressure and stretching in systolic heart failure patients [10–13]. This suggests that, if necessary (high resting heart rate, rapid rate atrial fibrillation, arrhythmias), or even as a routine protocol, beta blocker treatment, mainly with Metoprolol, can be initiated using higher doses than those recommended until now, without any major detrimental effect upon the haemodynamic status of the patient [15–20].

Thus, it will be possible to reach earlier the effective and optimal therapeutic dose to control heart rate and to improve the evolution and survival of the patients during long term treatment [5][6][12][13–15].

At the same time, the individual analysis of the results showed that, if in about 46% of the patients, Metoprolol did not increase the NT-proBNP plasmatic level, in 54% of them NT-proBNP significantly increased , probably due to the depression of the left ventricular systolic function caused by the beta blockade. This hypothesis is sustained by the significant increase of the NT-proBNP level in NYHA IV patients in comparison with NYHA III patients in whom NT-proBNP slightly decreases after Metoprolol.

Metoprolol did not significantly modify NT-proBNP in the 39 patients with LVEF> 30%, but increased it in the 19 of them with LVEF<30%. At a first view the above data are not sustained by the atrial fibrillation patients in whom the NT-proBNP plasmatic levels, before and after Metoprolol, are smaller than in patients in sinus rhythm. But it is necessary not to forget that atrial fibrillation increases mainly ANP synthesis and that lowering the heart rate in atrial fibrillation patients results in an improvement of cardiac performance [21–23]. Thus our data supports the recommendation of using high doses of beta blockers (100–150 mg/day for Metoprolol) in rapid rate atrial fibrillation, even in heart failure patients, without a high risk to develop acute heart failure [1][5][24].

In fact, the results suggest that in systolic heart failure patients with moderate depressed systolic function (LVEF between 30–40%) it is possible to initiate beta blocker treatment with higher doses than recommended until now. As for the patients with LVEF less than 30%, the classical protocol has to be used [1][2][5][26][27].

In conclusion, beta blockers do not have a severe depressant effect on left ventricular performance in all patients with systolic heart failure. A LVEF>30% suggests, but the lack of modification of NT-proBNP levels after administration of 50 mg Metoprolol confirm, that the treatment with beta blockers can be initiated with higher doses than those recommended until now.

BNP-ul este un marker sensibil şi specific al funcţiei ventriculare stângi (VS).

Efectul acut al betablocantelor asupra nivelelor plasmatice ale BNP-ului la pacienţii cu insuficienţă cardiacă cronică (ICC) a fost mai puţin studiat, dar este important datorită posibilului efect depresor iniţial asupra funcţiei VS.

Scop. De a studia efectul acut al Metoprololului administrat oral asupra nivelului plasmatic al BNP-ului la pacienţii cu ICC.

Metoda. Au fost incluşi 56 de pacienţi cu ICC, 38 cu cardiopatie ischemică şi 18 cu cardiomiopatie dilatativă idiopatică, 40 bărbaţi şi 16 femei, cu vârste cuprinse între 25 şi 65 de ani, care au fost comparaţi cu 19 indivizi sănătoşi, 12 bărbaţi şi 7 femei, de aceeaşi vârstă. Niciun pacient nu era sub tratament cu betablocant. Nivelul plasmatic al NT-proBNP a fost determinat à jeun, folosind metoda ELISA (VN<250fmol/ml). Apoi, fiecare pacient a primit 50 mg Metoprolol succinat şi s-au redeterminat nivelele plasmatice ale NT-proBNP la 3 ore (acesta fiind considerat peak-ul concentraţiei plasmatice).

Rezultate. NT-proBNP a fost crescut (1400±130 fmol/ml) la pacienţii cu ICC şi normal (187±17,2 fmol/ml) la subiecţii sănătoşi. După administrarea de

5 Effect of Metoprolol on congestive heart failure

39

Metoprolol nivelele plasmatice ale NT-proBNP nu au diferit semnificativ faţă de cele bazale, atât la pacienţii cu ICC (1419±133 fmol/ml), cât şi la subiecţii sănătoşi (162±13.3 fmol/ml). După Metoprolol NT-proBNP a scăzut (de la 1266±121 la 1120±107 fmol/ml, p>0,05) la pacienţii cu ICC NYHA III şi a crescut (de la 1457±142 la 1530±150 fmol/ml, p<0.05) la cei aflaţi în clasa NYHA IV. NT-proBNP a rămas nemodificat la pacienţii cu FEVS >30% (1384±140 vs 1389± 129 fmol/ml) şi a crescut (de la 1480±134 la 1690±161 fmol/ml, p<0.05) la cei cu FEVS <30%. De asemenea, nivelele NT-proBNP nu au fost semnificativ modificate la pacienţii cu fibrilaţie atrială (1348±132 vs 1516±168 fmol/ml) în comparaţie cu cei aflaţi în ritm sinusal.

În concluzie. În administrare acută betablocantele nu au un efect puternic depresor asupra performanţei VS la pacienţii cu ICC. O FEVS >30% sugerează, dar lipsa modificării nivelelor de NT-proBNP după administrarea a 50 mg Metoprolol confirmă faptul că tratamentul betablocant poate fi iniţiat cu doze mai mari decât cele recomandate până acum.

Corresponding author: D. Zdrenghea, MD, PhD

Rehabilitation Hospital, 46–50, Viilor St., Cluj-Napoca E-mail: [email protected]

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27. SHARMA V., DHILLON P., WAMBOLT R.B., PARSONS H.L., BROWNSEY R., ALLARD M.F. et al., Metoprolol improves cardiac function and modulates cardiac metabolism in the streptozotocin (STZ) diabetic rat. Am. J. Physiol. Heart Circ. Physiol., 2008 (Epub ahead of print).


Drug Secondary Prevention in Postmenopausal Women with Ischemic Heart Disease

D. ZDRENGHEA, DANA POP, ADELA SITAR-TĂUT, MIRELA CEBANU, V. ZDRENGHEA

Department of Cardiology, Rehabilitation Hospital, Cluj-Napoca, Romania

It is known that, in comparison with men, women with cardiovascular disease are undertreated, including drug treatment. This aspect was less studied with respect to drugs used for secondary prevention.

Methods. In an urban Romanian community there was studied a representative sample of 150 postmenopausal women with cardiovascular disease (62.7% of them with ischemic heart disease-IHD). We considered the secondary prevention by drugs. The results were compared with those registered in a similar sample of 160 men aged >55 years with cardiovascular disease (68.1% of them with IHD). The study was carried out using general practitioner’s files.

Results. According to the literature, there were considered as preventive drugs: antiplatelet agents, statins, beta blockers and ACEI. Aspirin was used in 56.4% of the women and 72.5% of the men (p<0.05), ACEI in 69.1% and 79.8 % (p>0.05), beta blockers in 69.1% and 74.3 % (p>0.05) and statins in 48.9% and 48.6% (p>0.05). The results show that antiplatelet drugs are underused in ischemic patients, but especially in women. The maximum use of preventive drugs in women was registered after acute myocardial infarction (beta blockers 85.7%, statins 50%, aspirin 60.7%, ACEI 75%). In turn, the myocardial revascularization by CABG and PCI is low in both groups, but much less in women than in men: CABG 1.1% in women, 4.6% in men p>0.05, PCI 5.3% in women and 13.8% in men p<0.05.

Conclusion. In postmenopausal women with ischemic heart disease the secondary prevention by drugs is similar with that applied in men, except aspirin and ACEI, which are underused.

Key words: cardiovascular disease, secondary prevention, women.

Even if in the past decades cardiovascular risk factors have changed, cardiovascular disease represents, in both sexes, the main cause of mortality and morbidity [1][2]. At the same time, population aging is responsible for an increase in the prevalence of cardiovascular disease [3]. Secondary prevention of ischemic heart disease is known to represent an important and achievable goal [4]. Regarding patients with ischemic heart disease, there are obvious arguments that treatment including aspirin, statins, ACE-inhibitors (ACEI) and betablockers reduces risk for future events [5][6]. It would be perfect for all patients to receive the four classes of drugs (unless some contra-indications are present) [7] and this is the reason to recommend and use “polypill” [8]. It had already been accomplished, in a slightly modified form – containing aspirin, statins and ACEI [9]. Although the treatment of ischemic heart disease has improved in a significant way, the number of patients that receive the proper medication is not exactly known [3], this being true also for Romania.

At the same time, it is very well known that women with cardiovascular disease are undertreated (in comparison with men), but this aspect was less studied regarding secondary prevention.

The purpose of the study was to assess the preventive medication used by postmenopausal women with cardiovascular disease, in comparison with men of the same age.


In an urban Romanian community, from Cluj-Napoca, the study was conducted on a representative sample of 310 subjects with cardio-vascular disease, 150 postmenopausal women with cardiovascular disease over 55 years of age being compared with 160 men over 55 years of age, also with cardiovascular disease. The study was carried out using general practitioner’s files, between February 2007 and April 2007. Data about blood pressure, weight, height, plasma lipids values, glycemia were collected. Uncomplicated hypertension was not

ROM. J. INTERN. MED., 2009, 47, 1, 41–45


42

registered as a cardiovascular disease, being considered a cardio-vascular risk factor.

Ischemic heart disease was defined as old myocardial infarction, unstable angina stabilized by drugs, or other forms. 203 patients with ischemic heart disease were identified: 91 patients with old myocardial infarction (28 women, 63 men), 31 with unstable angina stabilized by drugs (17 women, 14 men) and 81 patients with other forms of ischemic heart disease (arrhythmias, silent ischemia, stable angina, congestive heart failure – 49 women, and 32 men).

There was registered information about general practitioner’s prescribed medication and ACEI, statins, betablockers and antiplatelet agents were considered as secondary preventive drugs.

The data were analyzed using SPSS 8.0 for Windows and Minitab 15.0. We calculated mean and standard deviation for normal distributed quantitative variables. Differences between quantitative variables were examined using Student test (independent-sample T test), and for qualitative variables we used χ2 test. We also used logistic regression to describe the differences between sexes, the information being presented as odds ratio and confidence interval. A p value less than 0.05 was considered significant from the statistical point of view.

RESULTS

The mean age of women was 69.22±9.16 years, in comparison with the mean age of men

which was 65.9±8.09 years. The prevalence of ischemic heart disease was 65.5%, the values registered related to the sex of the patients being 68.1% (109 patients) in men and 62.7% (94 patients) in women (p>0.05).

No significant differences, no matter of age groups, were registered regarding the prevalence of ischemic heart disease between sexes, except the prevalence was higher in men over 75 years of age (80.8% in men vs 63% in women, p<0.05).

Also, for the whole sample, no significant difference was found between sexes (women vs men) regarding the prevalence of diabetes mellitus (32% vs 32.5% p>0.05), dyslipidaemia (46.7% vs 38.8 % p>0.05), obesity (29.6% vs 26.4% p>0.05). Hypertension was more frequent in women (97.3% vs 89.4% p=0.006), and only 9 patients (5.6%), all of them being males, were found to be smokers. Not a single woman was found to be smoker.

The data were similar with those registered in ischemic patients subgroup, no significant difference being registered between women and men regarding the percent of diabetic patients (35.1% vs 35.8% p>0.05), of dyslipidaemic patients (50% vs 38.5% p>0.05), of obese patients (35% vs 26.2% p>0.05). As for the whole sample, in ischemic patients, the hypertension was more frequent in women (96.8% women vs 89% men, p=0.02).

The differences between sexes regarding preventive drugs used in ischemic patients are presented in Table I.

Table I

Preventive drugs used in ischemic patients, depending on sex

Global

No. (%)

Men

No. (%)

Women

No. (%) OR* unadjusted (M:W)(95% CI+)

OR adjusted

(M:W) (95% CI+)

Statins 99 (48.8) 53 (48.6) 46 (48.9) 0.98

(0.56–1.71)

0.81

(0.45–1.47)

ACEI 152 ( 74.9) 87 (79.8) 65 (69.1) 1.83

(0.93–3.59)

2.10

(1.04–4.25)

Aspirin 132 (65) 79 (72.5) 53 (56.4) 2.10

(1.15–3.84)

1.83

(0.99–1.06)

β-blockers 146 (71.9) 81 (74.3) 65 (69.1) 1.30

(0.68–2.45)

1.11

(0.56–2.18)

*OR= odds ratio, +CI confidence interval; OR had been obtained using logistic regression (unadjusted, and after this adjusted according to the sex of patients)

3 Drug secondary prevention in post-menopausal women

43

Statins and betablockers were used in approximately equal proportion in women and in men, approximately 50% of the patients receiving statins and approximately 70% betablockers. Women are less frequently treated with ACEI and aspirin, these differences persisting even after age-adjustment. Irrespective of the preventive drug considered, the usage percent was less than 75%.

Older ischemic patients (over 75 years of age), in comparison with the younger ones, have received medication in a lower proportion: beta-blockers 60% vs 75.8% (p<0.05), statins 32% vs 54.2% (p<0.05), aspirin 58% vs 67.3% (p>0.05), the only exception being registered just for ACEI: 82.2% vs 72.5% (p>0.05).

Further, dividing the sample by sex we found that: older ischemic women (over 75 years of age), in comparison with those less than 75 years of age, have used medication in the following percentages: betablockers 58.6% vs 73.8% (p>0.05), statins 37.9% vs 53.8% (p<0.05), aspirin 51.7% vs 58.5% (p>0.05), IECA 75.9% vs 66.2% (p>0.05). The situation was approximately similar with that registered in men, the percentages in which medication was used by older (over 75 years of age), respectively younger (less than 75 years of age) patients being: for betablockers 61.9% vs 77.3% (p>0.05), for statins 23.8% vs 54.5% (p=0.01), for aspirin 66.7% vs 73.9% (p>0.05), for ACEI 90.5% vs 77.3% (p>0.05).

Considering the relationship between ischemic heart disease type and prevention by drugs, we observed that medication was used in a greater percentage in old myocardial infarction patients (with no significant differences between sexes). On the other hand, in patients with other types of ischemic heart disease, statins had been used in a greater proportion in women (46.9% in women vs 18.9% in men, p=0.008).

Regarding the utilization of PCI and CABG, this was very low in both sexes, especially in women: CABG 1.1% in women, 4.6% in men (p>0.05), PCI 5.3 % in women and 13.8% in men (p<0.05).

DISCUSSION

It is very well known that cardiovascular disease represents the main mortality cause [1][2] and that secondary prevention plays a great role in reducing mortality. Current guidelines do not make any difference between sexes concerning the

medication needed to be recommended, including the preventive one [10]. Current studies are conflicting, some of them sustaining that differences between sexes regarding medication used are real [11–14], while others do not describe such differences [1]–[15

The analysis of our sample showed differences between sexes regarding the medication used in secondary prevention, which are not fully explained by age differences. Men had received drugs in a greater proportion (as well as in the study made by Enriquez [16]), but the differences between sexes were not significantly statistic, excepting aspirin (this being in according to the literature [17]), even if it is well known that aspirin reduces mortality, no matter of sex, in patients with ischemic heart disease. At the same time, statins were used in approximately equal proportion in both sexes.

In comparison with EUROASPIRE studies, ACEI are used in a similar percent with that reported in EUROASPIRE III (74.6%) [18], betablockers and statins being used as in EURO-ASPIRE II (69%, respectively 57.3%) [19]. On the other hand, antiplatelet agents, including aspirin, are underused, being administered even less than in EUROASPIRE I. (81%) [19]. We cannot offer a clear explanation, because antiplatelet agents are not expensive. Anyway, the underuse of aspirin is even more obvious in women and can only partially be related with low gastric tolerability in women [17].

The medication was used in a lower percentage in patients over 75 years of age, especially beta-blockers and statins (as [6]), but the utilization of ACEI was inversely related to the age of the patients.

DeWilde [4], Ramsay [6] Capewell [7] had shown that older patients, especially those over 85 years of age, have less “chances” to receive adequate therapy. These inequalities are very frustrating, considering the fact that both risk and benefit are maximal in these patients [7]. The reasons for underutilization of secondary prevention, especially in older people, despite the benefits of drugs, are not fully understood [6].

Regarding the relationship between ischemic heart disease’s type and use of preventive medication in women, in our sample, those with old myo-cardial infarction had received medication in a greater percentage (as in other studies from literature [4][7]). At the same time, it is a fact that in women with ischemic heart disease (with lesions on coronary angiography), secondary preventive drugs are administered in a lower proportion [20].


44

Women rarely benefit from cardiac procedures (PCI or CABG) in Europe (for example in Finland [3]), but also in the United States or Canada [1]. The reasons “responsible” for this are related to the presence of comorbidities, the presence of small coronaries (which can determine technical difficulties, incomplete revascularization) [1]. Although with no direct relation with this study, we also found significant differences, in favour of men, regarding the percentage of patients subjected to revasculari-zation (PCI or CABG). This can be partially explained by the different nature and severity of cardiovascular disease in men, in comparison with women, [3][21], women with ischemic heart disease presenting lower degrees of obstruction of coronary vessels, suggesting the fact that other mechanisms are responsible for ischemia in women (such as dysfunction at microvascular level, vaso-motricity, erosions, dissection, thrombophilia) [22].

Limits of the study: the data have been collected from general practitioners’ files, reflecting prescribed medication, not necessary those taken

by the patients. Also, we did not register the contraindications for prescribed medication.

The study was performed on a sample from a urban community (academic city), and it is possible that the percentage to be even lower in rural communities.

CONCLUSION

Secondary drug prevention of ischemic heart disease in Romania follows the general tendency, but the proportion in which the treatment is taken is under the one recommended and achieved in developed countries, especially regarding statins and, surprisingly, antiplatelet agents. For women, the situation is even worse. That is why, at national level, it is necessary to create and apply programs for implementation of actual guidelines for treatment of cardiovascular disease, for effective secondary prevention in both sexes, but especially in women.

Scop. Este bine cunoscut că femeile cu boală cardiovasculară sunt subtratate (incluzând terapia medicamentoasă). Acest aspect a fost mai puţin studiat referitor la medicamentele care sunt utilizate pentru prevenţia secundară.

Metoda. Într-o comunitate urbană din România, a fost studiat, din punct de vedere al prevenţiei secundare medicamentoase, un lot reprezentativ de 150 femei în postmenopauză, cunoscute cu boală cardiovasculară. Rezultatele au fost comparate cu cele obţinute pe un eşantion similar de bărbaţi cu vârsta peste 55 ani, cu boală cardiovasculară. Studiul s-a desfăşurat utilizând fişele medicilor de familie.

Rezultate. În concordanţă cu literatura, au fost considerate ca făcând parte din categoria medicamentelor de prevenţie antiagregantele plachetare, statinele, betablocantele şi inhibitorii enzimei de conversie a angiotensinei (IECA). Aspirina a fost utilizată la 56.4% dintre femei, respectiv la 72.5% dintre bărbaţi (p<0.05), IECA la 69.1% şi 79.8 % ( p>0.05), betablocantele în 69.1% şi 74.3 % (p>0.05), iar statinele în 48.9% şi 48.6% (p>0.05). Rezultatele arată că antiagregantele plachetare sunt subutilizate la pacienţii ischemici, mai ales la femei. Procentul utilizării celorlalte categorii de medicamente este apropiat de cel raportat în literatura pentru Europa de Est, dar dacă utilizarea betablocantelor şi statinelor este aproximativ egală pentru bărbaţi şi femei, IECA sunt mai puţin utilizate la femei. Cel mai ridicat procent de utilizare a medicaţiei preventive la femei a fost înregistrat post infarct miocardic (betablocante-85.7%, statine-50%, aspirina-60.7%, IECA-75%). În schimb, revascularizarea miocardică prin PCI şi CABG este redusă la ambele sexe, în special la femei 1.1% comparativ cu 4.6% la bărbaţi (p>0.05), PCI 5.3 % la femei şi 13.8% la bărbaţi (p<0.05).

Concluzie. La femeile în postmenopauză cu cardiopatie ischemică prevenţia secundară medicamentoasă este similară cu cea utilizată la bărbaţi, exceptând aspirina şi IECA care sunt subutilizate.

5 Drug secondary prevention in post-menopausal women

45

Corresponding author: Prof. D. Zdrenghea Rehabilitation Hospital, Viilor 46–50, Cluj-Napoca E-mail: [email protected]

R E F E R E N C E S

1. PILOTE L., DASGUPTA K., GURU V., HUMPHRIES K.H., MCGRATH J., NORRIS C. et al., A compressive view of sex-specific issues related to cardiovascular disease. CMAJ., 2007, 176( 6), S1-S41.

2. STEVENSON J.C., Cardiovascular disease in women. Menopause International, 2008, 14, 5. 3. KATTAINEN A., SALOMAA V., JULA A., KESANIEMI Y.A., KUKKONEN-HARJULA K., KAHONEN M. et al., Gender

differences in the treatment and secondary prevention of CHD at population level. Scandinavian Cardiovascular Journal, 2005, 39, 327–333.

4. DeWILDE S., CAREY M., RICHARDS N., WHINCUP P.H., COOK D.G., Trends in secondary prevention of ischemic heart disease in the UK 1994–2005: use of individual and combination treatment. Heart, 2008, 94, 83–88.

