Volume 6, Issue 1, February Issue - 2018, Pages:211-219
|Authors: Zahran F, Gabr SA*, Abd El-Moneim AE, Sharoud MN,Hassanin.WF, Mesalam NM|
|Abstract: Present study has been carried out evaluate prophylactic effect of Camel milk on physiological and biochemical changes in CCl4-intoxicated rats. A total of 36 male albino rats were randomly divided into 6 groups viz. control group (G1), silymarin (SM) group (G2), camel milk (CM) group (G3), CCl4-intoxicated group (G4), silymarin prophylactic group (G5) and camel milk prophylactic group (G6). Each group has six rats. After completion of the study (8 wks), blood samples were collected and hematological parameters, liver contents of glutathione (GSH), malondialdhyde (MDA) , superoxide dismutaseactivity (SOD), serum urea, uric acid, creatinine, testosterone, estradiol were measured. The results of this study revealed that CCl4 toxicity significantly reduced Hb level, RBCs count and PCV, whereas it significantly increased the WBCs count with respect to normal control. Intoxication of CCl4 in G4 rats led to microcytic hypochromic anaemia, increased erythrocyte fragility and, leukocytosis was accompanied neutrophils increases and a decrease in lymphocyte counts. Also, CCl4 produced significant increase in serum urea, uric acid, creatinine and estradiol level and decrease in circulating testosterone level compared with normal rats. Pre-treatment with CM and SM brought significant restoration in hematological, renal function parameters and sexual hormones disturbance. Moreover, lipid peroxidation and oxidative stress were obviously noted in CCl4 intoxicated (G4) organism where a significant increase in liver MDA and decrease in its content of GSH and SOD activity. Contrarily, pre-treatment with CM and SM did not only decrease liver content of MDA but also increased hepatic GSH and SOD activity, suggesting that CM and SM attenuated CCl4-induced oxidative stress. In conclusion, CM and SM had a considerable prophylactic effectiveness against hematotoxicity, oxidative stress, lipid peroxidation, renal impairment and sexual hormones disturbances developed by intraperitoneal injection of rats withCCl4.|
|Full Text: |
Liver is the largest body organ and plays a vital role in detoxification of deleterious materials. It regulates numerous metabolic functions and maintains body homeostasis (Mayuren et al., 2010). Liver disorders are one of the most common problems throughout the world. Liver injury can be induced by certain xenobiotics and microbialin filtration via ingestion or infection (Hai et al., 2011). Carbon tetrachloride (CCl4) is well-known as xenobiotic agent. Liver is not the only the target organ of CCl4 but it also affects several body organs such as lungs, heart, testes, kidneys and brain (Ozturk et al., 2003). Evidence demonstrated that CCl4 activated highly reactive trichloromethyl radical in liver which initiates free radical mediated lipid peroxidation of cell membrane phospholipids (rich with polyunsaturated fatty acids) which are vulnerable to peroxidation. Accordingly, various functional and morphological changes are developed in liver cell membrane which caused an accumulation of lipid-derived oxidants and finally liver injury encountered (Singh et al., 2008). CCl4 is rapidly absorbed by liver tissue in humans and animals. Once it absorbed, it is widely spread among tissues, especially those with high lipid content, reaching peak concentrations in <1–6 hours, depending on exposure dose or its duration time (U.S. EPA. IRIS, 2010).
Oxidative stress and membrane damage in hepatocytes, mainly caused via CYP2E1 (Manibusan et al., 2007). Also, CCl4 alter the antioxidant profile of the liver by reacting with sulfhydryl groups of glutathione (GSH) and thiols group of protein, including the antioxidant enzymes as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione transferase (GST) (Knockaert et al., 2012; Yang et al., 2015).
Camel milk (CM) is an excellent source of well-balanced nutrients. It exhibits a range of biological activities that influence digestion, metabolism, growth and development of specific organs. These biological properties are mainly due to the presence of certain peptides and proteins in milk (Yagil et al., 1984; Korhonen & Pihlanto, 2001). Camel milk is different from other ruminant milk; it has low cholesterol, sugar and protein but high minerals such as sodium, potassium, iron, copper, zinc and magnesium. Besides this, presence of vitamins A, B2, C and E was also reported in camel milk. The presence of high insulin concentration was also reported in camel milk (Knoess, 1979). Along with this it can be consumed by lactase-deficient individuals because of non-allergic properties of camel milk. A series of metabolic and autoimmune diseases can be successfully cured by camel's milk. In India, camel's milk widely used therapeutically against dropsy, jaundice, problems of the spleen, tuberculosis, asthma, anemia, piles and diabetes (Rao et al., 1970). Further, antibacterial and antiviral activities of CM were also studied by El-Agamy et al., (1992).
