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Hyperhomocysteinemia in chronic alcoholism. Relationship to liver damage
Category 8: Nutrition, metabolism, alcoholic liver disease, pharmacology
Category 8: Nutrition, metabolism, alcoholic liver disease, pharmacology ~2-]
CHRONIC ORAL MISOPROSTOL AND HEPATIC AMINOACID-NITROGEN METABOLISM IN CIRRHOSIS
Giampaolo Bianchi 1, Andrea Fabbri 2, Nicoletta Brunetti I , Marco Zoli 1, Giulio Marchesini2. IDipartimento di Medicina Interna,
Cardioangiologia, Epatologia, University of Bologna, Bologna; 2Cattedra di Malattie del Metabolismo, Unversity of Bologna, Bologna, Italy Short-term intravenous infusion of a PGE1 analogue, reduces urea nitrogen synthesis rate (UNSR) resulting in nitrogen sparing and improves metabolic function. Ten cirrhotic patients were studied before and after 3050 days of misoprostol (400 micrograms, b.i.d.), an oral PGE1 analogue. Alpha-amino-nitrogen levels and UNSR were measured basally and in response to alanine infusion. We computed the functional hepatic nitrogen clearance (FHNC), as the slope of the regression of alpha-amino-N levels to UNSR, the alpha-amino-N corresponding to UNSR = 0, and the apparent nitrogen exchange (the balance between infused aminoacid-N and urea-N appearance rate) basally and at peak alanine concentrations. Misoprostol reduced both basal (from 23 [SD 11] mmol/hour to 5 [18]) and peak-alanine (from 135 [9] to 118 [23]; P = 0.023) UNSR. FHNC increased (from 19.9 [3.5] L/hour to 22.1 [3.3]; P = 0.03). The regression line shifted rightwards with an increase of the alpha-amino-N concentration to UNSR = 0 (from 1.16 [0.93] mmol/L to 2.05 [0.67]; P = 0.008). Misoprostol had no effects on insulin and glucagon. It significantly improved basal and peak (before: 16.2 [22.6] mmol/hour; after: 35.7 [27.2]) N exchange, mostly due to a decrease in plasma urea-N accumulation. Liver function tests improved mildly. Misoprostol increases the efficiency of the hepatic parenchyma to transform amino acids into urea. The reduced hepatic urea synthesis at any alpha-amino-N concentration indicates a metabolic state prone to nitrogen sparing; beneficial in clinical hepatology, given the increased catabolism of cirrhosis.
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THE EFFECT OF METHONINE ON BLOOD PLASMA ARGINASE ACTIVITY IN CHOLESTATIC RATS
Goran Bjelakovic 1, Aleksandar Nagorni 1, Gordana Bjelakovic 2, Gordana Kocic 2, Tatjana Jeftovic 2. 1Clinic of Gastroenterology and
Hepatology, Clinical Center Nis, Serbia; 2Institute of Biochemistry, Medical Faculty, University of Nis, Serbia, Yugoslavia Arginase (EC 3.5.3.1) is one of the essential enzymes in Krebs cycle of urea synthesis. This enzyme is present in many tissues and organs, although mammalian liver is the richest source. Providing ornithine, the chief precursor for biosynthesis of diamine putrescine, arginase is very important for polyamine biosynthesis, spermidine and spermine. Methionine as S-adenosylmethionine (SAM) is essential amino acid for polyamine biosynthesis. SAM is frequently used in the treatment of liver disease. This active form of methionine improves and normalizes liver function. (1) The aim of the study was to investigate the effect of methionine on arginase activity in blood of rats with experimental induced cholestasis. The experiment was done with 30 male Albino Wistar rats weighting 250-280 g. divided in 5 groups with 6 animals in each: control group (I), group that received methionine (II), sham operated animals (III), cholestatic animals (IV), cholestatic animals that received methionine (V). The duration of experiment was 7 days. Unoperated and cholestatic animals receive methionine through an intraperitoneal injection in daily dose of 50 mg per animal during the 7 days. Control and the sham operated animals received isotonic saline solution (0.9% NaC1) instead of the amino acid. Experimental cholestasis was done by ligation of the bile duct. At the seventh day, one hour before animals were sacrificed, the drugs were also applied.
