romanian journal of internal medicine

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3A. 5. DEMUTH D.G., MOLLEMAN A., Cannabinoid signaling. Life Sci., 2006; 78: 549–563. 5A. 6. PAGOTTO U. ...... androstanediol glucuronide, the index of the.
ROMANIAN JOURNAL OF INTERNAL MEDICINE Volume 47

No. 1, 2009

CONTENTS REVIEWS BRÎNDUŞA DIACONU, Risk factors in chronic pancreatitis .................................................................................................... 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 ............................................................................

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ORIGINAL ARTICLES I. SPOREA, ALINA POPESCU, ROXANA ŞIRLI, MIRELA DĂNILĂ, CORINA VERNIC, Current situation of colonoscopy in Romania – 3 years of colonoscopy performance ..................................................................................... 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 ........................................................................ 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 .................................................................. D. ZDRENGHEA, DANA POP, ADELA SITAR-TĂUT, MIRELA CEBANU, V. ZDRENGHEA, Drug secondary prevention in post-menopausal women with ischemic heart disease ................................................................................ A. FRANKE, M.V. SINGER, D.L. DUMITRAŞCU, How general practitioners manage patients with irritable bowel syndrome. Data from a German Urban Area .................................................................................................................... F. CASOINIC, D. SÂMPELEAN, CĂTĂLINA BĂDĂU, LUCHIANA PRUNĂ, Nonalcoholic fatty liver disease – a risk factor for microalbuminuria in type 2 diabetic patients .................................................................................................... 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 ............................................................................. D. BODA, DIANA PĂUN, ADRIANA DIACONEASA, Evaluation of 5-α reductase activity on cultured fibroblast in patients with hyperandrogenemia .....................................................................................................................................

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POINTS OF VIEW M. RIMBAŞ, MĂDĂLINA MARINESCU, M.R.VOIOSU, Bowel lesions in spondyloarthritides ............................................ C. BĂICUŞ, SIMONA CARAIOLA, ANDA BĂICUŞ, R. TĂNĂSESCU, M. RIMBAŞ, Involuntary weight loss: case series, etiology and diagnostic ..................................................................................................................................................... ROM. J. INTERN. MED., 2009, 47, 1, 1–100

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

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

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].

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

Toxic-metabolic

Idiopathic

Genetic

Autoimmune

Alcoholic Tobacco smoking Hypercalcemia Hyperlipidemia Organotin compounds Chronic renal failure Early onset Late onset Tropical Autosomal dominant Autosomal recessive/ modifier genes 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].

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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 mitochondrial 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 earlyonset 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

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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 earlyonset 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

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chromosome 7 and proved to be the cationic trypsinogen 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 recombinant trypsinogen observed an increased autoactivation 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 nonhereditary 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

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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, lymphoplasmocytic 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

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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]