5. YUSUF S. Two decades of progress in preventing vascular disease. Lancet, 2002, 360, 2–3. 6. RAMSAY S.E, MORRIS R.W., PAPACOSTA O., LENNON L.T., THOMAS M.C., WHINCUP P.H., Secondary prevention of

coronary heart disease in older British men: extent of inequalities before and after implementation of the National Service Framework. Journal of Public Health, 2005, 27(4), 338–343.

7. CAPEWELL S., O’FLAHERTY M., Maximising secondary prevention therapies in patients with coronary heart disease. Heart, 2008, 94, 8–9.

8. RASTEGARPANAH M., MALEKZADEH F., THOMAS G.N., MOHAGHEGHI A., CHENG K.K., MARSHALL T., A new horizon in primary prevention of cardiovascular disease, can we prevent heart attack by “heart polypill”? Arch. Iran Med., 2008, 11(3), 306–13.

9. FAHEY T., BRINDLE P., EBRAHIM S., The polypill and cardiovascular disease. BMJ, 2005, 330, 1035–6. 10. JACKSON G., Gender differences in cardiovascular disease prevention. Menopause International, 2008, 14, 13–17. 11. CLARKE K.W., GRAY D, KEATING N.A., HAMPTON J.R., Do women with acute myocardial infarction receive the same

treatment as men? Br. Med. J., 1994; 309, 563–6. 12. HIPPISLEY-COX J., PRINGLE M., CROWN N., MEAL A., WYNN A., Sex inequalities in ischemic heart disease in general

practice: cross sectional survey. BMJ, 2001, 322 (7290), 832. 13. CRILLY M., BUNDRED P., HU1 X., LECKEY L, JOHNSTONE F., Gender differences in the clinical management of patients

with angina pectoris: a cross-sectional survey in primary care. BMC Health Serv. Res., 2007, 142. 14. JOCHMANN N., STANGL K., GARB E., BAUMANN G., STANGL V., Female-specific aspects in the pharmacotherapy of

chronic cardiovascular diseases. Euro Heart J., 2005, 26, 1585–1595. 15. NILSSON P., BRANDSTROM H., LINGFORS H., ERHARDT L., HEDBACK B., ISRAELSSON B., SJOBERG G., for the

National Programme of Quality Assurance in Secondary Prevention in Sweden. Gender differences in secondary prevention of coronary heart disease: reasons to worry or not? Scand. J. Prim. Health Care, 2003, 21, 37–42.

16. ENRIQUEZ J.R., PRATAP P., ZBILUT J.P., CALVIN J.E., VOLGMAN A.S., Women tolerate drug therapy for coronary artery disease as well as men do, but are treated less frequently with aspirin, β-blockers, or statins. Gender medicine, 2008, 5(1), 53–61.

17. OPOTOWSKY A.R., McWILLIAMS J.M., CANNON C.P., Gender differences in aspirin use among adults with coronary heart disease in the United States. J. Gen. Intern. Med., 2007, 22(1), 55–61.

18. EUROASPIRE III. www.escardio.org Euro Heart Survey – Esc Congress, Vienna, September 2007. 19. EUROASPIRE I and II Group. Clinical reality of coronary prevention guideliness: a comparison of EUROASPIRE I and II in

nine countries. Lancet, 2001, 357, 995–1001. 20. STRAMBA-BADIALE M., FOX K.M., PRIORI S.G., COLLINS P., DALY C., GRAHAM I., JONSSON B., SCHENCK-

GUSTAFSSON K., TENDERA M., Cardiovascular disease in women: a statement from the policy conference of the European Society of Cardiology. Eur. Heart J., 2006, 27, 994–1005.

21. VITALE C., MICELI M., ROSANO M.C., Gender-specific characteristics of atherosclerosis in menopausal women: risk factors, clinical course and strategies for prevention. Climateric, 2007, 10 (suppl 2), 16–20.

22. ANDREOTTI F., MARCHESE N., Women and coronary disease. Heart, 2008, 94, 108–116.



46

How General Practitioners Manage Patients with Irritable Bowel Syndrome. Data from a German Urban Area

A. FRANKE1, M.V. SINGER1, D.L. DUMITRAŞCU2

1Department of Medicine II, University Hospital of Heidelberg at Mannheim, Germany 2Department of Medicine II, “Iuliu Haţieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania

Irritable bowel syndrome (IBS) is a common functional disorder in the general Western population. The majority of patients with IBS are managed by general practitioners (GP). Therefore, it is very important that GP are well trained in the approach to functional gastrointestinal disorders. There is the impression that the knowledge of diagnostic IBS criteria may be deficient in GPs. The present study is a survey of the knowledge and management of general practitioners in Germany regarding IBS.

Methods. All general practitioners (n=260) of an urban area with about 600,000 inhibitants were asked by mail to complete a multiple choice questionnaire concerning their knowledge on IBS and their management of IBS patients.

Results. A completed questionnaire was returned by 121 of the general practitioners (46.5% response rate). The majority of the responders were male (61%) and had an experience exceeding 10 years (75%). IBS was regarded as a functional disorder by 55% (49% as a motility disorder and 66% as a psychic disorder). The diagnosis of IBS was mainly based on the patient’s history, a colonoscopy was considered by 57%. Diagnostic criteria for IBS were used only by 22 GPs (18%). Referral to gastroenterologists was initiated by 26% of the GPs in all patients with suspected IBS. The majority referred patients only in case of unclear diagnosis or insufficient therapeutic results. Medical therapy was prescribed by 96% of the GPs. Psychotherapy and alternative therapies were additionally performed by 55% and 61%, respectively. The majority of the GPs (66%) estimated the percentage of IBS patients in their daily routine between 1 and 10% and stated that they have seen 1 to 5 patients during the past week.

Conclusions. Diagnostic IBS criteria such as Rome criteria are largely unknown among GPs in our area. Pathogenetic models of IBS are deficiently known in GPs. It is likely that the minority of patients who are referred to gastroenterologists have special problems. Their management should probably be different from that of the unreferred majority. Many therapeutic modalities in primary care have no medical evidence. Further studies on therapy options in IBS should also be made in primary care.

Key words: IBS, Irritable Bowel Syndrome, GP, general practitioner.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder of altered bowel habits and abdominal complaints and has a prevalence of up to 10–20% of the general Western population [1]. IBS patients have a significant impairment in health related quality of life compared to healthy individuals [2]. General practitioners diagnose and manage the majority of IBS patients [3]. However, most of the knowledge and research is made by specialists on the basis of a smaller percentage of patients with a higher score of intensity and severity of pain, frequency of complaints and number of consultations than those managed by general practitioners [4].

Since there is no specific physiological marker for diagnosis of IBS [5], symptom-based diagnostic criteria such as Manning [6], Kruis [7] and Rome criteria [8] have been developed over the years to define IBS more accurately. It has been shown that Rome criteria may have a positive predictive value for IBS of more than 98% when red flag symptoms are excluded [9]. In a study form the USA, the knowledge of Rome criteria in general practitioners was found to be less than 50% as assessed by a questionnaire, most of the physicians did not identify Rome II symptom criteria [10]. It has been shown that general practitioners in the UK and Italy predominantly diagnose IBS without knowledge and

ROM. J. INTERN. MED., 2009, 47, 1, 47–53

A. Franke et al. 2

48

use of the validated diagnostic criteria [3, 11, 12]. Inadequate knowledge may, on the one hand, reduce the accuracy for the diagnosis of IBS and, on the other hand, result in more diagnostic procedures, uncertainty in patients and doctors, and subsequent referral which may all negatively influence the prognosis and increase the costs [5]. Since data on the knowledge of IBS criteria and kind of management of IBS patients in German general practitioners is absent, we initiated a survey on this topic.

METHODS

SUBJECTS

An urban area around the university hospital of Mannheim with a population of about 600,000 inhabitants was chosen by the postal codes. All general practitioners (n=260) in this area were asked to participate in this survey by mail. The questionnaires were sent in autumn 2006.

The questionnaire contained 13 questions. GPs were asked to return the completed questionnaire anonymously by a provided stamped return envelope. The questionnaire included personal data such as numbers of working years as GP, age and gender, as well as multiple choice questions about the patho-genesis of IBS, diagnostic procedures, therapeutic approach and prevalence of IBS in the daily work of the GP. The questionnaire is presented in Appendix 1.

APPENDIX 1

Initials:Sex:Experience as GP (years): What is in your opinion IBS ? − motility disorder − functional disorder − psychic disorder How do you establish the diagnosis of IBS? − Based on history − Based on personal experience − Based on colonoscopy − Other way: explain Do you use diagnostic criteria for IBS?

Yes/No If yes, which one: − Manning − Kruis − Rome I − Rome II − Rome III

Do you know the Rome criteria for IBS? Yes/No

When do you refer IBS patients to gastroenterologists? − always − on demand of the patients − when the diagnosis is not clear − no effect by therapy − never In case of referral, how frequently is your diagnosis confirmed?

Always 75–99% 50–74% 25–49% <25%

Do you prescribe medical treatment? Continuously On demand Never

Do you suggest psychotherapy? Yes/No (if yes explain)

Do you prescribe alternative therapies? Yes/No (if yes explain)

Have you participated to courses, symposia, and other CME activities about IBS? Yes/ No

How frequently do you encounter patients with IBS? < 1% in my practice between 1–10% in my practice between 11–25% in my practice between 25–33% in my practice >33% in my practice

How many IBS patients did you see last week? None 1–5 6–10 11–20 >20

STATISTICS

Data analysis includes descriptive statistics calculated with commercially available software.

RESULTS

DEMOGRAPHIC DATA

From the 260 contacted general practitioners 121 returned a completed questionnaire. This corresponds to a response rate of 46.5%. The responders consisted of 39% females and 61% males. Over 75% of the responders had an experience as GP of more than 10 years (Fig. 1).

PATHOGENESIS OF IBS

IBS was regarded by 55% of GPs as a functional disorder. Most frequently a psychic disorder (66%) was considered as the cause of IBS. Almost half of the respondent GPs account IBS as a

3 Irritable bowel syndrome 49

<10 y

10-20 y

>20 y

43%

24%

33%

motility disorder (49%). The sum of the answers exceedes 100% since multiple answers were permitted.

<10 y

10-20 y

>20 y

43%

24%

33%

Fig. 1. – Experience in years (y) as general practitioner.

DIAGNOSIS OF IBS

Apart from 7, all GPs (94%) attached importance to the patient’s history. Additionally, a colonoscopy was demanded by 57% of the GPs to diagnose IBS. 44% of the GP stated that they diagnosed IBS using their impression of the patient. Addional examinations such as laboratory parameters, sonography and hydrogen breath tests were initiated by 23%.

Diagnostic criteria for IBS were used only by 22 GPs (18%). Of these, Manning and Rome II criteria were mainly applied (45% respectively). 6 GPs used Rome I (27%) and 2 GPs (9%) Rome III criteria. Only 15% of GPs recognized to know Rome criteria.

26% of the GPs stated to refer all their patients with suspected IBS to a gastroenterologist. The others referred only if the diagnosis was not clear (64%), therapeutic results were insufficient (26%) or the patient demanded referral (3%). 2 GPs (2%) stated not to refer IBS patients to gastroenterologists at all.

In case of referral the diagnosis IBS was in the opinion of the GPs predominantly approved by the gastroenterologist (Fig. 2)

Fig. 2. – Percentage of confirmation of the diagnosis IBS by a

gastroenterologist.

THERAPY OF GPS

A pharmacological therapy was prescribed by most of the GPs (96%). Most of them (85%) recommended an on demand therapy, whereas only 11% of the GPs recommended permanent drug therapy. 5 GPs (4%) did never use drug therapy in patients with IBS.

Psychotherapy was advised or implemented to IBS patients by more than half of the GPs (55%). At this behaviour therapy, relaxation therapy and conversational therapy prevailed.

Alternative therapies were used by 61% of the GPs to help IBS patients. Among these, homeopathy, acupuncture, phytotherapy, dietary treatment and probiotics were used most frequently.

PARTICIPATION IN IBS EDUCATION COURSES

46% of GPs stated that they participated in a medical education course on IBS.

PREVALENCE OF IBS IN DAILY ROUTINE

Two-thirds of the GPs estimated the percentage of IBS patients between 1 and 10%. Some GPs stated their percentage of IBS patients of < 1% or >10% (see Fig. 3 a). Two-thirds of the GPs have seen 1

Fig. 3. – Percentage of IBS patients based on all patients of the GP (a) and number of IBS patients seen during the last week

by the GP. (b).

A. Franke et al. 4

50

to 5 IBS patients during the last week, 27% have seen no IBS patients and only few GPs have seen more than 5 IBS patients (Fig. 3 b).

DISCUSSION

The majority of patients with IBS (Irritable Bowel Syndrome) are managed by general practitioners (GP) [3]. Therefore, it is very important that general practitioners are well trained in the approach to functional gastrointestinal disorders. In other countries there is the impression that the knowledge of diagnostic IBS criteria is deficient in GPs [3][10][11]. The present study is the first survey of the knowledge and management of general practitioners in Germany regarding IBS.

DEMOGRAPHIC DATA

Nearly half of the contacted general practitioners returned our questionnaire completed. The response rate was comparable to other studies using also a mailed questionnaire [13]. However, it was naturally lower compared to studies with an individual contact to the general practitioners [11][14].

PATHOGENESIS OF IBS

Only 55% of the GPs regard a functional disorder as the pathogenesis of IBS. More than 60% of the GP considered IBS as psychic disorder. This suggests the need for advanced training concerning pathogenetic concepts of IBS. Their knowledge is so important, since IBS patients need a satisfactory disease model in the form of a convincing explanation of their symptoms for coping with their disease [3].

DIAGNOSIS OF IBS

Physicians traditionally approach IBS as a diagnosis of exclusion. According to this principle more than half of the respondent GPs in our survey preferred a colonoscopy to rule out organic conditions. This may originate from the primary concern of general practitioners as well as of IBS patients to exclude serious organic diseases [3]. However, diagnostic workup can be extensive and may lead to risk and patient inconvenience in IBS. However, the use of diagnostic criteria may improve the accuracy of diagnosis, reduce unnecessary

examinations, and abbreviate the duration until the diagnosis is made [15]. The AGA recommends use of the Rome criteria in the initial evaluation of patients with IBS symptoms. If the criteria are satisfied and there are no red flag signs or symptoms, a limited laboratory investigation is suggested. This is supported by recent studies, which have demonstrated that ordering a standardized battery of diagnostic tests on patients with IBS symptoms is not likely to be useful, especially considering the high positive predictive value of the Rome criteria for IBS [16][17]. A European expert panel also recommends additional diagnostics only in certain patients group, e.g. patients with alarm symptoms, family history of colon cancer, or age over 45 years [18].

The knowledge and practice of diagnostic IBS criteria is only marginal in our group of respondent GPs. Nevertheless, most of them seem to be able to diagnose IBS with reasonable confidence, since the diagnosis was in case of referral predominantly confirmed by the gastro-enterologists. It has already been shown in a UK survey on the primary care management of IBS patients that GPs diagnosed IBS relatively accurate, although none used IBS criteria such as Manning or Rome [3].

Data from a similar study conducted in another area shown that GPs can be successfully trained in using diagnostic criteria for IBS [14].

REFERRAL

Only the minority of the GPs in our study said they refer all patients with suspected IBS to specialists. The majority referred patients only if they were uncertain of the diagnosis, had insufficient therapeutic effort or referral was demanded by the patient. This may explain the selection of more complicated and severe forms of IBS that are seen by specialists. In two UK surveys it was shown that one patient in every five was referred to secondary care, either of a gastroenterologist or of a surgeon [3][11]. The reasons for referral were comparable to those in our study: unsatisfied patient (54%), uncertainty of diagnosis (35%) and new ideas for management (7%) [3].

THERAPY OF GPS

The first step in the therapy of IBS should include the explanation of the nature of the disease


together with reassurance and advice [5]. Most patients in primary care do not need pharmaco-logical therapy [19]. However, almost all general practitioners in our study prescribed pharmaco-logical therapy in IBS patients. This may result from the fact that most patients expect a pharmacological therapy [20]. Pharmacological therapy should, if necessary at all, focus on the primary symptoms: diarrhea, constipation and abdominal pain [21]. The drug therapy was in our study predominatly used as an on demand therapy. This may suggest that mainly drugs against IBS symptoms such as anticholinergics, antispasmo-lytics, prokinetics or laxatives were used. The frequent use of pharmacological therapy is comparable to other studies where almost all GPs stated to use drugs in IBS therapy at some stage [3]. More advanced non-pharmacological treatment, such as behavioural treatment, hypnosis or psycho-analysis targeting underlying psychosocial factors, may at best positively influence complaints in a subgroup of patients and is normally not applied in primary care [5]. However, more than half of the GPs in our study applied psychotherapy in IBS patients. This may be evidence for an imprecise understanding of the pathogenesis of IBS. The high percentage of the use of alternative therapies in our study may be evidence of the difficulty to find a satisfactory therapy for IBS complaints.

PARTICIPATION IN IBS EDUCATION COURSES

Almost half of the respondent GPs stated to have visited medical education courses on IBS. However, the marginal knowledge and usage of IBS criteria among the GPs rather show increased need for advanced training.

PREVALENCE OF IBS IN DAILY ROUTINE

Most of the GPs in our study estimated the prevalence of IBS patients in their daily work between 1 and 10%. This seems to be under-estimated compared to reports of IBS prevalence rates in the general western populations of 10 to 20% [1]. However, the statement of our GPs that they saw 1–5 IBS patients last week corresponds to recent UK studies where IBS was responsible for 5

consultations per week of which one was a new case [3]. In another UK primary care study it was calculated that an average GP will see about 8 IBS patients each week [11].

LIMITATIONS

The current study does have some limitations. We did not validate our questionnaire concerning validity and reproducibility. It remains unclear if there are significant differences between the GPs who filled our questionnaire and those who did not answer. Moreover, our physician survey group was limited to a single urban centre. It has been shown that IBS is twice more prevalent in large cities than in rural areas [5]. However, our survey has a meaningful response rate of nearly 50% and admits some notable conclusions which may have the capability for some changes.

In summary, this study indicates four approaches to improve the management of IBS patients:

1. Most GPs manage IBS patients without knowledge of diagnostic IBS criteria such as Rome criteria. The spread of IBS criteria among GPs would simplify the decision which patients need further diagnostics and in which the diagnosis IBS can certainly be made without it.

2. Pathogenetic models of IBS are deficiently known in GPs. The training on that might help the GPs to elicit fear of serious disease in IBS patient by providing a convincing explanation of their complaints.

3. Most IBS patients are managed by GPs alone. It is likely that the minority of patients who are referred to gastroenterologist have special problems. Their management should probably be different from that of the unreferred majority. There may be the need for two different guidelines.

4. Many therapeutic modalities without any medical evidence are used by GPs. Further studies on therapy options in IBS should also be made in primary care.

Acknowledgement. D.L. Dumitrascu was funded by the Alexander von Humboldt Foundation, Bonn, Germany.

Sindromul de intestin iritabil (SII) este o tulburare funcţională frecventă în

populaţia vestică. Majoritatea pacienţilor cu SII sunt luaţi în grijă de către medici

A. Franke et al. 6

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generalişti. De aceea este foarte important ca medicii generalişti să fie bine pregătiţi în abordarea suferinzilor de tulburări funcţionale digestive. Este impresia că medicii generalişti au informaţii deficitare despre SII. Acest studiu este o analiză a cunoştinţelor şi comportamentului practic al generaliştilor germani faţă de SII.

Metode. Toţi generaliştii (n=260) dintr-o arie urbană cu apoximativ 600 000 locuitori au fost chestionaţi despre cunoştinţele şi abordarea SII printr-un chestionar trimis prin poştă, de tip multiple choice.

Rezultate. Chestionarul completat a fost returnat de 121 medici generalişti (rată de răspuns de 46,5%). Majoritatea respondenţilor erau de sex masculin (61%) şi aveau experienţă de peste 10 ani (75%). SII a fost privit ca o tulburare funcţională de 55% (49% ca tulburare de motilitate şi 66% ca tulburare psihică). Diagnosticul de SII s-a bazat mai ales pe istoricul bolii; colonoscopia a fost luată în considerare în 57% din cazuri. Doar 22 dintre medicii generalişti (18%) folosesc criterii de diagnostic. Un număr de 26% dintre generalişti trimit mai departe pacientul spre specialistul gastroenterolog. Majoritatea trimit pacienţii cu SII la gastroenterolog în caz de diagnostic neclar sau rezultate terapeutice insuficiente. Terapia medicală a fost prescrisă de 96% dintre medicii generalişti. Psihoterapia şi terapia alternativă au fost folosite adiţional de 55%, respectiv 61%. Majoritatea generaliştilor (66%) estimează procentul de SII în practica zilnică între 1 şi 10% şi afirmă că au văzut între 1 şi 5 pacienţi în săptămâna precedentă.

Concluzii. Criteriile diagnostice pentru SII precum criteriile Roma sunt în general necunoscute de medicii generalişti din aria studiată. Modelele patogenetice sunt cunoscute deficitar. Se pare că minoritatea pacienţilor trimişi la gastroenterolog au probleme speciale. Tratamentul este probabil diferit de cel al pacienţilor netrimişi la specialist. Multe modalităţi terapeutice nu se bazează pe dovezi medicale. Alte studii despre opţiunile terapeutice în SII ar trebui întreprinse în îngrijirea medicală primară.