Silymarin (SM), a polyphenolic flavonoid confined from milk and this is another antioxidant that has been found affective against liver injuries induced by various hepatotoxins including CCl4 (Shaker et al., 2011; Bektur et al., 2016). SM also prevents lipoperoxidation of membranes and scavenges ROS, thus increases GSH availability (Parveen et al., 2011; Vargas-Mendoza et al., 2014). The aim of this study was to investigate the prophylactic effect of camel milk and silymarin on hemotoxicity, oxidative stress, lipid peroxidation, renal function and sex hormone disturbances in CCl4-intoxicated rats.
2 MATERIALS AND METHODS
Carbontetrachloride (CCl4) obtained from LobaChemie, India. Solution of CCl4 prepared by dissolving in 50% olive oil V:V and injected intraperitoneally in to the experimental rats at a dose of 1 ml/kgb.w, once daily, 3 times weekly for four weeks to induce toxicity as described by Abdel-Moneim et al. (2015).
Raw silymarin was obtained from ElobourMedern Pharmaceutical Industries Co., Egypt. Rats were given silymarinorally at a dose of 150 mg/kgb.w suspended in distilled water (Chen et al., 2012). Recommended dose of silymarin were given once daily, 5 times in a week for 2 weeks and 3 times in a week for next 4 weeks.
Early morning, hand milking camel milk (CM) was collected daily from western desert camel farm in sterile screw capped containers and transported to the laboratory in cool boxes. CM was given to rats in a dose of 5 ml/ rat according to El Miniawy et al. (2014), once daily, 5 times in a week for 2 weeks and 3 times in a week for next 4 weeks.
2.2 Experimental animals
Total thirty six (36) adult male albino rats weighing about 180g in average were used for this study. They were selected among the animals bred in the small Animal House of the Nuclear Research Center, Atomic Energy Authority, Egypt. The animals were acclimatized for two weeks under ambient environmental conditions and housed in well aerated cages.
2.3 Animal grouping and treatment
Animals were randomly assigned into six equal groups viz. Normal control animals, without any treatment (G1), SM ingested (G2), CM drenched (G3), CCl4 intoxicated (G4), SM prophylactic group (G5 – SM treatment for first six weeks + CCl4 treatment started from fifth week of SM treatment and continue for the next 4 weeks) and CM prophylactic group (CM treatment for first six weeks + CCl4 treatment started from fifth week of CM treatment and continue for the next 4 weeks) and they were fed on a balanced rodent diet. They had free access to feed and drinking water from the beginning of the experiment until its termination
At the end of each treatment and after overnight fasting, animals were decapitated and trunk blood samples were collected in tubes with or without anticoagulant for assaying hematological indices, serum levels of renal function tests and sex hormones. Simultaneously liver was excised from each scarified rat. Promptly liver samples were rinsed in 0.1 M cold phosphate buffer (PH 7.4) and homogenized using a Teflon pestle to prepare 10% homogenates used for assessment of oxidative stress and peroxidation biomarkers.
2.4 Hematological Measurements
Blood samples were collected in EDTA coated tubes and used for determination of hematological parameters included hemoglobin (Hb) level, erythrocytes (RBCs), platelets (PLT) and leucocytes (WBCs) counts which were evaluated according to Dacie & Lewis (1993). Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated from the values of PCV, Hb and RBCs count as described by Jain (1986). For differential leucocyte counts, blood smear were stained with Giemsa.
2.5 TBARS and antioxidant enzymes assays
Lipid peroxidation biomarkers were expressed as malondialdhyde (MDA) and determined according to Satoh (1978). Reduced glutathione (GSH) was assayed as described by Beutler et al. (1963) and superoxide dismutase was measured by the procedure given by Nishikimi et al. (1972).