143
After that all animals were put under ketamine anesthesia (50 mg/kg intramuscularly). Blood was collected from abdominal vein with heparin as anticoagulant. Arginase activity in plasma was measured by colorimetric determination of released ornithine. (2) There was a thirty-fold increase in arginase activity in cholestatic animals compared with control group. (29.13 to 38.35 U/L in cholestatic animals versus 0 to 3.43 U/L in control group). Administration of methionine to cholestatic animals during seven days significantly decreases arginase activity. (mean -- 10.703 U/L). Application of methionine to unoperated animals did not change arginase activity, (mean = 2.25 U/L in methionine treated animals; mean = 2.045 U/L in control group). In laparotomised animals - sham operated rats, arginase activity did not change compared with control group. The augmentation of arginase activity in plasma of cholestatic animals implicit that heptocytes eliminate great amount of arginase by bile to intestine. The decrease of arginase activity by the application of methionine in cholestatic animals confirm the studies that methionine improve the clearance of bile compounds in liver diseases with cholestasis. References
[1] Corson B. The Role of S-Adenosylmethionine in Hepatic Structure and Function. by Internet -Netscape (2001). [2] Porembska Z, Kedra M. Early diagnosis of myocardial infarction by arginase activity determination. Clin Chim Acta 1975: 3; 355-61
~47
GENETIC POLYMORPHISMS OF ALCOHOL METABOLIZING ENZYMES DO NOT INFLUENCE THE DEVELOPMENT OF ALCOHOLIC LIVER DISEASE
Carmen Blasco, Juan Cabaileria, Albert Pares, Anna Lligona, Ramon Deulofeu, Josep Lluis, Lorenzo Caballeria, Antoni Gual, Juan Rod6s. Liver and Alcohol Units, Hospital Clinic, IDIBAPS,
Barcelona, Spain It has been suggested that genetic polymorphisms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and cytochrome P450 2El (CYP2E1) could play a role in the individual susceptibility to develop alcohol related liver disease. However, these polymorphisms have been only partially studied in white individuals and with no conclusive results. The aim of the study was to investigate the influence of these polymorphisms in the development and severity of liver damage in a large group of Spanish chronic alcoholics. We have analyzed the polimorphismsof ADH2, ADH3, ALDH2 and its promoter region, and of CYP2E1 in 327 chronic alcoholics classified into 4 groups according to the degree of liver damage: normal liver (n = 120), mild or moderate lesions (n = 100), alcoholic hepatitis (n = 16) and cirrhosis (n = 91), and in 97 healthy individuals. All the allele frequencies analyzed were similar in alcoholics and in controls: ADH2* 1/* 1: 98% and 98.5%; ADH3*l/*l: 59% and 55%; ALDH2*I/*I: 99.8% and 100%; promoter region of ALDH2: 86% and 77%; CYP2Elcl/cl: 98.2% and 97.8%, respectively. Moreover, there were no significant differences in the allele frequencies among the alcoholics classified according to the severity of liver damage. These results suggest that in the Spanish population the genetic polymorphisms of alcohol metabolizing enzymes do not influence in the development and severity of liver damage.
•
HYPERHOMOCYSTEINEMIA IN CHRONIC ALCOHOLISM. RELATIONSHIP TO LIVER DAMAGE
Carmen Blasco, Juan Caballeria, Ramon Deulofeu, Albert Pares, Jose Lluis, Aria Lligona, Lorenzo Caballeria, Antoni Gual, Juan Rod6s.