Corresponding author: A. Franke, M.D.

Department of Medicine II (Gastroenterology, Hepatology and Infectious Diseases) University Hospital of Heidelberg at Mannheim Theodor-Kutzer-Ufer 1–3 D-68167 Mannheim, GermanyTel.: (+49)-621-383-3359, Fax: (+49)-621-383-3805 E-mail: [email protected]

R E F E R E N C E S

1. LONGSTRETH G.F., THOMPSON W.G., CHEY W.D., HOUGHTON L.A., MEARIN F., SPILLER RC., Functional bowel disorders. Gastroenterology, 2006, 130: 1480–1491.

2. WHITEHEAD W.E., PALSSON O., JONES K.R., Systematic review of the comorbidity of irritable bowel syndrome with other disorders: what are the causes and implications? Gastroenterology, 2002; 122: 1140–56.

3. THOMPSON W.G., HEATON K.W., SMYTH G.T., SMYTH C., Irritable bowel syndrome: the view from general practice. Eur. J. Gastroenterol. Hepatol., 1997; 9:689–92.

4. VAN DER HORST H.E., VAN DULMEN A.M., SCHELLEVIS F.G., VAN EIJK J.T., FENNIS J.F., BLEIJENBERG G., Do patients with irritable bowel syndrome in primary care really differ from outpatients with irritable bowel syndrome? Gut, 1997; 41:669–674.

5. OBERNDORFF-KLEIN WOOLTHUIS A.H., BRUMMER R.-J.M., DE WIT N.J., MURIS J.W.M., STOCKBRÜGGER R.W., Irritable Bowel Syndrome in General Practice: An Overview. Scand. J. Gastroenterol., 2004; 39: Suppl 241:17–22.

6. MANNING A.P., THOMPSON W.D., HEATON K.W., MORRIS A.F., Towards positive diagnosis of the irritable bowel. Br. Med. J., 1978; 2:653–654.

7. KRUIS W., THIEME C., WEINZIERL M., SCHUSSLER, HOLL J., PAULUS W., A diagnostic score for the irritable bowel syndrome. Its value in the exclusion of organic disease. Gastroenterology, 1984; 87:1–7.


8. THOMPSON W.G., DOTEVALL G., DROSSMAN D.A., HEATON K.W., KRUIS W., Irritable bowel syndrome: guidelines for the diagnosis. Gastroenterol. Int., 1989; 2: 92–95.

9. VANNER S.J., DEPEW W.T., PATERSON W.G. et al., Predictive value of the Rome criteria for diagnosing the irritable bowel syndrome. Am. J. Gastroenterol., 1999; 94: 2912–17.

10. LONGSTRETH G.F., BURCHETTE R.J., Family practitioners attitudes and knowledge about irritable bowel syndrome: effect of a trial of physician education. Fam. Pract., 2003; 20: 670–74.

11. THOMPSON W.G., HEATON K.W., SMYTH G.T., SMYTH C., Irritable bowel syndrome in general practice: prevalence, characteristics, and referral. Gut, 2000, 46: 78–82.

12. BELLINI M., TOSETTI C., COSTA F., BIAGI S., STASI C. et al., The general practitioner’s approach to irritable bowel syndrome: From intention to practice. Dig. Liver Dis., 2005; 37:934–939.

13. YAWN B.P., LOCKE G.R. 3RD, LYDICK E., WOLLAN P.C., BERTRAM S.L., KURLAND M.J., Diagnosis and care of irritable bowel syndrome in a community-based population. Am. J. Manag. Care, 2001; 7:585–92.

14. DUMITRASCU D.L., DAVID L., SINGER M., What general practitioners know about irritable bowel syndrome. Preliminary data from a Romanian province. J. Gastrointest. Liver Dis., 2006; 14: 227–230.

15. OLDEN K.W., Diagnosis of irritable bowel syndrome. Gastroenterology, 2002; 122: 1701–14. 16. HAMM L.R., SORRELLS S.C., HARDING J.P. et al., Additional investigations fail to alter the diagnosis of irritable bowel

syndrome in subjects fulfilling the Rome criteria. Am. J. Gastroenterol., 1999; 94: 1279–1282. 17. CASH B.D., SCHOENFELD P., CHEY W.D., The utility of diagnostic tests in irritable bowel syndrome patients: a systematic

review. Am. J. Gastroenterol. 2002; 97: 2812–2819. 18. THOMPSON W.G., HUNGIN A.P., NERI M., HOLTMANN G., SOFOS S., DELVAUX M. et al., The management of

irritable bowel syndrome: a European, primary and secondary care collaboration. Eur. J. Gastroenterol. Hepatol., 2001; 13:933–939.

19. PATERSON W.G., THOMPSON W.G., VANNER S.J., FALOONN T.R., ROSSER W.W., BIRTWHISTLE R.W. et al., Recommendations for the management of irritable bowel syndrome in family practice. IBS Consensus Conference Participants. Can. Med. Assoc. J., 1999; 161:154–160.

20. BIJKERK C.J., DE WIT N.J., STALMAN W.A.B., KNOTTERUS J.A., HOES A.W., MURIS J.W.M., Irritable bowel syndrome in primary care; the patient’s and doctor’s view on symptoms, aetiology, and management. Can. J. Gastroenterol., 2003; 17: 363–368.

21. PACE F., COREMANS G., DAPOIGNY M., MULLER-LISSNER S.A., SMOUT A., STOCKBRUEGGER. et al., Therapy of irritable bowel syndrome: an overview. Digestion, 1995; 56: 433–442.

Received February 15, 2009

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Nonalcoholic Fatty Liver Disease – A Risk Factor for Microalbuminuria in Type 2 Diabetic Patients

F. CASOINIC1, D. SÂMPELEAN1, CĂTĂLINA BĂDĂU2, LUCHIANA PRUNĂ3

1University of Medicine and Pharmacy Cluj-Napoca, IVth Medical Clinic, Department of Diabetology 2Heart Institute “Niculae Stancioiu”, Cluj-Napoca

3County Hospital Baia Mare, Department of Diabetology

The aim of our study was to assess the presence of microalbuminuria in diabetic subjects with nonalcoholic fatty liver disease (NAFLD) compared with diabetic patients without NAFLD and to correlate this with inflammatory markers such as high sensitive C- reactive protein (hsCRP).

Material and Methods. The study was conducted on 75 diabetic subjects with ultra-sonographical NAFLD, in which alcohol consumption and other causes of chronic liver disease have been excluded. The exclusion criteria also included smoking, arterial hypertension, known renal disease. The control group consisted of 70 diabetic patients, matched for age and gender, without ultrasonographical evidence of NAFLD. In all subjects we measured height, weight, BMI, fasting glucose, HbA1c, total cholesterol, LDL and HDLcholesterol, triglycerides, serum transaminases, hsC-reactive protein and microalbuminuria. A p-value<0.05 was considered statistically significant.

Results. Microalbuminuria was significantly more frequent in subjects with NAFLD than in controls (12.7% vs 7.8%, p<0.05). Microalbuminuria was positively correlated with hsCRP levels.

In conclusion NAFLD is positively correlated with microalbuminuria-marker of early stage CKD, in diabetic patients. This seems to be related to higher levels of proinflammatory factors released by the liver, such as hsCRP.

Key words: nonalcoholic fatty liver disease, microalbuminuria, cardiometabolic risk, diabetes mellitus.

Nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome [1–3], is associated with obesity, prediabetes, type 2 diabetes mellitus, dyslipidaemia, insulinresistance and arterial hypertension [2][3]. In type 2 diabetic patients studies have shown that NAFLD is correlated with an increase in total cardiovascular risk independently of the other components of the metabolic syndrome [4–7].

Moreover, the presence of endothelial dys-function in patients with NAFLD has been demonstrated using the flow mediated vasodilatation method [8]. Microalbuminuria as an indicator of endothelial dysfunction is at present the best documented predictor for the development of diabetic chronic renal disease. Genetic susceptibility, metabolic abnormalities, hemodynamic changes, upregulated growth factors and cytokines may play a part in the development of diabetic micro-albuminuria [9].

The pathogenetic link between NAFLD in diabetic patient and microalbuminuria is supposed to be represented by proinflammatory cytokines secreted by the liver [10]. The presence of micro-

albuminuria in diabetic patients with NAFLD may increase the prediction of cardiovascular risk and that of chronic kidney disease, with concurrent important therapeutical management implications. It is thus possible to identify a subgroup of diabetic patients who require a more intensive treatment to reduce the risk of future cardiovascular events and chronic kidney disease [10].

The aim of our study was to determine the prevalence of microalbuminuria and to correlate it with chronic inflammation markers – such as high sensitive C reactive protein (hCRP) in diabetic patients with NAFLD versus diabetic patients without NAFLD.


SUBJECTS

The study was conducted on 75 diabetic patients with ultrasonographically diagnosed NAFLD. The

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control group included 70 diabetic patients without ultrasonographical evidence of NAFLD, matched for age, gender and the time from diagnosis of diabetes.

In both groups the exclusion criteria were: alcohol consumption, smoking, infection with hepatitic viruses B and C, more rare causes of chronic hepatopathy (autoimmune hepatitis, hemochro-matosis), known renal disease, urinary infection, arterial hypertension and severely unbalanced diabetes mellitus.

CLINICAL MEASUREMENTS AND LABORATORY PROCEDURES

Both groups were evaluated both clinically and paraclinically. Information regarding smoking, alcohol intake and current used medication were obtained by questionnaire. Body mass index was measured dividing weight in kilograms by height squared meters. Abdominal circumference was measured at the level of the umbilicus. Blood pressure was measured repeatedly using a mercury sfigmanometer.

Biochemical parameters were determined using morning drawn venous blood, after an overnight fast. We have determined serum transaminases, gamma

glutamyltransferase (with reference range of 10– 35 IU/L for ASAT, 10–40 IU/L ALAT, γGT), fasting glucose, HbA1c, total cholesterol, HDL cholesterol, hsCRP (reference range <=3 mg/dlL, serum creatinine.

Albumin excretion rate was measured from an early morning urine sample as the albumin/ creatinine ratio (ACR). Microalbuminuria was defined as albumin excretion rate of 30 to 299 g/mg creatinine.

Hepatic ultrasonography was performed by a single examiner blinded to patient clinical status. Ultrasonographical criteria for hepatic steatosis were diffuse echogenicity of liver relative to kidney and posterior attenuation of ultrasound beam [11].

Statistical analysis was performed using SPSS 11.0. The data are expressed in means ± SD. Unpaired t test for numerical variables and X2 test for categorical variables were used. A p value <0.05 was considered statistically significant.

RESULTS

The characteristics of patients are shown in Table I. Diabetic patients with NFLD were more likely to be men, older than controls, with a tendency towards obesity and a longer duration of diabetes.

Table I

Clinical and biochemical characteristics of the groups studied

Variables Diabetes mellitus with NAFLD

DM without NAFLD

P value

N 75 70 Sex (%males) 65.3 52.2 >0.05 Age (years) 63 ± 5 59 ± 3 >0.05 BMI (kg/m2) 29.4 ± 2 27.1 ± 4 <0.05 Diabetes duration (years) 9 ± 2 7 ± 3 <0.05 Systolic blood pressure (mmHg) 133 ± 5 130 ± 3 >0.05 Diastolic blood pressure (mmHg) 85 ± 5 80 ± 4 >0.05 HbA1c 7.4 ± 1.0 6.8 ± 0.7 <0.05 Triglycerides (mmol/L) 1.51 ± 0.5 1.42 ± 0.4 <0.05 HDL cholesterol (mmol/L) 1.38 ± 0.4 1.42 ± 0.3 <0.05 LDL cholesterol (mmol/L) 3.35 ± 0.5 3.33 ± 0.4 >0.05 ASAT (IU/L) 29 ± 10 22 ± 4 <0.05 ALAT (IU/L) 30 ± 9 23 ± 3 <0.05 γGT (UI/L) 28 ± 10 21 ± 5 <0.05 Creatinine (mg/dL) 0.91 ± 0.1 0.87 ± 0.2 >0.05 Metabolic syndrome (IDF criteria 2005)(%)

88 72 <0.05

Biologically the diabetics with NAFLD had a

poorer long term glycemic control, reflected by a significantly higher value of HbA1c than controls (7.4% vs 6.8%). The presence of NAFLD was also

3 Nonalcoholic fatty liver disease

57

7.14 %

12 %

0

2

4

6

8

10

12

14

DM with NAFLD DM without NAFLD

p <0,01

7.14 %

15 %

8.57 %

12%

02468

10121416

NAFLD

Simple stet

osis

Steatohep

atitis

Controls

associated with a more deleterious lipidic profile, with a tendency towards higher triglycerides and low HDL cholesterol levels, compared with controls.

Regarding liver enzymes, diabetic patients with NAFLD had higher levels of both transaminases and γGT, thus reflecting the presence in this group of patients with steatohepatitis.

The prevalence of microalbuminuria was significanty higher in diabetic patients with NAFLD, as shown in Fig. 1 (12 % versus 7.14%).

Fig. 1. – The prevalence of microalbuminuria in diabetic patients with NAFLD versus controls.

Using the elevated levels of serum transaminases as surrogate marker of hepatic injury, we sub-sequently divided the diabetic patients with NAFLD into two subgroups: simple steatosis (with normal levels of transaminases) and respectively steatohepatitis (with elevated serum transaminases). Comparing these two subgroups we found that patients with steato-hepatitis had a higher prevalence of microalbuminuria than those with simple steatosis (Fig. 2).

Fig. 2. – Prevalence of microalbuminuria in the two subgroups.

High sensitive C reactive protein levels were significantly higher in diabetic patients with NAFLD compared to controls, and were correlated with the presence of microalbuminuria, as shown in Fig. 3.

5.97.2

5.5 4.9 5.34.5

012345678

DM withNAFLD

DM withNAFLD

withmicroA

DMwithoutNAFLDwithoutmicroA

DMwithoutNAFLD

DMwithoutNAFLD

withmicroA

DMwithoutNAFLDwithoutmicroA

Fig. 3. – Mean values of hsCRP in the NAFLD diabetic patients with and without microalbuminuria, versus non

NAFLD with or without microalbuminuria.

DISCUSSION

In this study we have found that the presence of NAFLD in diabetic patients was associated with a higher prevalence of microalbuminuria and positively correlated with hsCRP levels. Our findings support the results reported by Targher et al., that suggested that the presence of NAFLD in diabetic patients increases the risk of microvascular complications, such as nephropaty and retinopathy [10].

Microalbuminuria is at present the best noninvasive independent predictor for endothelial dysfunction and also for cardiovascular morbidity and mortality. In our study the association between NAFLD and microalbuminuria in diabetic patients was independent of arterial hypertension.

The mechanism by which NAFLD could contribute to endothelial dysfunction is poorly understood. Our data suggest that NAFLD promotes inflammation, probably by releasing proinflammatory cytokines from injured hepatocytes, due to reactive oxygen species derived from steatosis stimulated fatty liver oxidation. A pathogenetic circle is thus promoted enhancing consequently both hepatic injury and subclinical systemic inflammation. In our study diabetic patients with elevated serum transaminases had the highest prevalence of microalbuminuria and the highest hsCRP levels.

Further studies are needed to establish the exact mechanism by which the deleterious effects of NAFLD are exercised.


58

In view of the emerging evidence that NAFLD is an independent cardiovascular risk factor (4–7), our data support the conviction that diabetic patients with NAFLD require a more attentive assessment of total cardiovascular risk and thus a more aggressive approach in order to diminish the number of future cardiovascular events.

The main limitation of our study is that the diagnosis of NAFLD was not performed by liver biopsy, and was based instead on clinical and

ultrasonographical data. Liver biopsy is not easily applied in epidemiological studies. Ultrasonography has a good sensitivity and specificity for moderate and severe steatosis, but a lower sensitivity for hepatic fat infiltration under 33% [11]. This tends to underestimate the cases of NAFLD.

In conclusion NAFLD in diabetic patients increases the risk for microalbuminuria and thus for future cardiovascular events and chronic kidney disease.

Scopul studiului de faţă constă din evaluarea prezenţei microalbuminuriei la pacienţii diabetici cu FGNA comparativ cu cei fără FGNA şi corelarea acesteia cu markeri ai inflamaţiei – cum este proteina C reactivă cu sensibilitate înaltă.

Material şi metodă. Studiul a fost desfăşurat pe un grup de 75 de pacienţi diabetici cu FGNA diagnosticat ultrasonografic, la care s-au exclus consumul de alcool precum şi alte cauze de boală cronică hepatică, fumatul, hipertensiunea arterială şi boala renală preexistentă. Grupul de control a fost constituit din 70 de pacienţi diabetici, fără dovezi ecografice de FGNA. La toţi pacienţii s-au determinat parametrii antropometrici, glicemia à jeun, HbA1c, colesterolul total, LDL şi HDL colesterolul, trigliceridele, transaminazele serice, hs PCR şi microalbuminuria. Analiza statistică a fost efectuată cu SPSS11.0. O valoare a p<0,05 a fost considerată semnificativ statistică.

Rezultate. Microalbuminuria a fost semnificativ mai frecventă la subiecţii cu FGNA decât la grupul de control (12,7% vs 7,8%, p<0,05). Microalbuminuria s-a corelat pozitiv cu hsCRP la pacienţii diabetici cu FGNA.

In concluzie FGNA la pacienţii diabetici, rezervorul cel mai mare de factori de risc din patologie – se corelează cu prezenţa microalbuminuriei-marker de boală cronică renală încă din stadiu precoce. Nivelele plasmatice crescute de hsCRP precum şi alte cytokine proinflamatorii eliberate de ficat par să joace un rol patogenetic în acest sens.

Corresponding author: F. Casoinic, MD

IVth Medical Clinic, CF University Hospital, Cluj-Napoca 18, Republicii St., 400015 Cluj-Napoca, Romania E-mail: [email protected]

R E F E R E N C E S

1. McCULLOUGH AJ., The clinical features, diagnosis and natural history of nonalcoholic fatty liver disease. Clin Liver Dis., 2004, 8:521–533.

2. MARCHESINI G., BUGIANESI E., FORLANI G., CERRELI F., LENZI M., Nonalcoholic fatty liver disease, steatohepatitis and the metabolic syndrome. Hepatology, 2003, 37:917–923.

3. ANGULO P., Nonalcoholic fatty liver disease. N. Engl. J. Med., 2002, 346:1221–1231. 4. TARGHER G., BERTOLINI L., POLI F., RODELLA S., SCALA L. et al., Nonalcoholic fatty liver disease and risk of future

cardiovascular events among type 2 diabetic patients. Diabetes, 2005, 54:3541–3546. 5. TARGHER G., BERTOLINI L., PADOVANI R. et al., Prevalence of nonalcoholic fatty liver disease and its association with

cardiovascular disease among type 2 diabetic patients. Diabetes Care, 2007, 27:1212–1218. 6. TARGHER G., BERTOLINI L., PADOVANI R., RODELLA S., ZOPPINI G, Relations between carotid artery wall thickness

and liver histology in subjects with nonalcoholic fatty liver disease. Diabetes Care, 2006, 29:1325–1330.

5 Nonalcoholic fatty liver disease

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7. TARGHER G., BERTOLINI L., RODELLA S. et al., Nonalcoholic Fatty Liver Disease Is Independently Associated With an Increased Incidence of Cardiovascular Events in Type 2 Diabetic Patients. Diabetes Care, 2007, 30:2119–2121.

8. VILLANOVA N., MOSCATIELLO S., RAMILLI S. et al., Endothelial dysfunction and cardiovascular risk profile in nonalcoholic fatty liver disease. Hepatology, 2005; 42(2)473–80.

9. VIBERTI G.C., HILL R.D., JARRETT R.J. et al., Microalbuminuria as a predictor of clinical nephropathy in insulin dependent diabetes mellitus. Lancet, 1982; 1:1430–2.

10. Targher G., Chonchol M., Bertolini L., Rodella S., Zenari L. et al., Increased risk of CKD among type 2 diabetics with nonalcoholic fatty liver disease. J. Am. Soc. Nephrol., 2008, 19(8):1564–70.

11. SAADEH S, YOUNOSSI ZM, REMER EM, GRAMLICH T, ONG JP, et al., The utility of radiological imaging in nonalcoholic fatty liver disease. Gastroenterology, 2002, 123:745–750.