2.6 Levels of serum testosterone and estradiol estimation
Levels of serum testosterone and estradiol were assayed by RIA kits (10227-Czch Republic purchased from IMMUNOTECH Company) by following manufacturer’s instructions.
2.7 Renal function tests
Renal function tests were determined by following the method of Fawcett & Scott, (1960) for urea, Barham &Trinder, (1972) for uric acid and Larsen, (1972) for creatinine.
2.8 Statistical Analysis:
The obtained results were expressed as means ± standard errors. The data were subjected to F test one way analysis of variance (ANOVA) according to Snedecor & Cochran (1982) followed by Duncan’s multiple range test (Duncan, 1955) to determine the significance of difference (P≤ 0.01) between means of treated groups.
Result of study presented in table 1, revealed that administration of CCl4 had negative effect on all studied hematological parameters. Significant reduction (P< 0.01) was reported in Hb level, RBCs numbers, PCV, MCHC, PLT and lymphocyte counts. While, an improvement (P< 0.01) was reported in MCV, MCH, WBCs and neutrophil counts in CM, SM treated animals as compared the control group. Similarly, when CM and SM were used as prophylactic agents against CCl4 toxicity, they nearly succeeded to bring back the values of the above mentioned parameters toward the basal figures of normal control groups.
Regarding to lipid peroxidation and oxidative stress, data presented in table 2 denoted that IP injection of rats with CCl4 (G4) led to a significant increase (P< 0.01) in MDA content of liver. While, concentration of GSH and SOD activity were significantly (P< 0.01) reduced as compared with their corresponding values in control group. Pre- treatments of CM and SM remarkably repaired the negative effects of CCl4 on hepatic MDA and GSH whereas the activity of SOD was completely restored and the level of GSH was partially returned close to the normal values of control group.
Data are expressed as Mean ± S.E, Mean values with different letters in the same column are significantly different at P< 0.01
Table 3 Effect of camel milk and silymarin treatment on serum urea, uric acid and creatinine of normal and CCl4 intoxicated rats
Abdel Kader SM (2017) Effect of Salvia aegyptiaca Aqueous Extract on Some Neurohormonal Disorders Induced by Carbon Tetrachloride in Adult Male Albino Rats. Arab Journal of Nuclear Sciences and Applications 50: 205-216.
Abdel- Rahman M, El-Nahary H (2004) Therapeutic and protective role of panaxgensing extract on some neurotransmitters in rats treated with CCl4. Bullettin Pure and Applied. Science 23: 71-86.
Abdel-Moneim AM, Al-Kahtani MA, El-Kersh MA, Al-Omair MA (2015) Free Radical-Scavenging, Anti-Inflammatory / Anti-Fibrotic and Hepatoprotective Actions of Taurine and Silymarin against CCl4 Induced Rat Liver Damage. PLoS ONE 10(: e0144509). DOI: https://doi.org/10.1371/journal.pone.0144509.
Adewole SO, Salako AA, Doherty OW, Naicker T ( 2007) Effect of melatonin on carbon tetrachloride-induced kidney injury in Wistar rats . BMC Public Health 10: 153-64.
Al-Shamsi M, Amin A, Adeghate E (2006) Effect vof vitamin c on liver and kidney functions in normal and diabetic rats. Annals of the New York academic of Sciences 10 : 371 – 390.
Althnaian T (2012) Protective Effect of Camel Milk Against Carbon Tetrachloride Hepatotoxicity in Rats. Global Veterinaria 9 : 564-570.
Baker HJ, Lindsey JR, Weisbroth SH (1979) Housing to control research variables. In: the laboratory rats. Biological and Diseases, Academic Press, New York.
Banfi G, Del Fabbro M (2006) Relation between serum creatinine and body mass index in elite athletes of different sport disciplines. British Journal of Sports Medicine 40:675–8.
Barbagallo M, Dominguez LJ, Tagliamonte MR, Resnick LM, Paolisso G (1999) Effects of Vitamin E and Glutathione on Glucose Metabolism Role of Magnesium. Hypertension 34:1002-6.
Barham D, Trinder P (1972) An improved colour reagent for the determination of blood glucose by the oxidase system. Analyst 97: 142–145.