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32. WITT H., LUCK W., HENNUS H.C., CLASSEN M., KAGE A. et al., Mutations in the gene encoding the serine protease inhibitor, Kazal type I are associated with chronic pancreatitis. Nat. Genet., 2000, 25, 213. 33. MOHAN V., PREMALATHA G., PITCHUMONI M.D., Tropical Chronic Pancreatitis. J. Clin. Gastroenterol., 2003, 36, 337. 34. WHITCOMB D.C., PRESTON R.A., ASTON C.E. et al., A gene for hereditary pancreatitis maps to chromosome 7q35. Gastroenterology, 1996, 110, 1975. 35. SAHIN-TOTH M., Biochemical models of hereditary pancreatitis. Endocrinol. Metab. Clin. N. Am., 2006, 35, 303. 36. KIRALY O., BOULLING A., WITT H., LE MARECHAL C., CHEN J.M. et al., Signal peptide variants that impair secretion of pancreatic secretory trypsin inhibitor(SPINK1) cause autosomal dominant hereditary pancreatitis. Hum. Mutat., 2007; 28, 469. 37. WHITCOMB D.C., PRESTON R.A., ASTON C.E. et al., A gene for hereditary pancreatitis maps to chromosome 7q35. Gastroenterology, 1996, 110, 1975. 38. SCHNEIDER A., PFÜTZER R., BARMADA M., SLIVKA A., MARTIN J., WHITCOMB D.C., Limited contribution of the SPINK1 N34S mutation to the risk and severity of alcoholic chronic pancreatitis. A report from the United States. Dig. Dis. Sci., 2003, 48, 1110. 39. HIROTA M., KUWATA K., OHMURAYA M., OGAWA M., From acute to chronic pancreatitis: the role of mutations in the pancreatic secretory trypsin inhibitor gene. JOP2003, 4, 83. 40. PFÜTZER R., BARMADA M., BRUNSKILL A., FINCH R., HART S. et al., SPINK1/PSTI Polymorphisms act as disease modifiers in familial and idiopathic chronic pancreatitis. Gastroenterology, 2000, 119, 615. 41. NOONE P., ZHOU Z., SILVERMANN L., JOWELL P.S., KNOWLES M., COHN J., Cystic fibrosis gene mutations and pancreatitis risk: relation to epithelial ion transport and trypsin inhibitor gene mutations. Gastroenterology, 2001, 21, 1310. 42. TRUNINGER K., AMMANN R.W., BLUM H.E., WITT H., Genetic aspects of chronic pancreatitis: insights into aethiopathogenesis and clinical implications. Swiss. Med. WKLY, 2001, 131, 565. 43. OKAZAKI K., CHIBA T., Autoimmune related pancreatitis. Gut, 2002, 51, 1. 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 1 Emergency Clinical County Hospital Craiova, Diabetes, Nutrition, Metabolic Diseases Clinic University of Medicine and Pharmacy Craiova, Diabetes, Nutrition, Metabolic Diseases Department 3 University of Medicine and Pharmacy Craiova, Internal Medicine - Nephrology Department

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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) ROM. J. INTERN. MED., 2009, 47, 1, 9–18

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 6q14q15 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 hypothalamic-pituitary-adrenal axis [4]. In the hypothalamus, 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 endogenous 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].

2 ENDOCANNABINOID BIOSYNTHESIS

Anandamide is synthesized at the level of nervous tissue through a condensation reaction ATP-independent of arahidonic acid and ethanolamine [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 phosphatidylethanolamine (PE), catalyzed by phospholipase D [21–23]. At the neuronal level there are two possibilities for 2-AG biosynthesis to occur: phospholipase C mediated hydrolysis of membrane phosphorlipids 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

11

CNR1 gene’s polymorphism 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].

ADIPOSE TISSUE

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]. Endocannabinoid 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, endocannabinoids 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

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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 nonobese 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].

4

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 gastrointestinal 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 circumference 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 polymorphisms rs806381 and rs2023239 from the introns level associate with an increased BMI. Continuing the analysis of polymorphisms, the same group of researchers shows two polymorphisms 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 goodhumored 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].

13

The same (AAT)n repetition can be observed also in Spanish population [66]. The same polymorphism seems to be present at different peoples in persons with schizophrenia [67][68], Parkinson disease [80]. Hamdani et al. analyzed rs1049353 polymorphism 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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6

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

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

(AAT)n

We found no studies

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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15

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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ORIGINAL ARTICLES

Current Situation of Colonoscopy in Romania – 3 Years of Colonoscopy Performance I. SPOREA1, ALINA POPESCU1, ROXANA ŞIRLI1, MIRELA DĂNILĂ1, CORINA VERNIC2 1 Department of Gastroenterology, University of Medicine and Pharmacy Timişoara, Romania Department of Biophysics and Medical Informatics, University of Medicine and Pharmacy Timişoara, Romania

2

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, p0.05), statins 37.9% vs 53.8% (p0.05), 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 (p0.05), 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%, aspirina60.7%, 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 (p20 STATISTICS

Data analysis includes descriptive statistics calculated with commercially available software.

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

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

49

Irritable bowel syndrome

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

10-2010-20 y y 43% 43% 33%

33%

>20 y

>20 y

24%

24%