Received December 20, 2008


60

Phospholipase A2 in Patients with Noncardioembolic Ischemic Stroke and Severe Inflammatory Reaction

INIMOARA MIHAELA COJOCARU1, M. COJOCARU2, R. TĂNĂSESCU1, IULIANA ILIESCU3, LAURA DUMITRESCU3, CECILIA BURCIN3, CAMELIA VIDIŢIA GURBAN4, FELICIA SFRIJAN4

1“Carol Davila” University of Medicine and Pharmacy, Department of Neurology, Colentina Clinical Hospital, Bucharest 2“Titu Maiorescu” University, Faculty of Medicine, Department of Physiology, Bucharest

3Clinic of Neurology, Colentina Clinical Hospital, Bucharest 4“Victor Babeş” University of Medicine and Pharmacy, Department of Biochemistry, Timişoara, Romania

The inflammatory reaction is characterized by increased circulatory levels of various indicators of the severity of inflammation. The objective was to investigate the value of lipoprotein-associated phospholipase A2 (Lp-PLA2) in patients with noncardioembolic ischemic stroke and severe proinflammatory reaction. There were investigated prospectively Lp-PLA2 levels in sera from 47 patients with ischemic stroke and severe inflammatory reaction (32 men and 15 women, mean age 63±4.23 years) as compared to 38 patients with ischemic stroke without inflammatory reaction (21 men and 17 women, mean age 61±5.52 years) and 114 healthy elderly controls. Lp-PLA2 levels were assessed using the diaDexus PLAC test (a noncompetitive ELISA). Out of 47 patients with ischemic stroke and severe inflammatory reaction 36 presented Lp-PLA2 high levels (79%). Lp-PLA2 was detected with high levels in 17 out of 30 patients with ischemic stroke without inflammatory reaction (45%). Patients with ischemic stroke and severe inflammatory reaction presented Lp-PLA2 with high levels more frequently than the healthy controls (RR 12.1 [95% CI. 6.22 to 19.333], p<0.0001). Levels of Lp-PLA2 were higher in subjects who experienced a stroke as compared to controls. Lp-PLA2 is a strong predictor of recurrent stroke risk and of increased risk of dying. The determination of Lp-PLA2 should be used to predict patient risk of cardiovascular disease and stroke; it does provide additional risk of inflammation when used in conjunction with the traditional markers. Lp-PLA2 might be used not only for risk stratification of stroke patients, but also as target for treatment.

Key words: lipoprotein-associated phospholipase A2 (Lp-PLA2), ischemic stroke, inflammatory marker.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a calcium independent enzyme that is associated with low-density lipoprotein (LDL) in the plasma.

(Lp-PLA2) is an enzyme produced by macrophages that appears to play a role in the atherosclerotic vessel wall. Several studies have documented the strong association of Lp-PLA2 with coronary heart disease and stroke in the general population. Lp-PLA2 is an enzyme produced by white blood cells that appears to play a role in the atherosclerotic vessel wall.

Phospholipase A2 (PLA2) plays an essential role in the metabolism of membrane phospholipids; it is related to inflammatory reactions [1].

Agents that block the activity of Lp-PLA2 might also reduce its proinflammatory effects.

(Lp-PLA2) is an enzyme that belongs to the superfamily of phospholipase A2 enzymes.

Lp-PLA2 is a proinflammatory enzyme secreted by macrophages that is primarly bound to LDL circulation. Information from some studies has provided clear evidence for the important role of Lp-PLA2 in regulating the physiological and pathological function of the central nervous system (CNS).

Lp-PLA2 can be used to determine cardio-vascular risk, both of coronary heart disease and cerebrovascular disease [2].

In addition, because many neurodegenerative diseases are associated with increased oxidative and inflammatory responses, an attempt was made to include studies of Lp-PLA2 in cerebral ischemia. Therefore, there is substantial interest in the role of Lp-PLA2 in the inflammatory responses in these cells [3].

The objective of this paper was to investigate the value of Lp-PLA2 in patients with non-cardioembolic ischemic stroke and severe inflam-matory reaction.

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MATERIALS AND METHODS

In the present study we investigated pros-pectively Lp-PLA2 levels in sera from 47 patients (32 men and 15 women, mean age 63±4.23 years) with ischemic stroke and severe inflammatory reaction as compared to 38 patients (21 men and 17 women, mean age 61±5.52 years) with ischemic stroke without inflammatory reaction and 114 healthy elderly controls.

For all eligible patients the informed consent was given for the use of their blood in this study. The research received approval by the ethical committee of the institution.

During routine venipuncture, 2–3 additional milliliters of blood was drawn, and centrifuged. Sera were then frozen and kept at –20°C, until the assays were performed.

Lp-PLA2 levels were assessed using the diaDexus PLAC test (a non-competitive ELISA), that employes two monoclonal antibodies to quantitatively measure the concentration of Lp-PLA2 in human serum or plasma, and is calibrated to a recombinant protein standard. A cutpoint of 235 mg/ml, which is the median of a healthy population, is recommended.

Cerebral ischemia was documented by an imaging technique, such as computerized tomography (CT scan) or magnetic resonance imaging (MRI).

Ultrasound examination of cervico-cerebral arteries and of heart was performed. Exclusion criteria were: infections, neoplasias, surgical interventions, vasculitis, concurrent major renal or hepatic diseases, major trauma in previous month.

Comparisons between patients and controls and patients group were expressed as relative risk with its 95% confidence interval (RR [95% CI]), where a lower limit >1.0 was considered significant.

All p values were determined by Fischer’s exact test. A value of p<0.05 was considered statistically significant.

The Canadian Neurological Stroke scale (CNSS) and Barthel Index (BI) assessed initial stroke severity and disability, respectively.

RESULTS

Out of 47 patients with ischemic stroke and severe inflammatory reaction 36 presented Lp-PLA2 levels (79%).

Lp-PLA2 was detected with high levels in 17 out of 30 patients with ischemic stroke without inflammatory reaction (45%).

Lp-PLA2 was detected with high levels in one of the normal controls (0.87%).

Patients with ischemic stroke and severe inflammatory reaction presented Lp-PLA2 with high levels more frequently than the healthy controls (RR 12.1 [95% CI. 6.22 to 19.333], p<0.0001).

Patients with ischemic stroke and severe inflammatory reaction presented Lp-PLA2 with high levels more frequently than ischemic stroke patients without inflammatory reaction (RR 1.97 [95% CI 0.9237 to 2.378], p<0.18).

All patients with ischemic stroke and severe inflammatory reaction were treated with statins.

Twenty-one patients died, 85% of vascular causes. At discharge the median CNSS score was 7 and the median BI score 50. Twelve patients (19%) were functionally independent and 52 patients (81%) functionally dependent.

High Lp-PLA2 levels were not associated with stroke severity.

DISCUSSION

A review of recent studies clearly demonstrates the important role of Lp-PLA2 in mediating normal and pathological functions in the CNS [4-8]. All major of Lp-PLA2 are present in the CNS [8].

Cessation of blood flow in cerebral ischemia (stroke) is known to trigger a number of physiological and biochemical changes, including rapid energy depletion, release of excitatory amino acid trans-mitters, neuronal membrane depolarization, and influx of Ca2+. Many of these changes are associated with an increase in oxidative stress, resulting in the production of ROS, which in turn, are important factors underlying delayed neuron cell death [9].

Astrocytes are the major cell type in the CNS and play multiple functional roles in providing nutrient support to neurons, modulating Ca2+ homeostasis, and regulating neurotransmission, as well as mediating host defense functions. Astrocytes have been shown to contain all major groups of PLA2. In addition to astrocytes, little is known about Lp-PLA2 in other types of glial cells, such as the microglial cells and the oligodendroglial cells. Microglial cells are immune-active cells and exhibit many properties similar to those of macrophages and astrocytes. In N9 microglial cells, Lp-PLA2 inhibitors could inhibit LPS-induced TNF-α release, suggesting an involvement of Lp-PLA2 in the cytokine pathway [3].

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Phospholipids in CNS membranes are enriched in polyunsaturated fatty acids (PUFAs). Metabolism of PUFA is stringently controlled by PLA2 and acyltransferases-known as the deacylation- reacylation cycle [9][10].

Research has suggested that Lp-PLA2 is involved in the oxidative modification of LDL that can lead to plaque formation and the generation of proinflammatory molecules. These molecules can recruit macrophages and contribute to the formation of atherosclerotic plaque [11].

Transient focal cerebral ischemia significantly increases Lp-PLA2 activity [12].

High levels of Lp-PLA2 trigger a cascade of inflammatory events.

Elevated levels of Lp-PLA2 are indicative of vascular inflammation associated with the formation of plaque within the arteries. Because 68% of heart attacks and strokes occur from blood clots, not narrowing of the arteries and 50% of all heart attacks occur in patients with normal cholesterol values, the Lp-PLA2 test is now used in conjunction with advanced lipoprotein tests [13].

Lp-PLA2 appears to be a specific marker of plaque inflammation that may play a direct role in the formation of rupture-prone plaque. In contrast traditional risk factors, lipid measurement, and most vascular imaging modalities do not direct assess the acute ischemic potential in the arterial wall [14].

The increase in Lp-PLA2, together with that of other lipid mediators in reactive astrocytes, is in agreement with the increased inflammatory response observed during this period of cerebral ischemia [15][16].

Lp-PLA2 may provide additional clinically relevant information that shows which patients have a high level of atherosclerotic disease activity as manifested by vascular inflammation, endo-thelial dysfunction, and increased risk for progression toward rupture-prone plaque [14].

Lp-PLA2 has been shown to be an independent risk factor for coronary heart disease (CHD) and ischemic stroke in several studies [5][17].

Individuals who have elevated Lp-PLA2 are twice as likely to suffer an ischemic stroke as similar individuals. Individuals with the highest

levels of Lp-PLA2 and systolic blood pressure are six times more likely to suffer an ischemic stroke, conferring an over six fold increase in stroke risk.

Lp-PLA2 is related to vascular inflammation and is not elevated in systemic inflammatory conditions, providing consistent and reliable results regardless of a patient’s other clinical conditions.

In 2005, the Lp-PLA2 blood test was approved by the US Food and Drug Administration (FDA) for assessing the risk of ischemic stroke and coronary artery disease. In epidemiologic studies low-density lipoprotein cholesterol and the lipid factors have not been shown to be consistent predictors of stroke risk [18]. Lp-PLA2 measures, on the other hand, have shown a consistent association with stroke risk conferring about a 2-fold increase in stroke occurrence [17–22].

Lp-PLA2 is a more specific marker than high sensitivity C-reactive protein (hs-CRP) which can be elevated from systemic infection, even when the vascular wall is healthy. High Lp-PLA2 in combination with hs-CRP increases the ability to determine risk level [23][ 24].

The American Heart Association/American College of Cardiology guidelines advocate for aggressive lowering of lipid and other factors to new targets for example, in patients with established atherosclerotic vascular disease [25–28].

CONCLUSION

1) Levels of Lp-PLA2 were higher in subjects who experienced a stroke as compared to controls.

2) Elevated serum inflammatory biomarkers, including Lp-PLA2 in patients who suffered a first ischemic stroke, indicate the likelihood of having another stroke or an increased risk of dying; Lp-PLA2 as a strong predictor of recurrent stroke risk.

3) The determination of Lp-PLA2 should be used in conjunction with clinical evaluation and patients risk assessment to assist in predicting patient risk of cardiovascular disease and stroke; it does provide additional risk inflammation when used in conjunction with the traditional markers.

4) Lp-PLA2 might be used not only for risk stratification of stroke patients, but also as target for treatment, statins for example, that reduce the proinflammatory effects of Lp-PLA2.


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Reacţia inflamatorie este caracterizată de niveluri sanguine crescute ale markerilor inflamaţiei.

Obiectivul lucrării a fost investigarea valorii fosfolipazei A2 asociată lipoproteinelor (Lp-PLA2) la pacienţi cu stroke ischemic fără cauză cardioembolică, dar cu reacţie inflamatorie severă.

S-au investigat prospectiv nivelurile de Lp-PLA2 în serul provenit de la 47 pacienţi cu stroke ischemic şi reacţie inflamatorie severă (32 bărbaţi şi 15 femei cu vârsta medie 63±4,23 ani) comparativ cu 38 pacienţi cu stroke iscemic fără reacţie inflamatorie (21 bărbaţi şi 17 femei cu vârsta medie 61±5,52 ani) şi 114 martori vârstnici.

Nivelurile de Lp-PLA2 au fost determinate folosind testul diaDexus PLAC (ELISA necompetitiv). Din cei 47 pacienţi cu stroke ischemic şi reacţie inflamatorie severă 36 au prezentat niveluri crescute de Lp-PLA2 (79%). S-a evidenţiat Lp-PLA2 cu niveluri crescute la 17 din 30 pacienţi cu stroke ischemic fără reacţie inflamatorie (45%).

Pacienţii cu stroke ischemic şi reacţie inflamatorie au prezentat niveluri de Lp-PLA2 crescute mai frecvent decât martorii (RR 12,1 [95% CI între 6,22 şi 19,333], p<0,0001).

Nivelurile de Lp-PLA2 au fost mai mari la subiecţii care au prezentat stroke comparativ cu martorii. Lp-PLA2 este un factor de predicţie sensibil al riscului de stroke recurent şi al riscului crescut de deces.

Determinarea Lp-PLA2 trebuie să fie folosită pentru predicţia riscului de boală cardiovasculară şi stroke; aceasta demonstrează riscul în plus al inflamaţiei când se foloseşte împreună cu markerii clasici.

Lp-PLA2 ar putea fi utilă nu numai pentru stratificarea pacienţilor cu stroke, dar şi pentru tratamentul adecvat.

Corresponding author: Inimioara Mihaela Cojocaru MD, PhD

Colentina Clinical Hospital 19–21 Şos. Ştefan cel Mare 020125 Bucharest, Romania E-mail: [email protected]

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26. SCHAEFER E.J., MCNAMARA J.R., ASZTALOS B.F. et al., Effects of atorvastatin versus other statins on fasting and postprandial C-reactive protein and lipoprotein-associated phospholipase A2 in patients with coronary heart disease versus control subjects. Am. J. Cardiol., 2005; 95: 1025–1032.

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Evaluation of 5-α Reductase Activity on Cultured Fibroblast in Patients with Hyperandrogenemia

D. BODA1, DIANA PĂUN1, ADRIANA DIACONEASA2

1“Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania 2“Gr.Alexandrescu” Emergency Children Hospital, Bucharest

The enzyme steroid 5-α reductase is responsible for the conversion of testosterone to dihydrotestosterone, the steroid that mediates the intracellular action of androgens in some target tissues. The goal of this study was to check the accuracy of three known biochemical methods of studying steroid 5-α reductase activity expressed by dermal fibroblasts, isolated from pubian skin. These methods were performed on cell lysates (spectrophotometric and spectrofluorimetric methods) and on cell culture media (Reversed Phase-HPLC) with the purpose of their use in diagnosis and monitoring of hyperandrogenic patients. We also optimized a molecular study of expression of 5-α reductase isoenzymes and used it in the analysis of patients diagnosed with polycystic ovary syndrome (PCOS) and hirsutism by comparison with normal women. There was noticed an increase of the isoenzyme expression level both in patients with PCOS and in the case of patients with hirsutism. In other experiments, dermal fibroblasts originating in 15 individuals were treated with androgen hormones (testosterone: 10–7–10–9 M) with the purpose of demonstrating the effect of hyperandrogenemia on the expression level of 5-α reductase isoenzymes. The study of 5-α reductase type 1 mRNA expression levels in fibroblasts resulted from 4 normal individuals, 3 patients with hirsutism and 6 patients with PCOS, demonstrated an increase with 108.3% at the patients with PCOS and 47.3% at the patients with hirsutism compared with normal women. We concluded that hyperandrogenemia is associated with high levels of expression of 5-α reductase type 1 and, to a less extent, of type 2 isoenzyme in pubian skin cultured fibroblasts.

Key words: 5-α reductase, fibroblast, hyperandrogenemia, spectrophotometry, spectrofluorimetry, RP-HPLC, mRNA expression.

Hyperandrogenemia represents the most frequent endocrine disease in women (up to 10% incidence). Polycystic ovary syndrome (PCOS) and its complications (infertility, diabetes mellitus, dys-lipidemia, endometrial cancer, ischemic heart disease) is by far the most frequent cause of hyperandrogenemia [1]. The correct diagnoses of those illnesses, their treatment and etiology have major impact in endocrinology, as well as in dermatology and pediatry.

A half of the plasma testosterone comes from peripheral conversion of androstendione, the fourth part from ovarian secretion and the other fourth part comes from secretion of adrenalin.

The sites of peripheral conversion are: liver, adipose tissue and target organs. Plasma dihydro-testosterone (DHT) results completely from peripheral conversion. In women the most important precursor of DHT is androstendione (60%). The next are testo-sterone (15%) and dehydroepiandrosterone (DHEA) together with delta 5 androstendiol (25%)[2].

The biological activity of testosterone is dependent on its peripheral conversion to DHT by 5-α reductase. On the other hand, the 5-α reductase activity is increased by testosterone. 5-α reductase and androgenic receptors are mainly localized in the sebaceous glands, dermal papilla and sweat glands. DHT is a more potent androgen than testosterone as a result of its high affinity for androgenic receptor [3].

Steroid 5-α reductase activity at pubian level is much higher at women with idiopathic hirsutism, in the absence of plasmatic hyperandrogenemy. This can lead to the increased peripheral use of androgens and at its turn can be responsive of hirsutism. The importance of 5-α reductase in hirsutism can be proved by high levels of urinary 3 alpha, 17 beta-androstanediol glucuronide, the index of the cutaneous androgenic metabolism. However, it is unclear which 5-α reductase isoform is responsible of alterations in hirsutism: the increased expression of the normal type 1 isoenzyme or abnormal expression of type 2 isoenzyme [4][5].

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OBJECTIVE

In this context, the objective of this assay was to assess a new diagnostic tool in hyperandrogenemia by measuring 5-α reductase and its isoenzymes levels in the skin from the pubian region.


CELL CULTURE

Human dermal fibroblasts (HDF) were isolated by explant technique from pubian skin biopsies, obtained from 15 patients with PCOS and/or hirsutism under local anaesthesia after informed consent was given. The patients were diagnosed on the basis of clinical observations and hormonal analyses realized at “C.I. Parhon” National Institute of Endocrinology, Bucharest. The cell culture medium was Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. HDF were incubated at 37oC in a humidified air with 5% CO2; culture medium was changed every 2–3 days. Cell viability was checked by Trypan Blue exclusion method. The mRNA expression and activity of steroid 5-α reductase were determined on cultured fibroblasts at passages 5–9.

ASSAYS OF STEROID 5-α REDUCTASE ACTIVITY

Three methods of studying steroid 5-α reductase activity were tested: 1) spectrophoto-metric dosing of NADPH consumed in the reaction of reduction of testosterone to DHT; 2) spectro-fluorimetric assay of NADPH used in the reaction catalysed by 5-α reductase, and 3) Reversed Phase-HPLC (high pressure liquid chromatography) measurement (RP-HPLC) of consumed testosterone. The first two tests were performed on cell lysates and the third one in culture media [5].

The cell lysates were obtained by trypsinizing fibroblast monolayers, centrifugation and lysis in extraction buffer (0.1M Tris, 0.001 EDTA-2Na, 0.3M MgCl2, 0.3M sucrose, pH 7.5).

Spectrophotometric method. The cell lysate was centrifuged for 10 min at 800g and 4oC and the enzymatic activity was determined in the supernatant by its incubation with different concentrations of NADPH (0–40µM) and testosterone (0–100µM).

The decrease of absorbance was monitored at 340 nm (NADPH) for 5–30 de minutes at a spectrophotometer Lambda 25 Perkin-Elmer with double fascicle.

Spectrofluorimetric method. The cell lysates obtained as above were incubated with different concentrations of NADPH (0–40µM) and testosterone (0–100µM) and the decrease of NADPH fluorescence was determined by a TECAN multiplate reader.

Reversed Phase-HPLC. This method was performed by using a PepRPC HR5/5 column and allowed the separation of testosterone and andro-stendione from culture medium. The method consisted of incubation of testosterone, NADPH and biological sample, extraction of steroids in a mix of ethyl cyclohexane acetate, the steroid separation by HPLC (high pressure liquid chromatography) using a column in reverse phase and an UV 254 nm detector. At this wavelength, DHT did not have a detectable absorption. The enzymatic activity of 5-α reductase was expressed in nmols of transformed testosterone/µg protein/min.

ASSAY OF STEROID 5-α REDUCTASE mRNA EXPRESSION

The expression of 5-α reductase type 1 and 2 is regulated by testosterone and DHT that act at cellular level through an androgen receptor. DHT and 4-androstendione can be converted to testosterone and subsequently to DHT. They can also play a part in the regulation of the two genes. Consequently, by its implication in conversion of testosterone to DHT, 5-α reductase intervenes in the regulation of the own gene. Cultured dermal fibroblasts preserve their features concerning the regulation of the two genes till the passages 9–10. Therefore, the cultures (passages 5–8) were treated with androgen hormones at concentrations of 10–7–10–9 M, reflecting their normal and pathological plasma levels. These treatments were realized for different time periods: 1h, 16h and 30h and the fibroblasts were subjected to the total mRNA extraction procedure. The RNA extraction was performed on the confluent cultures used by GenElute Mammalian Total RNA and RNAzol kits (Sigma). The RNA concentration and purity were determined by measuring OD at 260 and 280 nm. The RNA quality was also checked by denaturing electrophoresis and making evident ribosomal RNA 18 and 28S following the staining with

3 5-α reductase activity in cultured fibroblast 69

ethidium bromide and UV visualisation. If the DO260nm/DO280nm ratio was lower than 1.7 an additional RNA purification was done.