Liver and Alcohol Units, Hospital Clinic, IDIBAPS, Barcelona, Spain Homocysteine (Hcy) is an amino acid that is formed as an intermediary in methionine metabolism. Impairment of Hcy remethylation or transulfuration leads to hyperhomocysteinemia. Hypermomocysteinemia is considered as a risk factor for atherosclerotic vascular disease and stroke in chronic alcoholics. The aim of the study was to investigate the serum levels
144
Poster Sessions
of Hcy and its relationship to the presence of liver damage in a group of 287 chronic alcoholics (197 males and 89 females) in which serum Hcy was measured by HPLC. 116 patients (71 males and 45 females) had a normal liver and 170 (126 males and 44 females) had varying degrees of liver damage. The age of the patients of both groups was simlar. Serum Hey was significantly higher in patients with liver damage than in those with normal liver: males 11.6 -/- 8.6 vs 7.7 + 5.6 umol/l, p < 0.001; females: 10.6 -4- 9.5 vs 6.6 :t: 2.5 umol/l, p < 0.01. Moreover, the percentage of patients with liver damage and abnormal levels of Hcy was significantly higher than the percentage of patients with normal liver (24.1% vs 7.7%, p < 0.001). There were no differences in serum Hey between active alcoholics and those with a period of abstinence. In conclusion, serum levels of Hcy are frequently elevated in chronic alcoholics, being liver damage the main cause of hyperhomocysteinemia in these patients.
~ ' ~ 7 HEPATOPROTECTIVE AND ANTIOXIDANT EFFECT OF BORAGE OIL IN RATS WITH ALCOHOLIC STEATOSIS Vyacheslav Buko, Lubov Sushko, Alex Egorov, Yurji Popov. Institute of
Biochemistry, Grodno, Belarus, Europe Aims: The aim of the present study was to evaluate the effect of the oil extracted from Borago officinalis in rat alcoholic steatosis. Methods: Two groups of rats were treated with ethanol (4 g/kg per day, i.g., 30 days). One of the group received borage oil (120 mg/kg/day, i.g., 30 days). Results: The liver of ethanol-treated animals was characterized by fatty dystrophy. Liver triglyceride and cholesterol ester contents, activities of serum marker enzyme, alanine aminotransferase and gammaglutamyltransferase, were significantly increased. Ethanol increased chemiluminescence enhanced by luminol, hydroxyalkenals, malondialdehyde and cytochrome P-450 (CYP) content in microsomes as well as superoxide dismutase activity in cytosol. The increase in monoenoic fatty acids and the decrease in n-6 acid family in liver phospholipids was observed in ethanol-treated rats. The treatment with borage oil improved liver morphology, decreased triglyceride and cholesterol ester contents, normalized serum marker enzyme activities and fatty acid pattern in liver phospholipids. Borage oil developed the antioxidant effect decreasing all the above free radical-related parameters and normalizing CYP content compared to the ethanol-treated group. Conclusions: CYP is a main source of free oxygen radicals in the liver and we proposed that the antioxidant effect of borage oil is realized via the normalization of CYP content, where borage oil can improve lipid surrounding of CYP. In turn, hepatoprotection by borage oil in alcoholic steatosis is probably connected with its antioxidant properties.
~-]
EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN RAT HEPATOCYTE CULTURES AND DOWN-REGULATION BY CYCLOSPORIN A AND FKS06 OCCUR AT DIFFERENT LEVELS
Nikolina Canova, Eva Kmonickova, Petra Strestikova, Hassan Farghali.