Bektur NE, Sahin E, Baycu C, Unver G (2016) Protective effects of silymarin against acetaminohpen-induced hepatotoxicity and nephrotoxicity in mice. Toxicology and Industrial Health 32:589-600.
Beutler E, Duron O, Kellin BM (1963) Improved method for the determination of blood glutathione.Journal of Laboratory and Clinical Medicine 61: 882–888.
Cecen E, Dost T, Culhaci N, Karul A, Ergur B, Birincioglu M (2010) Protective effects of silymarin against doxorubicin-induced toxicity. Asian Pacific Journal of Cancer Prevention 12 : 2697–704.
Chen IS, Chen YC, Chou CH, Chuang RF, Sheen LY, Chirinu CH (2012) Hepatoprotection of silymarin against thioacetamide-induced chronic liver fibrosis. Journal of the Science of Food and Agriculture 92 : 1441-7.
Churchill DN, Finn A, Gault M (1983) Association between hydrocharbon exposure and glomerulonephritis. Anaraisal of the evidence. Nephron Journal, 33: 169-172.
Curney ME, Cutting FB, Zhai P (1996) Benefit of vitamin E, riluzole and gabapentin in a transgenic model of familial amyotrophic lateral sclerosis. Annual Neurology 39: 147–157.
Dacie JV, Lewis MS (1993) In practical hematology 6th ed., ch 5, Churchill Livings ton, London and N.Y., Pp. 37.
Duncan DB (1955) Multiple range and multiple F. test. Biometrics 11,1.
El Miniawy HMF, Ahmed KA, Mansour S, Khattab MMS (2017) In vivo antitumour potential of camel’s milk against hepatocellular carcinoma in rats and its improvement of cisplatin renal side effects. Pharmaceutical Biology 55:1513-1520.
El Miniawy HMF, Ahmed KA, Tony MA, Mansour S, Khattab MMS (2014) Camel milk inhibits murine hepatic carcinogenesis, initiated by diethylnitrosamine and promoted by phenobarbitone. International Journal of Veterinary Science and Medicine 2: 136–141.
El-Agamy SI, Ruppanner R, Ismail A, Champagne CP, Assaf RJ (1992) Antibacterial and Antiviral activity of camel milk protective proteins. Journal of Dairy Research 59: 169-175.
Farah Z, Rettenmaier R, Atkins D (1992) "Vitamin content of camel milk". International journal for vitamin and nutrition research 62:30-33.
Fawcett JK, Scott JE (1960) A Rapid And Precise Method For The Determination Of Urea. Journal of Clinical Pathology 13: 156–159.
Hai ZH, Bing W, Yong KL, Yong YB, Yan GU (2011) Hepatoprotective and Antioxidant Effects of Licorice Extract against CCL4-Induced Oxidative Damage in Rats. International Journal of Molecular Sciences 12: 6529-6543.
Hamed MA, Ali SA, El-Rigal NS (2012) Therapeutic Potential of Ginger against Renal Injury Induced by Carbon Tetrachloride in Rats. The Scientific World Journal : 1-12. doi: 10.1100/2012/840421.
Ibrahim MA, Wani FA, Rahiman S (2017) Hepatoprotective effect of olive oil and camel milk on acetaminophen-induced liver toxicity in mice. International Journal of Medical Science and Public Health 6: 186-194.
Jain NC (1986) Schalm’s Veterinary Haematology 4th ed. Lea and Febiger, Philadelphia.
Khan MR, Rizvi W, Khan GN, Khan RA, Shaheen S (2009) Carbon tetrachloride-induced nephrotoxicity in rats: protective role of Digeramuricata. Journal of Ethnopharmacology 122 : 91–99.
Khan MR, Afifa M, Shabbir M, Saeed N, Jasia B (2012) Antioxidant and hepatoprotective effects of oxalis corniculata against carbon tetrachloride (CCl4.) induced injuries in rats. African Journal of Pharmacy and Pharmacology 6 : 2255-2267.
Kim SH, Yang YP, Sung SH, Kim CJ, Kim JW, Kim YC (2003) Hepatoprtective of dibenzylbuyrolactoneligans of Torreyanuciferaagainst CC14- induced toxicity in primary cultured rat hepatocytes. Biological and Pharmaceutical Bulletin 26 :1202-1205.