SEMI-QUANTITATIVE RT-PCR METHOD

The total RNA obtained by an extraction procedure was subjected to Real Time-PCR. For each RT-PCR set of reactions were selected the points situated on the linear part of the amplification curve. The estimation of the expression of 5-α reductase type 1 and 2 genes was performed in relation to the constitutive actin gene expression level.

RESULTS

5-α reductase type 1 (isoenzyme 1 of 5-α reductase) is an enzyme constituted of 259 amino acids having an optimal pH of 6–9. It is the cutaneous type of enzyme, preponderantly localized in sebocytes, epidermal and follicular keratinocytes, dermal papilla, sweat glands and fibroblasts from the genital and non-genital tegument. It is coded by a gene localized on the 5p chromosome and composed of 5 exons and 4 introns.

5-α reductase type 2 (isoenzyme 2 of 5-α reductase) consists of 254 amino acids, with an optimal pH of 5.5. It is localized especially at the level of epididymis, seminal vesicles, prostate, fetal genital skin, the internal theca of the hair as well as in fibroblasts from adult normal genital tegument. It is coded by a gene situated on the 2p chromosome, with the same number of introns and exons as well as isoenzyme type 1.

The skin from the external region of genital organs expresses especially 5-α reductase type 2. Its activity is high and similar in the case of both sexes, even anterior to the puberty period. It is normal at the patients with total resistance to androgens as a result of androgenic receptor defects. This suggests that in the territories of primary sexual differentiation the enzyme is not regulated by androgens.

In other territories, such as the pubian region, type 1 isoform is preferentially expressed. Its level is low in the pre-pubertal period and, at puberty, it reaches levels two-three times higher at males than at females. This enzyme is present in low amounts at hypogonadal men and is re-established by androgenic treatment, indicating the androgen

dependence of these territories. This finding is also confirmed by the absence of the enzymatic activity at patients with androgen total resistance, contrasting with the high enzymatic activity at hyperandrogenic women.

The goal of our studies was to check the accuracy of three known methods of studying steroid 5-α reductase activity expressed by dermal fibroblast, isolated from pubian skin, with the purpose of their use in diagnosis and monitoring of hyperandrogenic patients. We also optimized a study of expression of 5-α reductase type 1 and used it in the comparative analysis of two patients: one diagnosed with PCOS and the other with hirsutism with a normal woman.

From each skin biopsy about four primary cultures were obtained (Fig. 1), each of them being propagated successively until the passages 3–4 and cryoconserved in liquid nitrogen.

Fig. 1. – Phase contrast micrograph of primary pubian skin fibroblast culture (×100).

The spectrophotometric method for determining the 5-α reductase activity expressed in cell lysates did not make evident a correlation between the testo-sterone level and the enzymatic activity (Fig. 2). Therefore, the method was abandoned.

The use of the spectrofluorimetric method pointed out the absence of a direct relationship between NADPH concentration in the reaction mix and the reaction velocity (expressed in α RFU/min, RFU= relative fluorescence units) (Fig. 3). Con-sequently, this method was also abandoned.

The third biochemical method of dosing 5-α-steroid reductase activity in fibroblasts was an original one, based on Reversed Phase-HPLC, but being laborious and expensive did not allow its application in a high number of cases.

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0,0 1 2 3 4 5 6 7 8 9 10,0min

A

Date: 15.07.2002 Time: 18:35:31 Method: 5AR.MKI Sample Type Range [min] dA/min Factor Activity Comments ----------------------------------------------------------------------------------- 5AR_30 Sample 0.000–15.000 –0.002 1.0000 0.0022 30µM NADPH Blank without Testosterone 5AR_31 Sample 0.000–15.000 –0.002 1.0000 0.0026 30µM NADPH, 10µM Testosterone 5AR_32 Sample 0.000–15.000 –0.002 1.0000 0.0025 30µM NADPH, 20µM Testosterone 5AR_33 Sample 0.000–15.000 –0.003 1.0000 0.0039 30µM NADPH, 30µM Testosterone 5AR_34 Sample 0.000–10.000 –0.006 1.0000 0.0041 30µM NADPH, 100µM Testosterone

Fig. 2. – The variation of 5-α reductase activity with testosterone concentration.

Date of measurement: fluor5AR-003 340nm–465nm GENios Measurement mode: Fluorescence Top NADPH RFU/min Excitation wavelength: 340 nm 1.3 D7 23.96 Emission wavelength: 465 nm 3.3 D8 5.4 Gain (Manual): 60 6.6 D9 6.08 Number of flashes: 3 13.3 D10 5.36 Lag time: 0 µs 26.6 D11 10.32 Integration time: 40 µs 39 D12 4.52 Plate definition file: GRE96fb.pdf Part of the plate: D7-D12 Number of kinetic cycles: 26 Kinetic interval: 60 s Shake duration (Orbital High): 25 s Shake settle time: 5 s Unit: RFU

Fig. 3. – Spectrofluorimetric dosing of 5-α reductase activity in fibroblast lysates.


Taking into account the difficulties of bio-chemical dosing of 5-α-steroid reductase activity, the second objective of the research was to study the enzyme mRNA expression in pubian skin fibroblasts. With this purpose, the following research steps were performed a) extraction and purification of total RNA; b) synthesis of cDNA specific to steroid 5-α reductase with reverse transcriptase; c) exponential amplification of cDNA by PCR reaction (denaturation, annealing and extension cycles); d) electrophoretic analysis of nucleic acids species (total ARN and cADN) and mARN quantification by densitometry of amplification products.

The study of 5-α-steroid reductase type 1 expression was realized, comparatively, on fibroblast samples coming from a patient with PCOS (F8), another with hirsutism (F6) and a normal woman (N). In each case 5-α reductase mRNA level (Fig. 4) was related to β-actin (Fig. 5). An increase of the isoenzyme expression level both in the case of patient with PCOS and in the case of patient with hirsutism was noticed (Fig. 6).

Fig. 4. – 5-α reductase type I mRNA expression level.

Fig. 5. – β-actin mRNA expression level.

In other experiments, dermal fibroblasts originating in 15 individuals were treated with androgen hormones (10–7–10–9 M) with the purpose of demonstrating the effect of hyperandrogenemia on the expression level of 5-α reductase isoenzymes.

As results from Fig. 6, the study of 5-α reductase type 1 mRNA expression levels in fibroblasts resulted from 4 normal individuals, 3 patients with hirsutism and 6 patients with PCOS, demonstrated an increase with 108.3% at the patients with PCOS and 47.3% at the patients with hirsutism compared with controls.

Fig. 6. – Expression level of 5-α reductase type 1 in pubian skin fibroblasts F6 (patient with hirsutism) and F8 (patient

with PCOS) in comparison with normal fibroblasts.

In these studies, quantification of the PCR products was done depending on the intensity (peak volume) of their specific electrophoretic bands (Fig. 7). Taking into account these results, all the RT-PCR reactions were realized by using the same quantities of RNA (50, 100, 150 ng) for all studied cases. The optimal RNA quantities for beta actin amplification were lower (5, 10, 15 ng) because this protein is constitutively expressed by fibroblasts and its mRNA level is much higher in the cell.

Fig. 7. – The electrophoretic profile of the RT-PCR amplification products specific to 5α-reductase type 1 – 362 bp (bands: 2–4) and beta actin – 530 bp (bands: 5–7); band 1: negative martor (does not contain RNA) and 8: φX174/Hae III marker.

In the same fibroblast cultures it was estimated the expression of 5-α reductase type 2 isoenzyme. It is much less expressed in the pubian dermal fibroblasts. Consequently, to obtain a detectable

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amplification, high amounts (200, 250, 300 ng) of total RNA were used for RT-PCR reactions (Fig. 8).

1 2 3 4 5 6 7 8

Fig. 8. – The electrophoretic pattern of the RT-PCR amplification products specific to 5α-reductase type 2 – 577 kD bp (bands: 2–4)

and beta actin – 477 bp (bands: 5–7); bands 1 and 8: φX174/Hae III marker.

CONCLUSION

The studies of molecular biology concerning the expression of 5-α-steroid reductase isoforms in dermal fibroblasts are characterized by a high accuracy. These methods imply extraction and purification of total RNA, synthesis of steroid 5-α reductase cDNA, exponential amplification of this cDNA, electrophoretic analysis and quantification by densitometry of amplification products.

We developed an original method of dosing of 5-α-steroid reductase activity in pubian skin fibroblasts originating in patients with hyper-androgenemia. However, being laborious and expensive, this method does not allow the enzyme evaluation in a high number of cases.

Enzima steroid 5-α reductaza este responsabilă de conversia testosteronului în dihidrotestosteron, steroid care mediază acţiunea intracelulară a androgenilor în anumite ţesuturi ţintă. Scopul acestui studiu a fost de a verifica acurateţea a trei metode biochimice cunoscute pentru studiul activităţii 5-α reductazei exprimate de fibroblastele dermale, izolate din pielea pubiană. Aceste metode au fost realizate pe lizate celulare (metode spectrofotometrice şi spectrofluorimetrice) şi pe medii de cultură celulară (Reversed Phase-HPLC) în scopul folosirii lor pentru diagnosticarea şi monitorizarea pacienţilor hiperandrogenici. De asemenea, am optimizat un studiu molecular de expresie a izoenzimelor 5-α reductazei şi l-am utilizat în analiza pacienţilor diagnosticaţi cu sindromul ovarului policistic (PCOS) şi hirsutism prin comparaţie cu femei normale. S-a constatat o creştere a nivelului de expresie al izoenzimelor, atât în pacientele cu PCOS, cât şi în cazul pacientelor cu hirsutism. În alte experimente, fibroblastele dermale ce provin de la 15 persoane au fost tratate cu hormoni androgeni (testosteron: 10–7–10–9 M) cu scopul de a demonstra efectul hiperandrogenemiei asupra nivelului de expresie al izoenzimelor 5-α reductazei. Studiul nivelului de expresie al mRNA 5-a reductazei de tip 1 în fibroblaste derivate din 4 persoane normale, 3 paciente cu hirsutism şi 6 paciente cu PCOS, a demonstrat o creştere cu 108.3% la pacientele cu PCOS şi 47.3% la pacientele cu hirsutism in comparaţie cu femeile normale. Noi am concluzionat că hiperandrogenemia este asociată cu valori crescute de expresie a 5-α reductazei tip 1 şi, în mai mică măsură, a izoenzimei de tip 2 în fibroblastele din pielea pubiană în cultură.

Corresponding author: D. Boda, MD

“Carol Davila” University of Medicine and Pharmacy E-mail: dan.boda @gmail.ro

R E F E R E N C E S

1. F.S. GREENSPAN, G.J. STREWLER, Basic&Clinical Endocrinology, 5th edition, 461–468. 2. EHRMANN D.A., BARNES R.B., ROSENFIELD R.L., Hyperandrogenism, Hirsutism and the Polycystic Ovary Syndrome.

N. Engl. J. Med., 1993; 57: 1345–1350.


3. CHEN W., ZOUBOULIS C.C., ORFANOS C.E., 5alfa reductase and its inhibitors. New perspectives in the treatment of androgen depending diseases. Dermatology, 193 (3): 177–84, 1996.

4. ISSELBACHER K.J., BRAUNWALD E., WILSON J.D., MARTIN J.B., FAUCI A.S., Harrison’s Principles of Internal Medicine, 14th edition, 2026–2027.

5. SATO T., TADOKORO T., SONODA T., ASADA Y., 5alfa reductase type I predominance in apocrine glands of patients with osmidrosis. Department of Dermatology, Oita Medical University, Japan; BJD 139(5):806–10, 1998 Nov.

6. SAWAYA M.E., PRICE V.H., Different levels of 5alfa reductase I/II, aromatase and androgen receptors in hair follicles of male and female patients with androgenic alopecia; Journal of Investigative Dermatology, 109 (3): 296–300, 1997 S.


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POINTS OF VIEW

Bowel Lesions in Spondyloarthritides

M. RIMBAŞ, MĂDĂLINA MARINESCU, M.R. VOIOSU

Gastroenterology Department, Clinic of Internal Medicine, Colentina Clinical Hospital, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania

The gut can play an important role in the pathogenesis of many rheumatic conditions; this is also true for spondyloarthropathies, ileocolonoscopic studies revealing the prevalence of histological gut inflammation in more than half of these patients.

Furthermore, in patients with spondyloarthritis, an evolution to clinical inflammatory bowel disease has been observed in 20% of patients with an initial subclinical chronic gut inflammation, indicating that joint and bowel inflammation are somehow connected. The presence of chronic gut inflammation can be the first sign of Crohn’s disease, and it is being speculated that early treatment of the gut inflammation could prevent this evolution when the appropriate drugs become available.

Moreover, the medication employed in treating these patients has the potential of influencing the inflammatory bowel lesions, in a negative (NSAIDs) or positive way (DMARDs, corticosteroids, biologic agents), and there are reports trying to prove that the spondylarthropathic ileo-colonic inflammation represents, at least in part, iatrogenic COX-2 driven damage.

Therefore, there are “obscure” fields regarding the issue of gut inflammation in spondylo-arthropathies, which need focused research, but taking into consideration the complex treatment strategies applied in patients with these kinds of diseases, this is not an easy task to perform.

Key words: spondyloarthritides, inflammatory bowel disease.

The spondyloarthritides (SpAs) are a group of similar diseases characterized by inflammatory involvement not only of the peripheral joints, but also of the axial skeleton and the entheses, the eye, the bowel, the urogenital tract, the skin and sometimes the heart, having an estimated prevalence in the general population of 1% [1].

Clinical characteristics of these diseases are the absence of rheumatoid factor and rheumatoid nodules, the presence of a typical pattern of inflammatory arthritis (pauci-articular, asymmetrical, involving large and small joints mostly of the lower limbs), the presence of inflammatory enthesiopathies (mostly of the feet), the presence of sacroiliitis and spondylitis, the clinical similarities between different diseases of the concept, the familial aggregation of these diseases, and the strong genetic relationship to human leukocyte antigen (HLA) B27 [2][3].

Diseases included within this concept of spondyloarthritis (SpA) are: ankylosing spondylitis (AS), psoriatic SpA (PsSpA), reactive SpA (ReSpA), SpA associated with inflammatory bowel disease (SpAIBD) and undifferentiated SpA (uSpA)[4]. Their clinical spectrum is much wider than previously realized, and some additional, less clearly defined

types are categorized as undifferentiated spondylo-arthritis [5]. Clear differentiation among these various forms, especially in their early stages, may not always be possible owing to overlapping clinical features; however, lack of differentiation does not usually affect treatment decisions.

Arthritis is recognized as part of the clinical picture of several illnesses and conditions, including inflammatory bowel disease (IBD)[6], bacterial and parasitic infections of the gut [7], gluten-sensitive enteropathy (celiac disease)[8], pseudomembranous colitis [9], and following intestinal bypass surgery [10]. Other illnesses have a propensity for causing inflammation of joints and the gut, such as Behcet’s [11] and Whipple’s diseases [12]. All these findings suggest that the gut can play an important role in the pathogenesis of many rheumatic conditions; this is also true for SpAs, ileocolonoscopic studies revealing the prevalence of histological gut inflammation in more than 50% of these patients [13].

An interplay between mucosal surface bacterial infections such as enteric and urogenital and HLA-B27 is apparent from the increased prevalence of HLA-B27-positive people among those with reactive

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arthritis, but an explanation for the association remains elusive, despite the increasing amount of data available in this field [14–20]. Within the concept of SpA, there is ample experimental, clinical, genetic, histopathologic and immunologic evidence for an important role of the gut in the pathogenesis of SpA and for overlap of SpA with IBD [21].

PREVALENCE OF GUT ABNORMALITIES IN SPONDYLOARTHROPATHIES

Since the first report on subclinical gut inflammation in SpA patients, revealed by ileo-colonoscopy, exploring the whole colon, cecum, and terminal ileum, several authors have confirmed these findings in different forms of spondylo-arthritis (SpA) [22–34].

In enterogenic ReSpA, macroscopic lesions were found in approximately 50% of patients, whereas the prevalence of histologic signs of inflammation ranged from 25% to 100% [28]. In urogenital ReSpA, macroscopic lesions are uncommon, but practically all authors found some patients presenting with histologic signs of gut inflammation [28].

In undifferentiated SpA, gut inflammation was present in a considerable proportion of patients (macroscopically, 24–38%; microscopically, 24–72%) [22].

In AS, macroscopic signs of gut inflammation and histologic inflammatory lesions are found in 29–49% and 25–62% of cases, respectively [26]. Prevalence of gut inflammation in AS was higher in patients with associated peripheral arthritis than in those only presenting axial involvement [29].

In juvenile-onset SpA, histologic evidence of gut inflammation was found in 75–80% of the patients [31[32].

In PsSpA, gut inflammation was found only in those groups related to the SpA concept, that is, in the axial and the pauci-articular group and not in the poly-articular group; however, the prevalence of gut lesions was lower than in the other SpA (26%) [33].

A systematic study analyzing a group of 27 patients with acute anterior uveitis with or without axial joint inflammation revealed gut inflammation in 66% of cases [34].

In control groups, consisting of patients with spastic colon or of patients with other inflammatory joint diseases, the prevalence of gut inflammation was significantly lower.

HISTOLOGIC PICTURES

The lesions were histologically different and two types of inflammation, acute and chronic, could be distinguished [35]. The acute type resembles acute bacterial enterocolitis. The mucosal architecture is well preserved. The ileal villi and crypt epithelial cells are infiltrated by polymorphonuclear cells. In the lamina propria, there is an increased number of inflammatory cells, consisting of a mixture of granulocytes, lymphocytes, and plasma cells [23][36].

The chronic type resembled chronic ileocolitis, mostly indistinguishable from Crohn’s disease (CD) [23][36]. Here, the mucosal architecture is clearly disturbed. The villi are irregular, blunted, and fused. The crypts are distorted and the lamina propria is edematous and infiltrated by mononuclear cells. Basal lymphoid follicles occur.

Acute lesions are mainly seen in patients with ReA [28]. In enterogenic ReA, 70% of patients presented with acute lesions, whereas only 26% showed chronic lesions. In urogenital ReA, chronic lesions were observed in 1 of 30 patients; all the other patients presenting with histologic gut inflammation had features of acute inflammation. In un-differentiated SpA chronic lesions (32–44%) were slightly more prevalent than acute lesions (29%) [37].

In AS, chronic inflammation lesions (39–52%) are significantly more frequent than acute lesions (10–15%), especially when peripheral arthritis is associated (46% versus 11%) [26].

In an ileocolonoscopic study on 354 patients with SpA in whom psoriasis and inflammatory bowel disease (IBD) were excluded at the start, microscopic gut mucosa morphology correlated with different clinical, biologic, and radiologic manifestations of the disease. Chronic inflammation lesions were significantly more frequent in patients with a family history of SpA, AS, or CD [38].

Chronic inflammation lesions were more prevalent in patients with reduced axial mobility, in patients with increased stool frequency, and in patients reporting several periods of diarrhea. Inflammatory serum variables (erythrocyte sedimentation rate and C-reactive protein) were significantly higher in SpA patients with chronic inflammatory gut lesions than in patients who presented with normal gut histology and those with acute lesions. Patients with chronic gut lesions presented with significantly more advanced stages of sacroiliitis (stage 2 or more) and more

3 Bowel lesions in spondyloarthritides 77

syndesmophytes, and a bamboo spine evolution was also more frequent. Destructive and erosive articular abnormalities of the small joints and hips were more prevalent in patients with SpA presenting with chronic gut lesions [39][40].

EVOLUTION OF THE INFLAMMATION IN THE GUT

In one study, 123 spondyloarthropathy patients who previously underwent an ileocolono-scopy were reviewed clinically after 2 to 9 years [37]. In total, 6.5% of the SpA patients who did not present with any clinical sign of gut abnormality developed IBD during the disease course (5% in the non-AS SpA group and 7.7% in the AS group), in comparison with 0.05% of the population when randomly screened [41]. All of these patients initially presented subclinical inflammatory gut lesions, and all but one had the features of chronic inflammation. These findings are responsible for the consideration that at the start these patients may have suffered from “subclinical CD” [42].

A number of “chronic” lesions, such as aphthoid ulceration, histiocytic microgranulomas, pseudopyloric metaplasia, and sarcoid-like granulomas, bear close resemblance to those found in proven CD and have been regarded as the earliest manifestations of the disease [43].

By comparing SpA patients with an evolution to IBD with those who did not evolve to IBD, risk factors could be evaluated. The experience of regular episodes of diarrhea early in the disease history, the persistence of raised inflammatory serum parameters, and the presence of chronic inflammatory gut lesions were found to be risk factors for the development of IBD. HLA-B27 negativity in the presence of sacroiliitis is another important risk factor, confirming the hypothesis that HLA-B27-negative patients with AS are at a greater risk for IBD [44].

None of the patients with an initially normal gut histology had gut inflammation at second examination. From this, the finding of a normal gut histology on ileocolonoscopy seems to be predictive of a good prognosis in patients with SpA. Evolution of non-AS SpA to full-blown AS and evolution of uncomplicated SpA to IBD was always associated with the presence of gut inflammation at disease onset, and one of five patients with SpA and chronic gut inflammation will develop IBD within the next 5 to 9 years [42].