Institute of Pharmacology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic Aim: To compare the effect of cyclosporin A (CsA) and FK506 on the expression ofiNOS in hepatocyte cultures exposed to inflammatory signals. Method: Isolated rat hepatocytes were cultured for 24 hours with or without LPS (5 microg/ml) or cytokine mixture (CM: TNF-alpha 500 U/ml, IL-lbeta 200 U/ml, IFN-gama 100 U/ml) in the absence or presence of CsA or FK506. Total RNA was isolated and the expression of iNOS was evaluated by RT-PCR or by NO2- production in culture supernatant. Urea synthesis and ALT leakage from hepatocytes were measured. Results: LPS or CM increased iNOS mRNA that was not affected by FK506 pre-treatment while CsA pre-treatment reduced its level. CsA (0.1 and 1.0 microg/ml) reduced the LPS- and CM-induced increase of NO2- by
21% and 32% respectively, ALT leakage was increased and urea synthesis was reduced. FK506 (0.1 and 1.0 microg/ml) decreased levels of NO2- by 8% and 21%, respectively. Conclusion: In hepatocytes CsA exhibits its inhibitory effect on a phosphatase at the transcriptional level, while iNOS expression down-regulation by FK506 occurred posttranscriptional. Moreover, NO produced during inflammatory signals might be protective to hepatocytes and the inhibition of iNOS by CsA and b7(506 should be considered in practice.
•
]
EVALUATION OF THE FUNCTIONALITY AND CYCLOSPORIN A BIOTRANSFORMATION IN LABORATORY SCALE HEPATOCYTE BIOREACTORS
Nikolina Canova, Eva Kmonickova, Dana Lincova, Hassan Farghali.
Institute of Pharmacology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic Background: Apparently hepatocyte culture models are metabolically less active, due to inefficient oxygenation and waste product buildup. Since several years, we are using immobilized and perfused hepatocytes in agarose gel as a prototype of hepatocyte bioreactor in various biomedical studies. Goal: The present study was directed to assess and compare the functionality of recently described flat membrane bioreactors [1] with hepatocyte in monolayer culture. Methods: Flat membrane bioreactors were prepared from isolated rat hepatocytes and compared to hepatocytes in primary culture. The functional and metabolic competence of hepatocytes in various models were tested by urea synthesis, ALT leakage and cyclosporin A (CsA) biotransformation (HPLC-assessed) during 48-72 hours. Results: Urea synthesis increased several-fold, ALT leakage was not increased 20 hours after the operation of the bioreactors and CsA concentration decreased to 20% of the initial value in perfusate. The use of hepatocyte bioreactors demonstrated that cells are more stable in a bioreactor models than in a conventional hepatocyte culture. Conclusion: Hepatocyte bioreactors may become popular in hepatology research due to the better performance of perfused hepatocytes as compared to conventional culture. Moreover their potentials as bioartificial liver for bridged therapy are now under extensive investigation. References [1] Langsch A and Bader A: Biotechnotogy and Bioengineering, 2001; 76, 115-125.
[-~
NORMAL HEPATIC IRON BUT INCREASED HEPATIC COPPER IN NASH
Blanka Cieslarova l , Jiri Horak 1, Jan Stritesky 2, Ivana Putova 3, Vladimir Palicka 4. 1Department of Medicine I., 3rd Faculty of Medicine,
Charles University, Prague; 2Institute of Pathology, 3rd Faculty of Medicine, Charles University, Prague; 3Institute of Biology, 3rd Faculty of Medicine, Charles University,Prague; 41stitute of Biochemistry, Faculty of Medicine Charles University, Hradec Kralove, Czech Republic On the ground of medical history, clinical laboratory examination and liver biopsy the diagnosis of nonalcoholic steatohepatitis (NASH) was established in 18 patients (15 males and 3 females). Obesity, NIDDM and hyperlipidemia as risk factors for steatosis/steatohepatitis were present in this group of patients either all or in various combinations. In females the ratio of AST:ALT was 1.02-2.6, in males 0.46-2.5. Enzyme activities roughly correlated with hepatocyte damage on liver biopsy. Serological markers of iron and copper metabolism were not suggestive of hemochromatosis or Wilson's disease. Liver biopsy specimens were measured for iron (18/18) and copper (7/18) contents. The hepatic levels of iron lay between 70.31345 mikrog/g of dry liver tissue (normal value < 1400 mikrog/g) and the hepatic iron index was 0.143-0.79 (normal value < 1.0). In 8/18 patients genetic examination of the HFE gene was performed. Two out of 8 were
