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Background & Aims Activation of hepatic stellate cells (HSC) and transdifferentiation to myofibroblasts following liver injury is the main culprit for hepatic fibrosis. Myofibroblasts show increased proliferation, migration, contraction, and production of extracellular matrix (ECM). In vitro , HMG-CoA reductase inhibitors (statins) inhibit proliferation and induce apoptosis of myofibroblastic HSC. To investigate the antifibrotic effects of atorvastatin in vivo we used bile duct ligated rats (BDL). Methods BDL rats were treated with atorvastatin (15mg/kg/d) immediately after ligation (prophylactically) or in on-going fibrosis (therapeutically). Fibrosis was assessed by hydroxyproline content and Sirius-red staining. The activation of HSC was investigated by analysis of αSMA expression. mRNA levels of cytokines and procollagen were analyzed by RT-PCR, and MMP-2 activity by zymography. Proliferation was assessed by expression of cathepsins (B and D), proliferating cell nuclear antigen (PCNA), and Ki67-staining. Apoptosis was characterized by caspase-3 activity, cleavage of PARP-1, and TUNEL assay. Hepatic inflammation was investigated by serum parameters and liver histology. Results Prophylactic and early therapy with atorvastatin significantly attenuated fibrosis and HSC activation. Later therapy lacked significant effects on fibrosis but reduced profibrotic cytokine expression and led to a more quiescent state of HSC with less proliferation and apoptosis, while hepatic inflammation did not change. Conclusions This study shows that very early atorvastatin treatment inhibits HSC activation and fibrosis in the BDL model in vivo , while late treatment reduces HSC turnover and activity. Our findings underline that long-term studies in humans are warranted.

The aim of this study was to investigate whether a decrease in carnitine body stores is a risk factor for valproic acid (VPA)-associated hepatotoxicity and to explore the effects of VPA on carnitine homeostasis in mice with decreased carnitine body stores. Therefore, heterozygous juvenile visceral steatosis (jvs)(+/-) mice, an animal model with decreased carnitine stores caused by impaired renal reabsorption of carnitine, and the corresponding wild-type mice were treated with subtoxic oral doses of VPA (0.1 g/g b.wt./day) for 2 weeks. In jvs(+/-) mice, but not in wild-type mice, treatment with VPA was associated with the increased plasma activity of aspartate aminotransferase and alkaline phosphatase. Furthermore, jvs(+/-) mice revealed reduced palmitate metabolism assessed in vivo and microvesicular steatosis of the liver. The creatine kinase activity was not affected by treatment with VPA. In liver mitochondria isolated from mice that were treated with VPA, oxidative metabolism of l-glutamate, succinate, and palmitate, as well as beta-oxidation of palmitate, were decreased compared to vehicle-treated wild-type mice or jvs(+/-) mice. In comparison to vehicle-treated wild-type mice, vehicle-treated jvs(+/-) mice had decreased carnitine plasma and tissue levels. Treatment with VPA was associated with an additional decrease in carnitine plasma (wild-type mice and jvs(+/-) mice) and tissue levels (jvs(+/-) mice) and a shift of the carnitine pools toward short-chain acylcarnitines. We conclude that jvs(+/-) mice reveal a more accentuated hepatic toxicity by VPA than the corresponding wild-type mice. Therefore, decreased carnitine body stores can be regarded as a risk factor for hepatotoxicity associated with VPA.

BACKGROUND/AIMS: It is postulated that nitric oxide (NO) is responsible for the hyperdynamic circulation of portal hypertension. Therefore, we investigated induction of fibrosis and hyperdynamic circulation in endothelial NO synthase knock-out (KO) mice. METHODS: Fibrosis was induced by bile duct ligation. Hemodynamic studies were performed after portal vein ligation. All studies were performed in wild-type (WT) and KO mice. RESULTS: Three to 4 weeks after bile duct ligation (BDL), both WT and KO groups had similar degrees of portal hypertension, 12 (9-14) and 11(8-15) mmHg, median (range), and liver function. Fibrosis increased from 0.0% in sham operated to 1.0 and 1.1% in WT and KO mice, respectively. Cardiac output was similar after portal vein ligation (20 and 17 ml/min in WT and KO mice, respectively). There was no difference in liver of mRNA for endothelin 1, inducible NO synthase (iNOS) and hem-oxygenase 1 (HO1); proteins of iNOS, HO1 and HO2; nor in endothelin A and B (EtA and EtB) receptor density between WT and KO mice after BDL. CONCLUSIONS: These results suggest that endothelial NO synthase is neither essential for the development of fibrosis and portal hypertension in bile duct ligated mice, nor for the hyperdynamic circulation associated with portal hypertension in the portal vein ligated mice.