RHEUMATOLOGIC INVOLVEMENT IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE (IBD)

The peripheral arthritis and the spondylitis occurring in patients with IBD are considered as enteropathic arthritides, ulcerative colitis and Crohn’s disease being the most frequently encountered types of idiopathic IBD. For several years, the entero-pathic arthritis in patients with IBD, unlike the arthritis in Whipple’s disease and celiac disease, has been included in the SpA concept.

Genetic factors are important in the patho-genesis of IBD and in its extraintestinal manifestations. This was illustrated in a study that included 54 families in whom one parent and at least one child had IBD (a total of 77 parent-child pairs) and 155 families in whom at least two siblings had IBD (a total of 190 affected sibling pairs). Similarity in extraintestinal manifestations was present in 70 and 84 percent in the parent-child and sibling pairs, respectively [45].

It is well known that the incidence of inflammatory bowel disease (IBD) is increased in patients suffering from ankylosing spondylitis (AS). On the other hand, there is an increased frequency of AS in IBD. Several findings in the analysis of a large series of patients suffering from both AS and IBD deserve emphasis. The large majority of the patients suffer from CD (76%) and not from UC. Classical features of idiopathic AS, such as early occurrence of rheumatologic symptoms, peripheral arthritis, and a family history of AS, occurs in about one third of these cases. Their rheumatologic disease often started many years before IBD symptoms developed. Alternatively, a smaller group of patients – about one quarter – have predominantly IBD symptoms, with intestinal fistulae, recurrent disease attacks, and multiple operations. In these patients classical AS features are less impressive. Finally, there is a large group of AS-IBD patients with simultaneous occurrence of rheumatologic and intestinal symptoms, often with characteristics less pronounced than in either of the 2 groups. Therefore, it is generally considered that the “syndrome of AS” in IBD patients indeed harbors both classical idiopathic AS and “secondary” forms [46].

The overall incidence of peripheral arthritis in patients with IBD ranges from 2% to 20% [47]. Peripheral arthritis may be acute and remitting (type I), often associated with flares of the bowel disease, occurring in 5 percent of patients with IBD,


or be a more chronic problem or have frequent relapses (type II), episodes of exacerbations and remissions continuing for years, affecting 3 to 4 percent of patients with IBD, and joint symptoms typically not parallelling the activity of bowel disease. The frequency and severity of the peripheral arthritis complicating IBD is correlated to the severity and extent of the bowel disease.

The prevalence of SpA in IBD was estimated at 11% in CD and at 14% in UC [48]. Two forms of axial arthropathy can be associated with IBD: (1) ankylosing spondylitis, radiologically and clinically indistinguishable from idiopathic AS; and (2) asymptomatic sacroiliitis. Symptomatic spondylitis is reported to occur in 1% to 20% of patients with IBD, although actual prevalence is probably higher. Asymptomatic bilateral sacroiliitis is seen in approximately 4% to 25% of patients with IBD, and progression to spondylitis is not necessary. IBD-associated AS is commonly found in patients with HLA-B27 (50–70%), but not as frequent as in idiopathic AS (>90%) [49]. Peripheral enthesiopathy, especially inflammation of the Achilles tendon and the insertion of the fascia plantaris, is a common manifestation in patients with SpA, but it is apparently less common in patients with IBD. Peripheral enthesiopathy occurs in 6% of the patients with IBD. In contrast to some forms of peripheral arthritis, the onset of axial involvement is independent of the bowel disease and may precede the intestinal symptoms by decades. Its subsequent clinical course is independent of the severity, extent, location, and duration of IBD and bowel surgery does not alter the course of associated spondylitis or sacroiliitis.

Furthermore, in patients with SpA, an evolution to clinical IBD has been observed in 20% of patients with an initial subclinical chronic gut inflammation, indicating that joint and tendon inflammation may be the first clinical manifestation of IBD. Noteworthy was the evolution of articular disease to AS in all patients evolving from subclinical gut inflammation to overt IBD [37].

On the other hand, the presence of peripheral arthritis is frequently associated with other extra-intestinal manifestations such as skin and eye involvement. Erythema nodosum occurs in 4% of patients with IBD, and it correlates with the disease activity. Arthritis is seen simultaneously in 70% of patients with IBD and erythema nodosum. Flare-ups of erythema nodosum and arthritis seem to parallel each other [47]. Pyoderma gangrenosum occurs in

2% of patients with IBD and is associated with arthritis in 38% of the patients [50]. Acute anterior uveitis (AAU) and panuveitis are common manifestations in patients with IBD and are associated with arthritis in 25% to 33% of patients [50].

CONSEQUENCES OF THERAPY

NSAIDs

Most patients with SpA rapidly respond to treatment with nonsteroidal anti-inflammatory drugs (NSAIDs). A number of studies using different methodologies have evaluated the potential deleterious effects of NSAIDs on the small bowel and colon. Considered together, they suggest that NSAID-related intestinal injury is common. About two-thirds of NSAID users demonstrate intestinal inflammation by indirect methods of 111-Indium-labeled leukocyte scintigraphy and 111-Indium fecal excretion [51]. However, the proportion of patients who develop clinically important NSAID-induced enteropathy or colopathy remains relatively small.

The gut is a potential site for a variety of NSAID-induced injuries including erosions, ulcers, strictures, perforation, and the formation of diaphragms, which can lead to bowel obstruction [52–54]. NSAIDs can also lead to colitis resembling inflammatory bowel disease (IBD), or exacerbate preexisting IBD [55][56]. The elderly and those on long-term NSAID therapy appeared to be at the highest risk.

An autopsy study evaluated 713 patients, 249 of whom had used NSAIDs in the six months prior to death. Nonspecific small intestinal ulceration (defined as ulcers >3 mm in diameter) was much more common in those who had taken NSAIDs (8.4 versus 0.6 percent in nonusers). Three of the 21 patients with small intestinal ulcers died from perforation of an intestinal ulcer [57]. A study involving double balloon enteroscopy found that 50 percent of NSAID-users had small bowel mucosal breaks compared with only 5 percent in controls [58].

In an attempt to decrease gastroduodenal side effects, the use of enteric-coated, sustained-release, or slow-release NSAIDs may have shifted the damage to the distal small intestine and colon. A high local concentration of an active NSAID following ingestion seems to be necessary to increase


intestinal permeability, which appears to be a prerequisite for NSAID-induced enteropathy [59].

At least five studies have used video capsule endoscopy (VCE) to assess small bowel injury, two of which had a randomized design [60–64]. One controlled trial included 120 healthy volunteers who were randomly assigned to celecoxib, naproxen plus omeprazole, or placebo [60]. After two weeks from the beginning of the drug(s) ingestion, all patients underwent VCE. Significantly more mucosal breaks were observed in the naproxen/omeprazole group compared with the celecoxib group. The proportion of patients with mucosal breaks was correspondingly higher (55 versus 16 and 7 percent in the naproxen/omeprazole, celecoxib, and placebo groups, respectively). Similar conclusions were reached in a second controlled trial that compared lumiracoxib to the combination of naproxen plus omeprazole or placebo [61]. By contrast, a later non-randomized cohort study found similar rates and types of small bowel injury with long-term use of COX-2 selective agents versus nonselective NSAIDs [64]. This finding is concordant with the observation that, when the COX-2-deficient mice were developed, some of these animals died from peritoneal sepsis. This is now known to be due to ileocecal perforation [65]. Indeed, about half of these animals have evidence of increased intestinal permeability and inflammation without detectable changes in mucosal prostaglandin levels. Many of these develop ileocecal ulcers that differ both in their location and histopathologic appearance from the acute damage seen in animals receiving conventional NSAIDs short term. This unexpected finding (for the COX believers) might be attributed to some dysregulation of compensatory pathways except for the fact that selective COX-2 inhibitors given long term cause the same damage in normal animals [66].

Interestingly, another long term study, assessing the effects of long term low dose indo-methacin, showed very similar ileocecal pathology [67]. Therefore, whether the relative safety of the COX-2 selective inhibitors on gastroduodenal injury will be observed in the small and large bowel remains to be established.

At the mucosal level, postulated mechanisms of injury have included inhibition of protective prostaglandins, alterations in blood flow, and increased small intestinal permeability with subsequent invasion by luminal factors [68]. Misoprostol has been shown to be effective long

term protection against NSAID associated gastro-intestinal side effects and is thought to be the preferred agent in this respect.

Inflammation is mild and atypical at histology [69]. The mucosal damage may lead to inflammation and ulceration, followed by reparative fibrosis and eventually stricture formation (the diaphragm-like strictures are characteristic for NSAID- induced injury).

Findings on capsule endoscopy [62], double-balloon enteroscopy [70], and/or colonoscopy may lend support to the diagnosis of NSAID-induced injury, although there is nothing endoscopically specific about NSAID-induced erosions, ulcers, or colitis. Histology is also nonspecific. The differential diagnosis should thus include infectious etiologies (e.g., Campylobacter, Yersinia, cytomegalovirus, TB) and IBD [71]. Characteristically, the NSAID-induced lesions (other than strictures and diaphragms) should improve or completely resolve upon with-drawal of the drug.

AMINOSALICYLATES

Sulfasalazine, the first drug of this class, contains a 5-aminosalicylic acid (5-ASA) moiety that is linked to sulfapyridine by an azo bond and is delivered intact to the colon. On entering the colon, the azo bond is cleaved by bacterial azo-reductase, releasing sulfapyridine and 5-ASA. The sulfa-pyridine is absorbed systemically and accounts for most of the drug’s toxicity and intolerance. The 5-ASA is the active anti-inflammatory compound and ultimately is excreted in the feces. Thus, sulfasalazine is mainly active in patients with colonic disease. Newer, better-tolerated 5-ASA preparations lack the sulfapyridine moiety and are classified as agents that contain an azo bond or are delayed or sustained release.

Nearly half of spondyloarthropathy patients who receive sulfasalazine achieve a peripheral locomotor remission with this drug. It is uncertain whether 5-aminosalicylic acid preparations would have the same effect as sulfasalazine itself, because it is the sulfapyridine moiety of sulfasalazine that is the active component in patients with other arthropathies such as rheumatoid arthritis [72], while 5-ASA compounds are primarily responsible for the efficacy of sulfasalazine in IBD [73].

The precise mechanisms responsible for the clinical efficacy of 5-ASA compounds are not


known. However, in vitro investigations have identified many antiinflammatory and immuno-suppressive properties of 5-ASA, suggesting a multifactorial basis of therapeutic action [74][75].

It is well-known that sulfasalazine and the 5-ASA compounds are useful for the treatment of mild to moderate ulcerative colitis, and for maintenance of remissions [76–78].

The effective dose range of sulfasalazine (between 2 to 6 g/day), or the equivalent of olsalazine (2 to 3 g/day), Asacol (2.4 to 4.8 g/day), or Pentasa (2 to 4 g/day) induces a remission in 50 to 80 percent of patients. Those receiving higher doses had a higher complete or partial response compared to placebo.

Oral 5-ASA agents are also effective for the treatment of active Crohn’s disease and for maintenance of remission, being more effective for the treatment of Crohn’s colitis, but less useful in patients with active ileitis [79][80].

Thus, sulfasalazine and 5-ASA are likely to down-regulate inflammation present in the bowel of patients with SpA. This was confirmed in studies in SpA patients with terminal ileitis [81].

OTHER DMARDs AND CORTICOSTEROIDS

Corticosteroids are used in treating patients with PsA [82]. The DMARD (disease-modifying anti-rheumatic drug) traditionally used in PsSpA with some benefit, besides sulfasalazine, is metho-trexate (MTX), although cyclosporine and azathio-prine are also used [83]. There are some data that suggest methotrexate can be effective in some patients with AS [84].

There is, however, a paucity of data regarding the use of corticosteroids, DMARDs, and immuno-suppressive drugs in the diseases included in the concept of spondyloarthropathy. Despite this, they are commonly used in the different spondylo-arthropathies. On the other hand, there is evidence that they favorably influence the bowel inflammation, when administered in different forms of IBD [85].

BIOLOGIC AGENTS

TNF-alpha blocking antibodies (infliximab) are highly effective in the treatment of severe CD. More recently, multiple double-blind controlled studies have demonstrated the effectiveness of these biological agents in different forms of the SpA concept.

Tumour necrosis factor a (TNF-a) is one of the most critical mediators in the pathogenesis of chronic inflammation. In recent years, therapeutic strategies aimed at blocking TNF-a have shown significant efficacy in the treatment of psoriatic arthritis and Crohn’s disease [86][87].

Infliximab and adalimumab are monoclonal antibodies to TNF-alpha made up of a chimeric and, respectively, human protein that directly inhibits the action of TNF-alpha and can bind to cells expressing TNF-alpha in membrane bound form. TNF inhibition with infliximab and adalimumab in patients with Crohn’s disease resulted in improvement of gastrointestinal manifestations in close to 80 and 60 percent, respectively, even if afterwards between 30 and 40 percent of patients eventually lose this acquired response to treatment despite continuous administration of the drug [88][89], and both these drugs are also highly effective in SpAs [90–94]. Moreover, new onset or flare of IBD is an overall rare event in AS patients on anti-TNF therapy [95].

A statistically significant increase in weight after treatment with infliximab was observed in one study in patients having severe AS. Several explanations could account for this observation, including inhibition of TNF-alpha-mediated weight loss and potential recovery from occult bowel inflammation, which has been shown to be highly prevalent during the course of AS [96][97].

Although there exists the theoretic concept regarding the favorable influence of all these antiinflammatory/disease-modifying agents on the associated gut inflammation, there is a striking paucity of controlled trials regarding this issue.

It is worth mentioning that adverse events associated with infliximab include, amongst others, non-Hodgkin’s lymphoma, serious infections and opportunistic infections (including tuberculosis, histoplasmosis, coccidiomycosis), all of which having the potential to produce bowel lesions, more or less superposed with the lesions produced by the disease itself or the treatment; however, it should be appreciated that these are rare events, and even relatively large studies may lack statistical power to detect significant differences between the control and treated groups [98].

DISCUSSION

It is now recognized that SpAs and IBD are related in some of their aspects. The chronic ileal


inflammation detected on the initial biopsy from the bowel of the patients with SpAs generally predicts that the patient has an aggressive form of SpA, which might represent an indication for early intervention with anti-TNF alpha therapy. Thus, as the ileitis provides prognostic information on the course of the arthritis and can guide treatment, there is a rationale for patients with active ankylosing spondylitis to undergo ileocolonoscopic examination.

Presence of chronic gut inflammation can be the first sign of CD, and in the future, it seems possible that early treatment of the gut inflammation could prevent this evolution when the appropriate drug becomes available.

On the other hand, the extensive ileocolono-scopic studies that have been carried out in patients with SpAs, especially reactive arthritis and ankylosing spondylitis, enrolled subjects who were on or had received long-term conventional NSAIDs. And the finding was that 30 to 70% of these patients have macro- and microscopic ileitis with a variable proportion of patients having concurrent ileo-colonic inflammation. However, it is interesting that the

prevalence of the lesions and the microscopic features are very similar to those found in mice subjected to long term COX-2 inhibition or absence.

Therefore, it seems possible that the spondylo-arthropathic typhlitis represents iatrogenic COX-2 driven damage as described in animals, but taking into consideration the complex treatment strategies in patients with SpAs (including medication that also inhibits the inflammatory lesions in the bowel), it is probably very hard to prove it in specially designed controlled trials. And if this is true, then the most worrying aspect is that the severity and prognosis of the spondyloarthropathy is in part dependent on the histopathologic features of this inflammation.

Acknowledgement. This paper was supported through a research grant PN-II IDEI, contract no. 320/01.10.2007, entitled “The characterization of small bowel and colonic involvement in patients with seronegative spondylo-arthritides”, financed by the Romanian Ministry of Education and Research – Executive Unit for Financing Higher Education and Scientific University Research (MEdC – UEFISCSU).

Intestinul poate juca un rol important în patogeneza multor boli reumatice; acest lucru este adevărat şi în cazul spondilartropatiilor, studiile ileocolonoscopice efectuate demonstrând prezenţa inflamaţiei demonstrate histologic la nivelul intestinului la mai mult de jumătate din numărul pacienţilor.

Mai mult decât atât, în cazul pacienţilor afectaţi de o formă sau alta de spondilartropatie, o evoluţie către o boală inflamatorie intestinală clinic manifestă a fost remarcată la 20% din cei cu inflamaţie cronică intestinală subclinică, indicând faptul că inflamaţia articulară şi intestinală sunt oarecum legate. Prezenţa inflamaţiei intestinale cronice poate fi primul semn al unei boli Crohn, şi se speculează în acest moment că tratamentul precoce al acestei inflamaţii intestinale ar putea preîntâmpina această evoluţie, în momentul în care medicamentele necesare ar fi disponibile.

În plus, medicaţia utilizată în tratamentul acestor pacienţi poate influenţa leziunile inflamatorii intestinale, în sens negativ (AINS) sau pozitiv (medicamente modificatoare ale evoluţiei bolii reumatice, corticosteroizi, agenţi biologici), fiind communicate date care încearcă să dovedească faptul că inflamaţia ileo-colonică spondilartropatică ar reprezenta, cel puţin în parte, lezare iatrogenă iniţiată de deficitul de COX-2.

Astfel, sunt zone “obscure” în legătură cu problematica inflamaţiei intestinale la aceşti pacienţi care ar necesita cercetare aprofundată, dar luând în considerare tratamentul complex aplicat în acest tip de boli, sarcina cercetătorului nu este nicidecum uşoară.

Corresponding author: M. Rimbaş, MD.

Gastroenterology Department Clinic of Internal Medicine, Colentina Clinical Hospital 19–21, Ştefan cel Mare Street, 020125, Bucharest, Romania E-mail: [email protected]


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Involuntary Weight Loss: Case Series, Etiology and Diagnostic

C. BĂICUŞ1,3, SIMONA CARAIOLA1,3, ANDA BĂICUŞ2,3, R. TĂNĂSESCU3, M. RIMBAŞ1,3

1Colentina Hospital, Clinic of Internal Medicine, Bucharest, Romania 2INCDMI Cantacuzino Bucharest, Romania

3Réseau d’Epidémiologie Clinique International Francophone (RECIF)

Involuntary weight loss is an important clinical condition that has not been extensively covered in the medical literature. Searching MEDLINE, we identified twelve case series in different regions of the world, mostly in developed countries. Three series included ambulatory patients, while nine studied patients from secondary care hospitals. A quarter of patients with involuntary weight loss had a cancer, and many had psychiatric diseases.

Organic causes of involuntary weight loss are usually revealed by a basic evaluation, and a normal result is generally reassuring. In this case it is recommended a watchful surveillance instead of blind investigations, because the prognosis of IWL of undetermined cause is good.

On the contrary, patients with involuntary weight loss caused by cancers have a severe prognostic due to the advanced stage of the disease. On the other way, these patients are easy to diagnose.

Key words: involuntary weight loss, unintentional weight loss, cancer.

DEFINITION

The involuntary weight loss (IWL) is an important health problem, because 3–5% of the patients admitted in the Internal Medicine Department come for this problem [1–3]. In our previous study [4], more than 400 patients were admitted for involuntary weight loss during a 9 months period, and a quarter of them had cancer.

The most used definition of IWL important enough to necessitate etiological investigations is the weight loss of at least 5% of the habitual weight during the last 6–12 months [1].

However, we know that, in many instances, the patient does not know the initial weight, making the amount of weight loss impossible to compute. Considering this, it is amazing how the authors of the most series (half of them being retrospective) succeeded in the inclusion of patients, having this definition as unique criterion. Only in two studies are mentioned additional criteria in order to solve this problem: Hernandez [5] requested for proofs of weight loss represented by changes of clothes measure or confirmation by a relative/friend, while Băicuş [4] needed only weight loss to be among the first three reasons for hospitalisation. As we know, self estimation of weight loss differs from person to person, but not randomly: patients with cancer as the cause of their weight loss underestimate the amount of weight

loss, while those with a psychiatric disorder overestimate it [6]. Moreover, in one study, half of the patients referred for weight loss did not have any weight loss at all [7].

ETIOLOGY

IWL is a non-specific symptom, very common in the chronic diseases. IWL, together with asthenia, anorexia and fever are constitutional symptoms.

The most frequent causes of IWL belong to three categories of illnesses: malignant neoplasms, chronic inflammatory/infectious diseases, or psychiatric diseases; there are, also, other frequent diseases as possible etiology, as hyperthyroidism or diabetes. However, the most important step in the etiological diagnosis is the early difference between the first category and the others, in order to save the life of the patient. Meanwhile, we have no answer, yet, to the question how far have we to proceed with more and more invasive/expansive investigations on the way of the etiological diagnosis.

Generally, the patient with this condition loses his appetite, excepting a few causes of weight loss in which the appetite is normal, or even increased: un-controlled diabetes mellitus, hyperthyroidism, pheochro- mocytoma, malabsorption and dentition problems.

IWL is a clinical condition that was not well covered in the medical literature [2][3]. There are

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not evidence based protocols or algorithms for an efficient and cost-effective diagnosis and management [2], probably because IWL has many possible causes, although not so many as fever of unknown origin has [8].