Knockaert L, Berson A, Ribault C, Prost PE, Fautrel A, Pajaud J, Lepage S, Lucas-Clerc C, Bégué JM, Fromenty B, Robin MA (2012) Carbon tetrachloride-mediated lipid peroxidation induces early mitochondrial alterations in mouse liver. Laboratory Investigation 92: 396–410.
Knoess KH ( 1979) Milk Production of the Dromedary. In the Proceeding of the 1st (IFS ) International Symposium on Camels, (ISC’79), Sudan, Pp:201-202.
Korhonen H, Pihlanto A (2001) Bioactive peptides opportunities for designing future foods. Current Pharmaceutical Design 9: 1297–1308.
Kraus A, Roth HP, Kirchgessner M (1997) Supplementation with vitamin C, vitamin E or beta-carotene influences osmotic fragility and oxidative damage of erythrocytes of zinc-deficient rats. Journal of Nutrition 127 : 1290-1296.
Kumarappan CT, Vhand BS, Mandal SC, Sengottuvel T (2010) Hepatoprotective effect of the poly phenolic extract from ichnocarpus fruits scens leaves. Deccan Journal of Pharmacology 1 : 1-16.
Larsen K (1972) Creatinine assay by a reaction-kinetic principle. Clinica Chimica Acta 41:209-17.
Latif R, Lodhi GM, Aslam M (2008) Effects of amlodipine on serum testosterone, testicular weight and gonado-somatic index in adult rats,” Journal of Ayub Medical College, Abbottabad 20: 8–10.
Makni M, Yassine C, Hamadi F, El Mouldi G, Mohamed B, Chama M, Choumous K, Najiba Z (2012) Erythrocyte oxidative damage in rat treated with CCl4 protective role of vanillin. Saga Journal 28: 908-916.
Mandal A, Karmakar R, Bandyopadhyay S, Chatterjee M (1998) Antihepatotoxic potential of Trianthema portulacastrum in carbon tetrachloride-induced chronic hepatocellular injury biochemical characteristics. Archives of Pharmacal Research 21: 223-230.
Manibusan MK, Odin M, Eastmond DA (2007) Postulated carbon tetrachloride mode of action: A review. Journal of Environmental Science and Health 25: 185–209.
Maxine M, Benjamin BS (1985) Outline of veterinary clinical pathology (3rdEdn) Rakha printers PVT. LTD. New Delhi.
Mayuren C, Reddy VV, Priya SV, Devi VA (2010) Protective effect of Livactine against CCl4 and paracetamol induced hepatotoxicity in adult Wistar rats. North American Journal of Medical Sciences 2:491- 495.
McDowell L (2000) Vitamins in Animal and Human Nutrition .2nd ed. Iowa State Univ. Press. Iowa, USA., Pp: 155-225.
Nishikimi M, Roa NA, Yogi K (1972) The occurrence of superoxide 728 anion in the reaction of reduced phenazinemetho sulfate and molecular oxygen. Biochemical and Biophysical Research Communications, 46: 849–854.
Omar TY, Hmmam FH (2014) Effect of Camel Milk on Hepatic Antioxidant Enzymes in Rabbits Intoxicated with Carbon Tetrachloride. International Journal of Biological & Pharmaceutical Research 5: 461-468.
Ozturk F, Ucar M, Ozturk IC, Vardi N, Batcioglu K(2003) Carbon tetrachloride-induced nephrotoxicity and protective effect of betaine in Sprage-Dawley rats. Urology 62:353-356.
Parveen R, Baboota S, Ali J, Ahuja A, Vasudev SS, Ahmad S (2011) Effects of silymarinnano emulsion against carbon tetrachloride-induced hepatic damage. Archives of Pharmacal Research 34:767–774.
Perez AJ, Courel M, Sobrado J, Gonzalez L (1987) Acute renal failure after topical application of carbon tetrachloride. Lancet 1: 515-516.
Rao MB, Gupta RC, Dastur NN (1970) Camels' milk and milk products. Indian Journal of Dairy Science 23: 71-78.
Sahreen S, Khan MR, Khan RA (2013) Ameliorating Effect of Various Fractions of Rumex hastatus Roots against Hepato- and Testicular Toxicity Caused by CCl4. Oxidative Medicine and Cellular Longevity . 2013:1-11. http://dx.doi.org/10.1155/2013/325406.