Category 8: Nutrition, metabolism, alcoholic liver disease, pharmacology ~2-]
CHRONIC ORAL MISOPROSTOL AND HEPATIC AMINOACID-NITROGEN METABOLISM IN CIRRHOSIS
Giampaolo Bianchi 1, Andrea Fabbri 2, Nicoletta Brunetti I , Marco Zoli 1, Giulio Marchesini2. IDipartimento di Medicina Interna,
Cardioangiologia, Epatologia, University of Bologna, Bologna; 2Cattedra di Malattie del Metabolismo, Unversity of Bologna, Bologna, Italy Short-term intravenous infusion of a PGE1 analogue, reduces urea nitrogen synthesis rate (UNSR) resulting in nitrogen sparing and improves metabolic function. Ten cirrhotic patients were studied before and after 3050 days of misoprostol (400 micrograms, b.i.d.), an oral PGE1 analogue. Alpha-amino-nitrogen levels and UNSR were measured basally and in response to alanine infusion. We computed the functional hepatic nitrogen clearance (FHNC), as the slope of the regression of alpha-amino-N levels to UNSR, the alpha-amino-N corresponding to UNSR = 0, and the apparent nitrogen exchange (the balance between infused aminoacid-N and urea-N appearance rate) basally and at peak alanine concentrations. Misoprostol reduced both basal (from 23 [SD 11] mmol/hour to 5 [18]) and peak-alanine (from 135 [9] to 118 [23]; P = 0.023) UNSR. FHNC increased (from 19.9 [3.5] L/hour to 22.1 [3.3]; P = 0.03). The regression line shifted rightwards with an increase of the alpha-amino-N concentration to UNSR = 0 (from 1.16 [0.93] mmol/L to 2.05 [0.67]; P = 0.008). Misoprostol had no effects on insulin and glucagon. It significantly improved basal and peak (before: 16.2 [22.6] mmol/hour; after: 35.7 [27.2]) N exchange, mostly due to a decrease in plasma urea-N accumulation. Liver function tests improved mildly. Misoprostol increases the efficiency of the hepatic parenchyma to transform amino acids into urea. The reduced hepatic urea synthesis at any alpha-amino-N concentration indicates a metabolic state prone to nitrogen sparing; beneficial in clinical hepatology, given the increased catabolism of cirrhosis.
~
THE EFFECT OF METHONINE ON BLOOD PLASMA ARGINASE ACTIVITY IN CHOLESTATIC RATS
Goran Bjelakovic 1, Aleksandar Nagorni 1, Gordana Bjelakovic 2, Gordana Kocic 2, Tatjana Jeftovic 2. 1Clinic of Gastroenterology and
Hepatology, Clinical Center Nis, Serbia; 2Institute of Biochemistry, Medical Faculty, University of Nis, Serbia, Yugoslavia Arginase (EC 3.5.3.1) is one of the essential enzymes in Krebs cycle of urea synthesis. This enzyme is present in many tissues and organs, although mammalian liver is the richest source. Providing ornithine, the chief precursor for biosynthesis of diamine putrescine, arginase is very important for polyamine biosynthesis, spermidine and spermine. Methionine as S-adenosylmethionine (SAM) is essential amino acid for polyamine biosynthesis. SAM is frequently used in the treatment of liver disease. This active form of methionine improves and normalizes liver function. (1) The aim of the study was to investigate the effect of methionine on arginase activity in blood of rats with experimental induced cholestasis. The experiment was done with 30 male Albino Wistar rats weighting 250-280 g. divided in 5 groups with 6 animals in each: control group (I), group that received methionine (II), sham operated animals (III), cholestatic animals (IV), cholestatic animals that received methionine (V). The duration of experiment was 7 days. Unoperated and cholestatic animals receive methionine through an intraperitoneal injection in daily dose of 50 mg per animal during the 7 days. Control and the sham operated animals received isotonic saline solution (0.9% NaC1) instead of the amino acid. Experimental cholestasis was done by ligation of the bile duct. At the seventh day, one hour before animals were sacrificed, the drugs were also applied.