Searching MEDLINE for “involuntary weight loss”, 137 articles are retrieved, while using “unintentional weight loss”, one finds 141. Among them, the vast majority are reviews and case reports, and only a few case series. Totally, directly on MEDLINE and in the references of the studies found on MEDLINE, we gathered 12 case series, 6 retrospective [4][5][7][9–11] and 6 prospective

[12–17]. A retrospective study was performed in Romania [4]. Most of the studies were done in secondary or tertiary care hospitals, excepting three [7][11][16], performed in ambulatory care. Nine studies were performed on samples of more than 100 de patients [4][5][7][9–11][14][16][17]. Concerning the geographical distribution, 5 studies were performed in the USA, two in Spain and one in each of Germany, France, Belgium, Mexico and Romania.

Among the patients admitted for IWL, a quarter have cancer, and among them, most have gastrointestinal cancer (Table I).

Table I

The most important case series of involuntary weight loss – etiology

Case series Marton 1978

Rabinovitz 1979

Huerta 1985

Thomson NS

Levine NS

Lankish 1996

Hernandez 1996

Băicuş 2003

Metalidis 2006

Country US Israel Mexico US US Germany Spain Romania Belgium Patient number 91 154 50 45 107 158 276 431 101 Cancer 20 36 10 16 6 24 38 24 22 Nonmalignant gastrointestinal diseases

14 17 16 11 6 19 10 32 15

Organic, nonmalignant

36 13 22 29 30 30 25 23 19

Psychiatric 9 10 42 20 22 11 23 12 16 Unknown 26 23 10 24 36 16 5 0 28 Mortality (months of follow-up)

25 (12)

38 (30)

NS 9 (24)

11 (15)

32 (22)

NS NS 16 (6)

From the point of view of the family

physician or gastroenterologist, if a patient with dyspepsia has the so-called “alarm symptoms” among which there is weight loss, an endoscopic evaluation must be begun, although age or the presence/absence of these alarm symptoms have a limited value in the diagnosis of gastrointestinal cancer [18][19].

Lankish [14] evaluated a two stage diagnostic algorithm for the etiology of IWL similarly to the studies concerning fever of unknown origin. He proposed, for the first stage, history and physical examination, chest radiography, electrocardiography, abdominal ultrasonography, standard laboratory tests, and tests for hyperthyroidism and occult blood in the faeces. Patients undiagnosed after the first step received secondary diagnostic tests determined on the basis of results from the first step: tests included gastroscopy, colonoscopy, faecal weight and fat estimations, and for cause of

malabsorption (duodenoscopy, secretin pancreozymin testing, and enteroclysis when necessary). During admission, the 2 step procedure achieved a diagnosis for 132 patients (84%). During follow-up, causes were determined for 7 of the 26 patients without a diagnosis from the 2 step procedure.

Other studies tried to forecast the presence of cancer as a cause of IWL, based on a score consisting of clinical and biological variables as age, erythrocyte sedimentation rate (ESR), white cell count, albumin, alkaline phosphatases and lactate dehydrogenases (LDH) [4][5][15]. Hernandez [5] found that a patient with normal serum albumin, LDH, alkaline phosphatases and white blood cells has only an 8% risk to have a cancer, while a patient with all these parameters abnormal has a 56% risk to have a cancer (the prevalence of cancer was 38% in his series), while in Baicus’ retrospective series [4], a patient with age>62 years, high ESR and anaemia had a 91% risk of

3 Involuntary weight loss 89

cancer, while a patient with age ≤62, with normal ESR and without anaemia had a risk of 8% of cancer (the prevalence of cancer was 24%).

The conclusion was that clinical examination and basic laboratory values in normal range can assure us that IWL was not caused by a serious disease [17].

PATHOGENESIS OF WEIGHT LOSS IN CANCER

It is a common place the fact that patients with cancer lose weight. An important weight loss (> 5 kg from baseline weight) is an independent factor of bad prognostic [20], and in some patients this is the direct cause of death [21].

Few studies were dedicated to the mechanisms of cachexia and they suggest a potentially important role of the cytokines and other substances whose target are the skeletal muscles.

Tumour necrosis factor-alpha (TNF)-alpha, interleukin (IL)-1 beta and IL-6 were correlated with weight loss in patients with lung cancer [22], prostate cancer with metastases [23] and pancreatic cancer [24]. In animals, on one way, the injection of any of these cytokines induced cachexia and on the other way the administration of anticytokine antibodies lessened it. All these studies were performed during the first half of the years 1990 and were not continued.

The proteolysis induction factor (PIF), a proteoglycan highlighted in a murine model of adenocarcinoma [25], was identified afterwards in the urine of the patients having cancer cachexia, unlike those without weight loss [26] or those with nonneoplastic weight loss. The results are conflicting, however, and other studies could not succeed in demonstrating a link between PIF and neoplastic cachexia in humans [27–30].

ANAEMIA IN CANCER; FERRITIN AND IRON DEFICIENCY ANAEMIA IN GASTROINTESTINAL

CANCER

In cancer, most patients may have two kinds of anaemia, that of chronic diseases and iron deficiency. While the first appears in any cancer and in inflammatory diseases that produce weight loss too, iron deficiency anaemia appears in gastrointestinal cancer due to occult haemorrhage.

In every eight patients with iron deficiency anaemia endoscopically investigated, one patient

has cancer [31–34]. Looking at the problem from the opposite side, a high proportion of the patients with gastrointestinal cancer (50%) have not anaemia when the tumour is found [35][36], so anaemia as a diagnostic test for gastrointestinal cancer is not sensitive at all.

Anaemia is a prediction factor for the neoplastic etiology of IWL in all the studies, those concerning the etiology of IWL as in those concerning the diagnosis of gastrointestinal cancer which showed, on one way, that the risk of cancer is higher in patients aged more than 50, with weight loss and iron deficiency anaemia [37][38], and on the other way that the levels of anaemia and serum iron and ferritin correlate with the dimensions, localisation and the stage of the tumour [39]. Therefore, anaemia appears only at a certain moment in the evolution of cancer and cannot be used for the early diagnosis.

In the succession of the events which lead to iron deficiency anaemia, the ferritin diminution appears early, immediately after the waning of the iron deposits in the bone marrow, and before the appearance of anaemia (and before the appearance of all the other changes from iron deficiency anaemia: diminutions of the tranferrin saturation, of serum iron, of the red cell volume and of the haemoglobin) [40], and this allows us to hope that ferritin would have a higher sensitivity for the diagnosis of the gastrointestinal cancer. In fact, ferritin is the test with the highest accuracy in the diagnosis of iron deficiency anaemia, comparing with the other enumerated parameters [41–43].

Ferritin was assessed in a few studies for the diagnosis of gastrointestinal cancer, and the results showed that it has no value as screening test [44], probably because it lowers only when the tumour is big enough [45]. However, in more than half of the patients having a ferritin less than 50 ng/ml serious lesions were found at endoscopy [46], in two patients without anaemia, a low ferritin (<18 ngm/l) lead to the discovery of colon cancer [47], and in patients with anaemia, a ferritin higher than 100 ng/ml excluded gastrointestinal cancer [48].

Ferritin was assessed in only one of the 11 studies concerning weight loss, in the most recent [17], but it showed only that ferritin was globally higher in patients with organic diseases (malignant or not) than in those without organic diseases, probably because ferritin is a marker of inflammation, too, and sometimes a neoplastic marker. However,


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ferritin was not specifically assessed for the prediction of gastrointestinal cancer.

In conclusion, in most series, approximately a quarter of patients with IWL had cancer, and a third another organic disease; the remaining had mostly a psychiatric disease, or the cause remained obscure.

An organic cause of IWL is usually revealed by a basic evaluation, and a normal evaluation is generally reassuring. Moreover, in this case it is recommended a watchful surveillance instead of blind investigations, because the prognosis of IWL of undetermined cause is good.

The patients with IWL caused by cancers have a severe prognostic (short survival time), due to the advanced stage of the disease. Therefore, they are easy to diagnose.

Acknowledgement. This paper was supported through a research grant PN-II IDEI (ID_10/2008), entitled “The assessment of the accuracy of certain biological and clinical parameters as tests for cancer diagnosis in patients with involuntary weight loss and the elaboration of a prediction model”, financed by the Romanian Ministry of Education and Research – Executive Unit for Financing Higher Education and Scientific University Research (MEdC-UEFISCSU).

Scăderea ponderală involuntară este o problemă care nu prea a fost acoperită în literatura medicală. Căutând pe MEDLINE, am găsit douăsprezece serii de cazuri din diferite regiuni ale lumii, cele mai multe din ţări dezvoltate. Trei serii au inclus pacienţi din ambulator, iar nouă au studiat pacienţi internaţi în spitale de îngrijire secundară. Un sfert dintre pacienţii cu scădere ponderală involuntară aveau cancer, şi mulţi au avut afecţiuni psihiatrice.

Cauzele organice ale scăderii ponderale involuntare sunt de obicei descoperite după o evaluare simplă, iar un rezultat normal este în general liniştitor. În acest caz se recomandă mai degrabă urmărirea decât continuarea orbească a investigaţiilor, cu atât mai mult cu cât prognosticul scăderii ponderale involuntare de cauză necunoscută este bun.

Pacienţii cu scădere ponderală determinată de cancer au, dimpotrivă, un prognostic sever, din cauza stadiului avansat al bolii. Pe de altă parte, aceşti pacienţi sunt uşor de diagnosticat.

Correspondence author: C. Băicuş

Colentina Hospital, Clinic of Internal Medicine 19–21, Şos. Ştefan cel Mare, 0202125 Bucharest, Romania E-mail: [email protected]

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CASE REPORTS

Invasive Pneumococcal Infections: Austrian Syndrome

ADRIANA HRISTEA1†, DANIELA NICOLAE2, A.I. LUKA3, RUXANDRA MOROTI CONSTANTINESCU1, VICTORIA ARAMĂ1, R. TĂNĂSESCU4†

1“Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania “Professor Dr. Matei Balş” National Institute of Infectious Diseases, Bucharest, Romania

2Târgovişte County Infectious Diseases Hospital, Romania 3“Professor Dr. Matei Balş” National Institute of Infectious Diseases, Bucharest, Romania

4“Carol Davila” University of Medicine and Pharmacy “Colentina” Clinical Hospital, Department of Neurology; Bucharest, Romania

†Réseau d’Epidémiologie Européen Francophone (RECIF)

Austrian syndrome or Osler’s triad represents an association of pneumonia, meningitis and endocarditis caused by Streptococcus pneumonia. It is a clinical rarity, with a severe prognosis, mainly due to the very aggressive evolution of endocarditis, together with the underlying pathology of the patients.

We present the case of a 54 years old female with several risk factors for invasive pneumococcal infections (post-traumatic splenectomy, type II DM and hepatic cirrhosis) that was admitted to our service with Austrian syndrome and emphasize some important clinical aspects of the disease and treatment recommendations described in the literature.

Our aim is to increase awareness and allow a correct and early treatment which would improve the high mortality observed in Austrian syndrome. We also emphasize the need for antipneumococcal vaccination, especially in high risk patients.

Key words: endocarditis, meningitis, Streptococcus pneumoniae, Austrian syndrome.

Streptococcus pneumoniae is one of the most common Gram positive pathogenic bacteria in humans and it causes infections of the middle ear, upper airways and lungs by local spread from nasopharyngeal colonization. Invasive pneumococcal infections are rare clinical occurrences which are the result of haematogenous spread to various sites leading to pneumococcal sepsis, meningitis, endo-carditis, septic arthritis, peritonitis, etc., most of them occurring in patients with predisposing factors. The morbidity and mortality associated with invasive pneumococcal infections, although it is significantly lower than in the pre-antibiotic era, is still increased, due to a certain extent by the growing prevalence of penicillin resistant strains (PRP).

Our aim is to highlight the association of pneumonia, endocarditis and meningitis caused by Streptococcus poneumoniae (described by Osler in 1881, known as Osler’s triad or Austrian syndrome) in order to increase awareness and allow a correct and early diagnosis and treatment which would improve its bad prognosis. We also emphasize the importance of antipneumococcal vaccination, especially in high risk patients.

CASE REPORT

A 54 year old female with a past medical history significant for post-traumatic splenectomy, type II diabetes mellitus and hepatic cirrhosis (following a chronic HCV infection) was admitted for fever, vomiting and right upper quadrant pain of 2 days duration.

On physical exam the patient was afebrile, oriented to time, place and person, slightly drowsy, with no meningeal syndrome, no focal neurologic signs, scleral jaundice, abdominal tenderness on palpation of the right upper quadrant, normal cardio-pulmonary exam and stable vital signs.

Blood tests showed leucocytosis with an elevated neutrophil count (WBC 32.000/mm3, neutrophils 95%), thrombocytopenia (PLT: 95.000/mm3), prothrombin concentration: 45%, positive fibrin degradation products, erythrocyte sedimentation rate: 50 mm/1h, hyperbilirubinemia (total bilirubin: 5.6 mg/dL with direct bilirubin: 3.4 mg/dl), hyperglycemia (180 mg/dL).

The chest X-ray on admission revealed a slight opacity of the base of the right lung.

Because the clinical picture was dominated by digestive symptoms, an abdominal ultrasound

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was performed, which showed a thickened gallbladder wall, possible microcalculi in the main biliary duct and dilated intrahepatic ducts.

The diagnosis of acute cholecystitis, right lower lobe pneumonia and disseminated intravascular coagulation was considered and treatment with Augmentin iv 1.2g tid, fresh frozen plasma and hydration was initiated.

On the third day after admission the patient became increasingly drowsy and meningeal syndrome developed. The cerebrospinal fluid (CSF) analysis showed: 300 leucocytes (70% neutrophils, 30% lymphocytes), albumin: 996 mg/dL, glucose: 10 mg/dL, chloride: 650mg/dL. Bacteriological analysis of the CSF revealed encapsulated Gram positive diplococci on Gram stain, positive pneumococcal antigen on latex agglutination test, but negative CSF cultures. The blood cultures were also negative.

Treatment with Ceftriaxone iv 2g bid was started, together with Mannitol and a short course of Dexamethasone, with clinical improvement of the meningeal syndrome.

On the ninth day after admission the patient developed arthritis of the left ankle and a mitral holosystolic murmur. Levofloxacin 750 mg qd was added to the treatment regimen with clinical improvement of the arthritis but progressive worsening of the mitral murmur and development of congestive heart failure. Cardiac ultrasound revealed posterior mitral valve vegetations, anterior mitral valve abscess, grade III mitral regurgitation and grade IV aortic regurgitation but no vegetations on the aortic valve (the aortic regurgitation being probably the result of a ruptured non-coronary cusp).

Valvular replacement surgery was considered, but the patient was not deemed to be a suitable surgical candidate after the cardiovascular surgery consults. At this moment Amikacin 500mg bid was associated to the previous beta-lactam and quinolone regimen (continued for 3 weeks). Antibiotic treatment was continued subsequently with Ampicillin and Levofloxacin for 3 more weeks with a favorable outcome. Before discharge the patient was vaccinated with the polysaccharidic pneumococcal vaccine.

DISCUSSION

Austrian syndrome or Osler’s triad represents an association of pneumonia, meningitis and endocarditis caused by Streptococcus pneumoniae

and it is a clinical rarity – the incidence of pneumococcal endocarditis decreased from 10–15% in the pre-antibiotic era to 1–3% [1] at present and only 0.8–3.4% of patients with pneumococcaemia develop endocarditis [2]. It still has a very high mortality, which is mainly due to the very aggressive evolution of the endocarditis (62% mortality without surgery) [3][4], together with the underlying pathology of the patients and the emergence and increasing prevalence of penicillin resistant pneumococcal strains (PRP).

Like all invasive pneumococcal infections, Austrian syndrome occurs after haematogenous dissemination of bacteria from a localized infection, the most common site being pneumonia and sinusitis.

Some well recognized predisposing factors cited in the literature are alcoholism (21–37% of cases), splenectomy (7.5% of cases), diabetes mellitus (6.3% of cases), other causes of immuno-suppression, including HIV infection (8.8% of cases), dural fistulas (18.8% of cases), ear ,nose and throat infections (sinusitis, otitis) (50% of cases). [3][5] Invasive pneumococcal infections also appear with a higher frequency in IV drug users [6] but there have been cases described in immunocompetent patients [7] as well.

Patients with HIV infection that present with pneumococcal bacteraemia have a higher mortality than non-HIV patients [8] and they also have a higher risk of being infected with penicillin resistant pneumococcal strains 29.7% vs 8.6%, in non-HIV patients [9].

Our patient had several of these risk factors (splenectomy, diabetes mellitus, hepatic cirrhosis), the most probable site for the source of the bacteremia being the pulmonary infection.

The pneumococcal endocarditis, although a rare occurrence, has an acute and extremely aggressive course, usually involving native valves (valvular or congenital heart disease present as a predisposing factor in only 12% of patients). [10] The rate of local (perforated valves, perivalvuar abscesses, ruptured cusps) and systemic complications (septic emboli, glomerulonephritis, septic arthritis) is high even with adequate antibiotics, requiring surgical treatment of the valvular lesions in the majority of cases [3][11]. Several of these complications were present in our patient (perivalvular abscess, ruptured cusps, septic arthritis). The most common valve involved is the native aortic [3][12].

3 Austrian syndrome 95

The endocarditis can also have a subacute evolution which is more frequently encountered in cases with involvement of the mitral, [7] especially in the elderly. It can also present with bivalvular involvement (mitral and aortic) [13] as was the case in our patient.

In the clinical evolution of Austrian syndrome there is often an important delay to the diagnosis of valve involvement and its presence is usually evidenced when clinical signs of valvular dysfunction and/or heart failure become evident [11][14]. Austrian reports a delay of 3 to 55 days between admission and diagnosis [14]. This delay may be due in some cases to the fact that endocarditis can appear late in the evolution of the disease, even after initiation of correct antibiotic therapy and the apparent recovery from the pneumonia and meningitis [1]. In our report the delay was 9 days, which emphasizes the necessity of early consideration of cardiac involvement in every patient with documented invasive pneumococcal infection and active seeking of early signs and symptoms of valvular dysfunction. Some authors consider performing cardiac ultrasound in patients presenting with pneumococcal pneumonia and meningitis [6] to detect as soon as possible any signs of cardiac disease.

Initiating an effective antibiotic treatment as soon as possible is crucial for the favorable outcome of the disease. Since there is an increasing emergence of PRP (44–58% in some European countries and about 24% in the USA), [1] many authors recommend initiating therapy which is active against these strains. Every effort should be made to isolate the etiologic agent and determine the antibiotic susceptibility (in particular the MIC to penicillin or cephalosporins) [7] to help guide the subsequent therapy, which is why several hemoculture should be drawn even in the afebrile patients.

Current recommendations for the empiric antibiotic treatment of PRP are cefotaxime or ceftriaxone with vancomycin [1][15]. Other empiric treatment regimens consist of combinations of rifampin with vancomycin (which should be used in cases when the strains show full resistance to cephalosporins), third generation cephalosporins or the new generation quinolones [13] (levofloxacin and moxifloxacin). Due to its limited crossing of the blood-brain barrier, vancomycin as a single agent can be used only if supplemented with intrathecal doses.

If these recommendations are followed penicillin resistance of the organism does not seem to be associated with a worse outcome [1][5][7].

The evolution of our patient was favorable with an association of ceftriaxone (later replaced by amoxicillin) and levofloxacin (that was introduced after evidence of articular involvement) in spite of the fact that we had no initial coverage for highly penicillin resistant pneumococcal strains. Unfortunately, we had no positive cultures that would have allowed us to determine the susceptibility of the strain and we maintained a combination of beta-lactam with levofloxacin for the entire duration of the treatment except for the first 9 days. Following current recommendations, the initial empiric regimen would have probably included vancomycin as well.

Corticosteroids have also been shown to have a favorable impact on the outcome of the meningitis, dexamethasone improving the mortality rates in pneumococcal meningitis from 34% to 14% and also having a favorable impact on the overall outcome as judged by the Glasgow Outcome Scale [16]. Some authors suggest that corticosteroids (hydrocortisone) can also promote hemodynamic stability in case of septic shock [17]. Clinical improvement of the meningeal syndrome was also noticed in our patient after a short course of dexamethasone together with the antibiotic treatment.

Surgical management has a major role in the treatment of pneumococcal endocarditis, significantly lowering the mortality from 62% in non-operated patients to 32% in those who undergo surgery [3]. Early valvular replacement should be considered when the aortic valve is involved to avoid septic shock and multiple organ failure alone can be adequate in subacute cases that involve the mitral valve [3][7][11].

Due to the severe evolution and high mortality of invasive pneumococcal disease and in particular of Austrian syndrome, prophylaxis should be essential in the patient groups with risk factors for invasive pneumococcal infections. The indication for prophylaxis include all patients older than 65 years and those more than 2 months of age with anatomic or functional asplenia, chronic respiratory disease, heart failure, chronic renal failure, chronic liver disease, diabetes mellitus, immunosuppression, cochlear implants, dural fistulas and children with a history of invasive pneumococcal infections.

Our patient had several of these indications (anatomic asplenia, diabetes mellitus and chronic liver disease) and yet she had not received anti-pneumococcal vaccination before this episode.