Satoh K (1978) Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clinica Chimica Acta 90:37-43.
Shaker ME, Zalata KR, Mehal WZ, Shiha GE, Ibrahim TM (2011) Comparison of imatinib, nilotinib and silymarin in the treatment of carbon tetrachloride-induced hepatic oxidative stress, injury and fibrosis. Toxicology and Applied Pharmacology 252:165–175.
Singh N, Kamath V, Narasimhamurthy K, Rajini PS (2008) Protective effect of potato peel extract against carbon tetrachlorideinduced liver injury in rats. Environmental Toxicology and Pharmacology 26 : 241-246. doi: 10.1016/j.etap.2008.05.006.
Snedecor GW, Cochran WG (1982) statistical methods. 7th ed., the lawa state university Press, Ames, Lawa, USA, Pp.215.
Soliman MM, Attia HF, Nassan MA (2016) Protective effect of camel milk on genetic and histopathological changes retated to fertility and vision in diabetic wistar rats. lucr?ri?tiin?ifice vol. 59 medicin?veterinar?partea 1 : 33-54.
Swenson MJ (1993) Physiological properties and cellular and chemical constituent of blood. In: Dukes’ Physiology of domestic animals. Comstock Publishing Associates, Ithaca and London Pp. 29-32.
Tung HT, Cook FW, Wyatt RD, Hamilton PB (1975) The anemia caused by aflatoxin. Poultry science 54: 1962-1969.
US EPA IRIS (2010) Toxicological review of carbon tetrachloride (External Review Draft). U.S. Environmental Protection Agency, Washington, DC, EPA/635/R-08/005A. Available at www.epa.gov/iris/toxreviews/0020tr.pdf accessed on 25th September 2012.
Vargas-Mendoza N, Madrigal-Santillán E, Morales-González A, Esquivel-Soto J, Esquivel-Chirino C, García-Luna Y, González-Rubio M (2014) Hepatoprotective effect of silymarin. World Journal of Hepatology 6 :144–149.
Veldink JH, Kalmijn S, Groeneveld GJ, Wunderink W, Koster A, de Vries JHM, Van der Luyt J, Wokke JHJ, Van den Berg LH (2007) Intake of polyunsaturated fatty acids and vitamin E reduces the risk of developing amyotrophic lateral sclerosis. Journal of Neurology, Neurosurgery and Psychiatry 78: 367 – 371.
Venkatanarayana G, Sudhakara G, Sivajyothi P, Indira P (2012) Protective effects of curcumin and vitamin E on carbon tetrachloride-induced nephrotoxicity in rats. EXCU Journal 11: 641-650.
Videla LA, Valenzuela A (1982) Alcohol ingestion, liver glutathione and lipoperoxidation: metabolic interrelations and pathological implications. Life Sciences - Journal 31: 2395-2407.
Yacout GA, Elguindy NM, EI Azab EF (2012) Hepatoprotective effect of basil (Ocimum basilicum L.) on CCI.-induced liver
Yagil R, Saran A, Etzion Z (1984) Camel’s milk: for drinking only? Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology 78: 263-266.
Yang BY, Zhang XY, Guan SW, Hua ZC (2015) Protective effect of procyanidin B2 against CCl4-induced acute liver injury in mice. Molecules 20: 12250–12265.
Young DS, Thomas DW, Friedman RB, Pestaner LC (1972) Effects of drugs on clinical laboratory tests. Clinical Chemistry 18:1041- 1303.
Yousef MI, Awad TI, Mohamed EH (2006) Deltamethrin – induced oxidative Damage and biochemical alterations in rat and its attenuation by Vitamin E. Toxicology 29: 240 – 247.
Youssef WI, Mullen KD (2002) The liver in other (nondiabetic) endocrine disorders. Clinical Liver Disease 6: 879-89.
Zietz B, Lock G, Plach B, Drobnik W, Grossmann J, Schölmerich J, Straub RH (2003) Dysfunction of the hypothalamic- pituitary-glandular axes and relation to Child-Pughclassification in male patients with alcoholic and virus-relatedcirrhosis. European Journal of Gastroenterology & Hepatology. 15: 495-01.