143
After that all animals were put under ketamine anesthesia (50 mg/kg intramuscularly). Blood was collected from abdominal vein with heparin as anticoagulant. Arginase activity in plasma was measured by colorimetric determination of released ornithine. (2) There was a thirty-fold increase in arginase activity in cholestatic animals compared with control group. (29.13 to 38.35 U/L in cholestatic animals versus 0 to 3.43 U/L in control group). Administration of methionine to cholestatic animals during seven days significantly decreases arginase activity. (mean -- 10.703 U/L). Application of methionine to unoperated animals did not change arginase activity, (mean = 2.25 U/L in methionine treated animals; mean = 2.045 U/L in control group). In laparotomised animals - sham operated rats, arginase activity did not change compared with control group. The augmentation of arginase activity in plasma of cholestatic animals implicit that heptocytes eliminate great amount of arginase by bile to intestine. The decrease of arginase activity by the application of methionine in cholestatic animals confirm the studies that methionine improve the clearance of bile compounds in liver diseases with cholestasis. References
[1] Corson B. The Role of S-Adenosylmethionine in Hepatic Structure and Function. by Internet -Netscape (2001). [2] Porembska Z, Kedra M. Early diagnosis of myocardial infarction by arginase activity determination. Clin Chim Acta 1975: 3; 355-61
~47
GENETIC POLYMORPHISMS OF ALCOHOL METABOLIZING ENZYMES DO NOT INFLUENCE THE DEVELOPMENT OF ALCOHOLIC LIVER DISEASE
Carmen Blasco, Juan Cabaileria, Albert Pares, Anna Lligona, Ramon Deulofeu, Josep Lluis, Lorenzo Caballeria, Antoni Gual, Juan Rod6s. Liver and Alcohol Units, Hospital Clinic, IDIBAPS,
Barcelona, Spain It has been suggested that genetic polymorphisms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and cytochrome P450 2El (CYP2E1) could play a role in the individual susceptibility to develop alcohol related liver disease. However, these polymorphisms have been only partially studied in white individuals and with no conclusive results. The aim of the study was to investigate the influence of these polymorphisms in the development and severity of liver damage in a large group of Spanish chronic alcoholics. We have analyzed the polimorphismsof ADH2, ADH3, ALDH2 and its promoter region, and of CYP2E1 in 327 chronic alcoholics classified into 4 groups according to the degree of liver damage: normal liver (n = 120), mild or moderate lesions (n = 100), alcoholic hepatitis (n = 16) and cirrhosis (n = 91), and in 97 healthy individuals. All the allele frequencies analyzed were similar in alcoholics and in controls: ADH2* 1/* 1: 98% and 98.5%; ADH3*l/*l: 59% and 55%; ALDH2*I/*I: 99.8% and 100%; promoter region of ALDH2: 86% and 77%; CYP2Elcl/cl: 98.2% and 97.8%, respectively. Moreover, there were no significant differences in the allele frequencies among the alcoholics classified according to the severity of liver damage. These results suggest that in the Spanish population the genetic polymorphisms of alcohol metabolizing enzymes do not influence in the development and severity of liver damage.
•
HYPERHOMOCYSTEINEMIA IN CHRONIC ALCOHOLISM. RELATIONSHIP TO LIVER DAMAGE
Carmen Blasco, Juan Caballeria, Ramon Deulofeu, Albert Pares, Jose Lluis, Aria Lligona, Lorenzo Caballeria, Antoni Gual, Juan Rod6s.