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Sindromul Austrian sau triada Osler reprezintă asocierea pneumoniei cu

meningita şi endocardita, produse de Streptococcus pneumonia. Prognosticul acestei entităţi clinice rare este sever, mai ales datorită evoluţiei foarte agresive a endocarditei, pe fondul patologiei subiacente ce favorizează această infecţie. Prezentăm cazul unei paciente în vârstă de 54 de ani cu câţiva factori de risc pentru infecţie invazivă pneumococică (splenectomie post-traumatică, diabet zaharat de tip II şi ciroză hepatică) care a fost internată cu sindrom Austrian, subliniind aspectele clinice importante ale bolii, precum şi recomandările de tratament. Scopul prezentării este să atragem atenţia asupra acestei asocieri în scopul unui diagnostic corect şi precoce, care să permită ameliorarea mortalităţii ridicate a sindromului Austrian. Subliniem totodată necesitatea vaccinării antipneumococice a pacienţilor cu factori de risc pentru infecţii pneumococice invazive.

Corresponding author: Adriana Hristea

“Professor Dr. Matei Balş” National Institute of Infectious Diseases, E-mail: [email protected]

R E F E R E N C E S

1. MUNOZ, P.J., SAINZ1 M. RODRIGUEZ-CREIXEMS et al., Austrian syndrome caused by highly penicillin-resistant Streptococcus pneumonia, Clin. Infect. Dis., 1999, 29: 000–000.

2. LINDBERG J., FANGEL S., Recurrent endocarditis caused by Streptococcus pneumoniae, Scand. J. Infect. Dis., 1999, 31(4): 409–10.

3. ARONIN S.I., MUKHERJEE S.K., WEST J.C. et al., Review of pneumococcal endocarditis in adults in the penicillin era, Clin. Infect. Dis., 1998, 26: 165–71.

4. VELAZQUEZ C., ARAJI O., BARQUERO J.M. et al., Austrian syndrome: a clinical rarity, Int. J. Cardiol., 2008, 127(2): 36–38. 5. AUBURTIN M., PORCHER R., BRUNEEL F. et al., Pneumococcal meningitis in the intensive care unit: prognostic factors of

clinical outcome in a series of 80 cases, Am. J. Respir. Crit. Care Med., 2002, 165: 713–7. 6. BEADSWORTH M.B., WOOTON D., CHENZBRAUN A. et al., Austrian’s syndrome: The first described case of

pneumococcal meningitis pneumonia and endocarditis in an injecting drug user, Eur. J. Intern. Med., 2007, 18(8): 605–6. 7. Du CHEYRON D., LESAGE A., LE PAGE O. et al., Corticosteroids as adjunctive treatment in Austrian’s syndrome

(pneumococcal endocarditis, meningitis, and pneumonia): report of two cases and review of the literature, Clin. Pathol., 2003, 56: 879–81.

8. PESOLA G.R, CHARLES A., Pneumococcal bacteremia with pneumonia. Mortality in acquired immunodeficiency syndrome, Chest., 1992, 101: 150–5.

9. CREWE-BROWN H.H., KARSTAEDT A.S., SAUNDERS G.L. et al., Streptococcus pneumoniae blood culture isolates from patients with and without human immunodeficiency virus infection: alterations in penicillin susceptibilities and in serogroups or serotypes, Clin. Infect. Dis., 1997, 25: 1165–72.

10. TAYLOR N., SANDERS V., Unusual manifestations of invasive pneumococcal infection, Am. J. Med., 1999, 107 (1A) 12S-27S. 11. LEFORT A., MAINARDI J.L., SELTON-SUTY C. et al., Streptococcus pneumoniae endocarditis in adults. A multicenter study

in France in the era of penicillin resistance (1991–1998), Medicine, 2000, 79: 327–37. 12. GRANSDEN W.R., EYKYN S.J., PHILLIPS I., Pneumococcal bacteraemia: 325 episodes diagnosed at St Thomas’s Hospital,

BMJ (Clin. Res. Ed.), 1985, 290: 505–8. 13. SIEGEL M., TIMPONE J., Penicillin-resistant Streptococcus pneumoniae endocarditis: a case report and review, Clin. Infect.

Dis., 2001, 32: 972–4. 14. AUSTRIAN R., Pneumococcal endocarditis, meningitis and rupture of the aortic valve, Arch. Intern. Med., 1957, 99: 539–44. 15. SEWLALL N.H., TIKLY M., Invasive pneumococcal infection presenting as septic arthritis and Austrian-like syndrome

involving the tricuspid valve in a patient with underlying HIV infection, Joint Bone Spine, 2005, 72: 86–8. 16. De GANS J., VAN DE BEEK D., Dexamethasone in adults with bacterial meningitis, N. Engl. J. Med., 2002, 347: 1549–56. 17. ANNANE D., SEBILLE V., CHARPENTIER C. et al., Effect of treatment with low doses of hydrocortisone and fluorocortisone

on mortality in patients with septic shock, JAMA, 2002, 288: 862–71.


Rectal Leiomyoma – Report of Two Cases Originating in Muscularis Mucosae

SABINA ZURAC1,2, IRINA TUDOSE2,, GIANINA MICU2, ALEXANDRA BASTIAN2, ELIZA GAMADA2, FLORICA STĂNICEANU1,2, CRISTIANA POPP2, D. SIMEANU2, A. HAIDAR3

1Carol Davila” University of Medicine and Pharmacy 2Department of Pathology, Colentina Clinical Hospital

3Department of Gastroenterology, Colentina Clinical Hospital, Bucharest, Romania

We present two rare cases of rectal leiomyoma originating in muscularis mucosae. The lesions were incidentally discovered in a 58 years old woman and 53 years old man during colonoscopy performed for unrelated symptoms. Both presented as small polyps and were removed by conventional colonoscopic snare polypectomy without clinical recurrence. Gross examination revealed pedunculated polyps covered by non-ulcerated mucosa. Histologically the lesions represent smooth muscle cells proliferation originating in muscularis mucosae. Both lesions were uniformly positive for SMA and DESM and negative for CD34, CD117 and S100. These tumors have a different clinical course than gastrointestinal stromal tumors; since morphologic appearance is sometimes confusing, CD117 staining is mandatory for an accurate diagnostic. We decided to present these cases because of the rarity of this condition concerning both the location and the origins of tumors. We stress the importance of the proper classification of this rare lesion, considering its outmost importance.

Key words: rectal leiomyoma, gastrointestinal stromal tumors, muscularis mucosae, CD117.

Note: Zurac and Tudose should be regarded as first author in equal contribution.

The most frequent mesenchymal tumors of gastrointestinal tract (GI) are myogenic, neurogenic and gastrointestinal stromal tumors (GIST). Myogenic tumors of GI are generally divided into benign lesions (leiomyomas) and malignant counterpart (leiomyo-sarcomas). True leiomyomas are smooth muscle neoplasms that appear more common in the upper part of GI tract and occasionally in the colon and rectum. Those arising from colon constitute only 3% of GI leiomyomas and about 1% of all GI tumors [1]. The sigmoid and transverse segments of the colon appear to be the most frequent sites of tumor occurrence in the colon [1]. There are almost 200 colonic and rectal

leiomyomas and leiomyosarcomas described in the literature, but these studies antedate the routine use of CD117 as a useful marker in distinguishing between true smooth muscle tumors and GIST [2][3].

CASES REPORT

We report two cases of rectal asymptomatic leiomyomas incidentally discovered by colonoscopy performed for leiomyoma-unrelated reasons. The clinical, colonoscopic, treatment and follow-up data are summarized in Table I.

Table I

Clinical, colonoscopic, treatment and follow-up data

Case 1 Case 2

Sex Female Male Age (yrs) 58 53 Clinical examination Unremarkable Unremarkable Lab findings Positive for blood in stool Normal Indication for colonoscopy Ocult blood in stool Colonic carcinoma in familial history Colonoscopic examination Rectal polyp (10cm from anus)

Internal haemorrhoids Rectal polyp

Treatment polypectomy by conventional colonoscopic snare

polypectomy by conventional colonoscopic snare

Follow-up Free of disease 6 months Free of disease 3 months

ROM. J. INTERN. MED., 2009, 47, 1, 97–100

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Gross examinations of the polypectomy specimen were similar: firm, pedunculated polyps covered by non-ulcerated mucosa; on the cut surface well circumscribed, yellow-grey nodules were present.

Histologically the well circumscribed nodules consist in spindle cells fascicles with elongated and cigar-shaped nuclei with perinuclear vacuolisation, minimal nuclear pleomorphism and no mitotic activity (Fig. 1). The immunoreactivity showed uniform positivity for SMA and DESM and negativity for CD34, CD117 and S100 (Figs 2 and 3). The results for negative markers were confirmed based on the presence of internal positive control (endothelial cells, mast cells, and nervous fibers, respectively). The nodules were inserted on the superficial aspect of the muscularis mucosae, completely merging with it; the tumoral nodules grew into the lamina propria of the mucosae; the overlying epithelium presented alterations consisting with mild pressure atrophy; no superficial ulceration was noted in any case. The resection margins consisted in small band of submucosal connective tissue in one case and smooth muscle fibers with orderly arrangement (parallel to the superficial epithelium) in the other case (remnants of muscularis mucosae). Based on these findings the lesions were classified as leiomyomas, completely resected.

DISCUSSION

Smooth muscle neoplasms are the second most common mesenchymal neoplasms of GI tract after GISTs. The vast majority of previously reported leiomyomas occur in the upper part of GI tract. In fact, the large bowell leiomyomas are quite rare (less than 200 cases reported to date), the rectal ones being exccedingly rare [4]. In our hospital almost 300 colonoscopic examinations are yearly performed, most of them for epithelial malignancies. The two cases of rectal leiomyoma we report here were diagnosed in 2008. These cases represent the first and only cases of endoscopically resected leiomyomas of GI tract in our department since 1996, when endoscopic techniques were available in our hospital. Since 1996 we encountered other three cases of GI leiomyomas (one solitary leiomyoma of the oesophagus, one case of oesophagus leiomyomatosis and one case of gastric leiomyoma), all of them incidentally discovered during autopsy, all of them originating from muscularis propria. No open

surgery was ever performed in our hospital for GI tract leiomyomas.

Most cases of GI tract leiomyomas are sessile intraluminal or intramural tumors that could cause bleeding, mechanical obstruction or perforation [5]. Both our cases reported here were pedunculated polyps incidentally discovered during endoscopic examination without any lesion-related symptoms. One of our patients had occasional presence of blood in stool and the colonoscopy revealed internal haemorrhoids. Considering the lack of histologically-proven ulceration of the underlying mucosa in the leiomyoma, the haemorrhoids were established as the cause of the bleeding. The other patient did not have a specific colonic semeiology and the rectal tumor was discovered pure incidentally on routine colonoscopy.

Histologically GI tract leiomyomas usually derive from the muscularis propria, tumors originating in muscularis mucosae or in the vascular system being uncommon [2]. Our cases were lesions derived from muscularis mucosae of the rectum. Considering the presence of lax, edematous or even lipomatous submucosa beneath the lesion, leio-myomas of the muscularis mucosae are easily resected. This anatomic peculiarity ensures a relatively facile resection with high degree of confidence in complete excision, the prognosis of the patient being unexpectedly favorable even in the case of a dramatic clinical onset.

The histopathologic differential diagnosis of rectal leiomyoma includes the establishing of two main issues: the leiomyomatous nature of the tumor and the benign character of the lesion (i.e. leiomyoma versus leiomyosarcoma).

The first issue requires differentiation between various spindle cell proliferations of the rectum. All these lesions are rare, but some of them are more familiar to the examiner like GIST or schwannoma, while others are very uncommon (most of them isolated case reports) as solitary fibrous tumor, inflammatory myofibroblastic tumor, endometrial stromal sarcoma or fibromatosis of the colon.

The overall frequency of sarcomas within colon and rectum is below 0.1%. GISTs (tumors originating in interstitial cells of Cajal) are malignant in almost 50% of the cases in this location, thus representing the most frequent sarcomas of the colon and rectum (the most frequent type of GISTs – the gastric counterparts – are malignant in less than one third of cases). The histopathologic appearance of the GIST is more often malignant with high cellular proliferation of

3 Rectal leiomyoma 99

spindle plump cells with elongated cigar shaped nuclei. The cellular pleomorphism is high, many atypia and frequent mitoses are present. The cells are arranged in various patterns (more frequent interlacing fascicles or whorls). Both GIST and leiomyomatous lesions may present perinuclear vacuolisation (focal or sometimes extensive) and/or epithelioid morphology. The immunohistochemical phenotype of the tumors represents the base of the differential diagnosis:

− GIST are positive for CD117 (c-kit), CD34, vimentin; almost one third of the cases present smooth muscle actin positivity and few cases (5%) are S100 protein positive; desmin is negative

− Leiomyoma/leiomyosarcoma are positive for smooth muscle actin and desmin and negative for CD117, CD34, S100 protein [6–9]

The variant of the GIST of colon with ultra-structural neural differentiation (gastrointestinal autonomic tumor – GANT) may have similar morphology as leiomyoma but the muscular markers (actin and desmin) are invariably negative [10].

Colonic schwannoma is much rare than GIST; its occurrence is not associated with neurofibromatosis of the colon. It presents as spindle cell proliferation with focal nuclear palisading and occasional cellular atypia, appearance that prone to confusion more frequent with GIST than with leiomyoma; however, since palisading may occur in various degree in leiomyomatous tumors, immuno-histochemical stainings are very useful in order to establish the correct diagnosis: schwannoma is positive for S100 protein and negative for CD117, CD34, smooth muscle actin and desmin [11].

Inflammatory myofibroblastic tumor (inflammatory pseudotumor) is a benign often recurrent lesion that may occur in the colon; its neoplastic nature is supported by clonality for cytogenic rearrangements for ALK receptor tyrosine kinase gene. Histo-pathologic appearance is that of spindle cells proliferation with mild nuclear pleomorphism and prominent nucleoli; the tumor stroma is variable infiltrated with lymphocytes and plasma cells. Cases with mild inflammatory infiltrate may be misdiagnosed as leiomyomas. Immunophenotype is not very different – as leiomyoma, inflammatory myofibroblastic tumor is positive for desmin and actin and negative for CD117, CD34, S100 protein. However, ALK is never positive in leiomyomas [12].

Solitary fibrous tumor of the colon is exceedingly rare; it was reported more often as “parasitic” tumor, arising in the mesenchymal tissue of peritoneum or retroperiotneum and subsequently adhering to the colon. It consists in a spindle cell proliferation with “patternless pattern” with immunohistochemical positivity for CD34, CD99 bcl2 and smooth muscle actin; mild focal postitivity for S100 protein and desmin was reported; the lesion is negative for CD117 [13].

Also adherent to the colonic wall, mesenteric or retroperitoneal fibromatosis consists in spindle cells proliferation arranged in fascicles. No pleo-morphism and very low mitotic activity (4 mitoses/ 50 HPF) are recorded. The tumor cells are positive for vimentin and in some cases for CD117, smooth muscle actin, desmin and beta-catenin and negative for CD34 and S100 protein. When involvement of the muscularis propria is present, the differential diagnosis is based more on the infiltrative character and overall morphology than on immunophenotype; beta-catenin is very helpful in the differential diagnosis, since it was never reported in leiomyomas, GISTs or peripheral nerve sheath tumors and only rarely in solitary fibrous tumor and endometrial stromal sarcoma [14].

Endometrial stromal sarcoma of the colon occurs as a rare event in case of colonic endo-metriosis and consists in spindle cells proliferation similar to proliferative endometrium; the tumoral cells express estrogen and progesteron receptor and CD10 while CD117, CD34, S100 protein, desmin, actin are negative [15].

The second issue, of establishing the benign or malignant character of the leiomyomatous proliferation, follows the same routine as in other organs, based on cellularity, pleomorphism, atypia, mitotic index or presence of tumor necrosis. A particular aspect should be noted concerning a benign lesion that may be parasitic – subserosal myometrial leiomyomas with secondary adherence to the colonic serosa. The lack of continuity with muscularis propria and the positivity for estrogen and progesteron receptors should settle the diagnosis. Luckily, this issue has importance for the academic aspect of the problem and not for the treatment and the prognostic of the patient. Meanwhile, the differentiation between leiomyoma and GIST has outmost importance in order to establish the proper treatment.

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Prezentăm două cazuri de leiomiom rectal cu origine în musculara mucoasei. Leziunile au fost descoperite incidental la o femeie de 58 de ani şi un bărbat de 53 de ani în cursul colonoscopiei efectuate pentru simptome fără legătură cu afecţiunea în cauză. Examenul macroscopic relevă polipi pedunculaţi acoperiţi de mucoasă neulcerată. Histologic, leziunile constau în proliferare de celule musculare netede cu punct de plecare în musculara mucoasei. Ambele leziuni au fost uniform pozitive pentru actină de muşchi neted şi desmină şi negative pentru CD34, CD117 şi S100. Aceste tumori au evoluţie clinică diferită faţă de tumorile gastrointestinale stromale; deoarece aspectul morfologic poate uneori genera confuzii, efectuarea CD117 este obligatorie pentru un diagnostic de precizie. Am decis să prezentăm aceste cazuri datorită rarităţii tumorii, atât în ceea ce priveşte localizarea, cât şi originea acestora. Subliniem importanţa clasificării corecte a acestei leziuni rare, luând în considerare importanţa deosebită a diagnosticului.

Corresponding author: Sabina ZURAC, MD

Colentina Hospital, 19–21, Şos Ştefan cel Mare 020125 Bucharest Romania Tel (004 021) 317 2354/5612 fax (004 021) 316 55 12 E-mail: [email protected]

R E F E R E N C E S

1. HUSAIN N., BOTCHU R., SCHAHABDEEN M.M., SCHOFIELD J., SOUTH L.M., Leiomyoma of the rectosigmoid junction in an adult. The Internet Journal of Surgery, 2005, 7.

2. MARTIN FERNANDEZ J., CARBAJO VINCENTE M., GARCIA ROJO M. et al., Tumors originating in the muscularis mucosae of the recto-sigma. Rev. Esp. Enferm. Dig., 1996, 88, 868–72.

3. MIETTINEN M., SARLOMO-RIKALA M., SOBIN L.H., Mesenchymal tumors of muscularis mucosae of colon and rectum are benign leiomyomas that should be separated from gastrointestinal stromal tumors – a clinicopathologic and immuno-histochemical study of eighty-eight cases. Mod. Pathol., 2001, 14, 950–6.

4. AGAIMY A., WUNSCH P.H., True smooth muscle neoplasms of gastrointestinal tract: morphological spectrum and classification in a series of 85 cases from a single institute. Langenbecks Arch. Surg., 2007, 392, 75–81.

5. CHOW W.H., KWAN W.K., NG W.F., Endoscopic removal of leiomyoma of the colon. Hong Kong, Med. J., 1997, 3, 325–7. 6. GREENSON J.K., Gastrointestinal stromal tumors and other mesenchymal lesions of the gut. Mod. Pathol., 2003, 16, 366–75. 7. HASSAN I., YOU Y.N., DOZOIS E.J. et al., Clinical, pathologic, and immunohistochemical characteristics of gastrointestinal

stromal tumors of the colon and rectum: implications for surgical management and adjuvant therapies. Dis. Colon Rectum., 2006, 49, 609–15.

8. KUHLGATZ J., SANDER B., GOLAS M.M. et al., Differential diagnosis of gastrointestinal leiomyoma versus gastrointestinal stromal tumor. Int. J. Colorectal. Dis., 2006, 21, 84–8.

9. INSABATO L., DI VIZIO D., CIANCIA G., PETTINATO G., TORNILLO L., TERRACCIANO L., Malignant gastrointestinal leiomyosarcoma and gastrointestinal stromal tumor with prominent osteoclast-like giant cells. Arch. Pathol. Lab. Med., 2004, 128, 440–3.

10. WANG B.Y., BRANDWEIN M.S., GORDON R.E., CHAHINIAN P.A., STRAUCHEN J.A., HARPAZ N., Gastrointestinal autonomic nerve tumor in the colon. Am. J. Surg. Pathol., 2002, 26, 396.

11. MIETTINEN M., SHEKITKA K.M., SOBIN L.H., Schwannomas in the colon and rectum: a clinicopathologic and immunohistochemical study of 20 cases. Am J Surg Pathol. 2001, 25, 846–55.

12. IHEDIOHA U., COLDEWEY J., MURPHY D., Inflammatory pseudotumour of the caecum: a case report. Scott. Med. J., 2004, 49, 157–8.

13. NAKATANI T., TAMADA S., IWAI Y., TANIMOTO Y., Solitary fibrous tumor in the retroperitoneum: a case with infiltrative growth. Hinyokika Kiyo, 2002, 48, 637–41.

14. EREN S., A sporadic abdominal desmoid tumour case presenting with intermittent intestinal obstruction. Eur. J. Pediatr. Surg., 2005, 15, 196–9.

15. KOVAC D., GASPAROVIĆ I., JASIC M., FUCKAR D., DOBI-BABIĆ R., HALLER H., Endometrial stromal sarcoma arising in extrauterine endometriosis: a case report. Eur. J. Gynaecol. Oncol., 2005, 26, 113–6.

Received March 9, 2009

Rectal leiomyoma--report of two cases originating in muscularis mucosae

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