Liver and Alcohol Units, Hospital Clinic, IDIBAPS, Barcelona, Spain Homocysteine (Hcy) is an amino acid that is formed as an intermediary in methionine metabolism. Impairment of Hcy remethylation or transulfuration leads to hyperhomocysteinemia. Hypermomocysteinemia is considered as a risk factor for atherosclerotic vascular disease and stroke in chronic alcoholics. The aim of the study was to investigate the serum levels
144
Poster Sessions
of Hcy and its relationship to the presence of liver damage in a group of 287 chronic alcoholics (197 males and 89 females) in which serum Hcy was measured by HPLC. 116 patients (71 males and 45 females) had a normal liver and 170 (126 males and 44 females) had varying degrees of liver damage. The age of the patients of both groups was simlar. Serum Hey was significantly higher in patients with liver damage than in those with normal liver: males 11.6 -/- 8.6 vs 7.7 + 5.6 umol/l, p < 0.001; females: 10.6 -4- 9.5 vs 6.6 :t: 2.5 umol/l, p < 0.01. Moreover, the percentage of patients with liver damage and abnormal levels of Hcy was significantly higher than the percentage of patients with normal liver (24.1% vs 7.7%, p < 0.001). There were no differences in serum Hey between active alcoholics and those with a period of abstinence. In conclusion, serum levels of Hcy are frequently elevated in chronic alcoholics, being liver damage the main cause of hyperhomocysteinemia in these patients.
~ ' ~ 7 HEPATOPROTECTIVE AND ANTIOXIDANT EFFECT OF BORAGE OIL IN RATS WITH ALCOHOLIC STEATOSIS Vyacheslav Buko, Lubov Sushko, Alex Egorov, Yurji Popov. Institute of
Biochemistry, Grodno, Belarus, Europe Aims: The aim of the present study was to evaluate the effect of the oil extracted from Borago officinalis in rat alcoholic steatosis. Methods: Two groups of rats were treated with ethanol (4 g/kg per day, i.g., 30 days). One of the group received borage oil (120 mg/kg/day, i.g., 30 days). Results: The liver of ethanol-treated animals was characterized by fatty dystrophy. Liver triglyceride and cholesterol ester contents, activities of serum marker enzyme, alanine aminotransferase and gammaglutamyltransferase, were significantly increased. Ethanol increased chemiluminescence enhanced by luminol, hydroxyalkenals, malondialdehyde and cytochrome P-450 (CYP) content in microsomes as well as superoxide dismutase activity in cytosol. The increase in monoenoic fatty acids and the decrease in n-6 acid family in liver phospholipids was observed in ethanol-treated rats. The treatment with borage oil improved liver morphology, decreased triglyceride and cholesterol ester contents, normalized serum marker enzyme activities and fatty acid pattern in liver phospholipids. Borage oil developed the antioxidant effect decreasing all the above free radical-related parameters and normalizing CYP content compared to the ethanol-treated group. Conclusions: CYP is a main source of free oxygen radicals in the liver and we proposed that the antioxidant effect of borage oil is realized via the normalization of CYP content, where borage oil can improve lipid surrounding of CYP. In turn, hepatoprotection by borage oil in alcoholic steatosis is probably connected with its antioxidant properties.
~-]
EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN RAT HEPATOCYTE CULTURES AND DOWN-REGULATION BY CYCLOSPORIN A AND FKS06 OCCUR AT DIFFERENT LEVELS
Nikolina Canova, Eva Kmonickova, Petra Strestikova, Hassan Farghali.
Institute of Pharmacology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic Aim: To compare the effect of cyclosporin A (CsA) and FK506 on the expression ofiNOS in hepatocyte cultures exposed to inflammatory signals. Method: Isolated rat hepatocytes were cultured for 24 hours with or without LPS (5 microg/ml) or cytokine mixture (CM: TNF-alpha 500 U/ml, IL-lbeta 200 U/ml, IFN-gama 100 U/ml) in the absence or presence of CsA or FK506. Total RNA was isolated and the expression of iNOS was evaluated by RT-PCR or by NO2- production in culture supernatant. Urea synthesis and ALT leakage from hepatocytes were measured. Results: LPS or CM increased iNOS mRNA that was not affected by FK506 pre-treatment while CsA pre-treatment reduced its level. CsA (0.1 and 1.0 microg/ml) reduced the LPS- and CM-induced increase of NO2- by
21% and 32% respectively, ALT leakage was increased and urea synthesis was reduced. FK506 (0.1 and 1.0 microg/ml) decreased levels of NO2- by 8% and 21%, respectively. Conclusion: In hepatocytes CsA exhibits its inhibitory effect on a phosphatase at the transcriptional level, while iNOS expression down-regulation by FK506 occurred posttranscriptional. Moreover, NO produced during inflammatory signals might be protective to hepatocytes and the inhibition of iNOS by CsA and b7(506 should be considered in practice.
•
]
EVALUATION OF THE FUNCTIONALITY AND CYCLOSPORIN A BIOTRANSFORMATION IN LABORATORY SCALE HEPATOCYTE BIOREACTORS
Nikolina Canova, Eva Kmonickova, Dana Lincova, Hassan Farghali.
Institute of Pharmacology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic Background: Apparently hepatocyte culture models are metabolically less active, due to inefficient oxygenation and waste product buildup. Since several years, we are using immobilized and perfused hepatocytes in agarose gel as a prototype of hepatocyte bioreactor in various biomedical studies. Goal: The present study was directed to assess and compare the functionality of recently described flat membrane bioreactors [1] with hepatocyte in monolayer culture. Methods: Flat membrane bioreactors were prepared from isolated rat hepatocytes and compared to hepatocytes in primary culture. The functional and metabolic competence of hepatocytes in various models were tested by urea synthesis, ALT leakage and cyclosporin A (CsA) biotransformation (HPLC-assessed) during 48-72 hours. Results: Urea synthesis increased several-fold, ALT leakage was not increased 20 hours after the operation of the bioreactors and CsA concentration decreased to 20% of the initial value in perfusate. The use of hepatocyte bioreactors demonstrated that cells are more stable in a bioreactor models than in a conventional hepatocyte culture. Conclusion: Hepatocyte bioreactors may become popular in hepatology research due to the better performance of perfused hepatocytes as compared to conventional culture. Moreover their potentials as bioartificial liver for bridged therapy are now under extensive investigation. References [1] Langsch A and Bader A: Biotechnotogy and Bioengineering, 2001; 76, 115-125.
[-~
NORMAL HEPATIC IRON BUT INCREASED HEPATIC COPPER IN NASH
Blanka Cieslarova l , Jiri Horak 1, Jan Stritesky 2, Ivana Putova 3, Vladimir Palicka 4. 1Department of Medicine I., 3rd Faculty of Medicine,
Charles University, Prague; 2Institute of Pathology, 3rd Faculty of Medicine, Charles University, Prague; 3Institute of Biology, 3rd Faculty of Medicine, Charles University,Prague; 41stitute of Biochemistry, Faculty of Medicine Charles University, Hradec Kralove, Czech Republic On the ground of medical history, clinical laboratory examination and liver biopsy the diagnosis of nonalcoholic steatohepatitis (NASH) was established in 18 patients (15 males and 3 females). Obesity, NIDDM and hyperlipidemia as risk factors for steatosis/steatohepatitis were present in this group of patients either all or in various combinations. In females the ratio of AST:ALT was 1.02-2.6, in males 0.46-2.5. Enzyme activities roughly correlated with hepatocyte damage on liver biopsy. Serological markers of iron and copper metabolism were not suggestive of hemochromatosis or Wilson's disease. Liver biopsy specimens were measured for iron (18/18) and copper (7/18) contents. The hepatic levels of iron lay between 70.31345 mikrog/g of dry liver tissue (normal value < 1400 mikrog/g) and the hepatic iron index was 0.143-0.79 (normal value < 1.0). In 8/18 patients genetic examination of the HFE gene was performed. Two out of 8 were