Background/aims Nitric oxide synthase (NOS) 3-deficient (NOS-3 KO) mice have an increased systemic arterial pressure but develop portal hypertension to the same extent as wildtype (WT) mice. We hypothesized that other vasodilators in the portal circulation compensate for the lack in NOS-3 activity. We used quantitative PCR as a screening method to identify mediators that possibly compensate for NOS-3 in NOS-3 KO mice. Methods Mean arterial pressure (MAP) and portal venous pressure (PVP) were measured in the anaesthetized animal. mRNA levels in whole liver tissue were determined by quantitative RT-PCR. Results NOS-3 KO mice had a significantly higher mean arterial pressure than WT mice, but portal venous pressure did not differ. Bile duct ligation (BDL) induced a drop in MAP and a rise in PVP in both groups. Bile duct ligation induced a significant increase in mRNA levels of the cannabinoid receptor (CB)-1, adrenomedullin and NOS-2 in the liver of NOS-3 KO and WT mice. Nitric oxide synthase-1 and NOS-3 mRNA levels were elevated in BDL WT mice compared with sham-operated WT mice. Higher mRNA levels of CB-1, NOS-1 and the adrenomedullin receptor were found in sham-operated NOS-3 KO mice compared with sham-operated WT mice. Conclusions We used quantitative PCR as a screening method to identify vasodilative mediators that might be involved in the compensation for the lack of NOS-3 activity in NOS-3 KO mice. Elevated mRNA levels in sham-operated NOS-3 KO mice compared with sham-operated WT mice were demonstrated for CB-1, NOS-1 and the adrenomedullin receptor.

Background The contractile response of human splanchnic vessels to different vasoconstrictors is attenuated in cirrhosis. Functional studies indicate a cellular signalling defect upstream of the G-protein level. The aim of the present study was to analyze expression and mRNA levels of the following most relevant vasopressor receptors in the smooth musculature of human hepatic arteries: α1 adrenoceptor (AR) subtypes a, b and d, angiotensin II type 1 receptor (AT1), arginine vasopressin receptor type 1a (V1a), endothelin receptor type A (ETA) and B (ETB). Materials and methods Hepatic arteries were collected from 10 donors (noncirrhotic) and 14 recipients (cirrhotic) at liver transplantations. Real-time-PCR was performed to quantify steady-state levels of receptor mRNAs. Results α1aAR mRNA levels showed no significant difference between the cirrhotic arteries and the controls while the mRNA levels of the other vasoactive receptors were significantly higher in the cirrhotic hepatic arteries (α1bAR: 4-fold, P = 0·013; AT1: 16-fold, P = 0·024; V1a: 23-fold, P = 0·001; ETA: 4-fold, P = 0·02; ETB: 8-fold, P = 0·008). No mRNA for the α1dAR was detected either in the donor or recipient hepatic arteries. Conclusion We conclude that vascular hyporeactivity to the most relevant endogenous vasoconstrictors of cirrhotic hepatic arteries is not caused by a receptor down-regulation at mRNA levels. In contrast they were up-regulated.

Recent controlled trials on the efficacy of an amantadine/interferon combination in treatment-naive patients with chronic hepatitis C yielded contradictory results. We therefore conducted a large, double-blind, placebo-controlled, multicenter trial in naive patients with chronic hepatitis C: 246 patients were randomized to receive interferon alfa-2a (6 MIU sc thrice weekly for 20 weeks, then 3 MIU sc thrice weekly) and either amantadine sulphate (2 × 100 mg po QD) or placebo. Treatment continued for a total of 52 weeks, if HCV-RNA in serum polymerase chain reaction (PCR) had fallen below detection limit (1,000 copies/mL) at treatment week 10, and stopped otherwise. All patients were followed for 24 weeks off therapy. After 10 weeks of treatment, 66/121 patients treated with amantadine (55%) and 78/125 treated with placebo (62%) had lost HCV-RNA (n.s.). After 24 weeks of follow-up, 25 patients in the amantadine (21%) and 17 (14%) in the placebo group remained HCV-RNA negative (n.s.). During therapy, virologic breakthroughs occurred less often in the amantadine than in the placebo group [14 (12%) vs. 27 (22%) patients; P = .04]. Multivariate logistic regression analysis revealed genotype, viremia level, age, and amantadine therapy [risk ratio 0.4 (95%CI 0.2-1.0), P = .05] as predictors of sustained virologic response. Adverse events and impact of therapy on quality of life were similar in amantadine and placebo treated patients. Compared with current standard treatment (interferon/ribavirin), the interferon/amantadine combination was not cost-effective. In conclusion, amantadine does not add to a clinically relevant extent to the treatment of naive patients with chronic hepatitis C. (H EPATOLOGY .)

BACKGROUND: During clinical trials bosentan, the first orally active endothelin receptor antagonist, caused asymptomatic transaminase elevations in some patients. In this study we investigated whether inhibition of the hepatocanalicular bile salt export pump (rodents, Bsep; humans, BSEP ABCB11) could account for bosentan-induced liver injury. METHODS: We reanalyzed the safety database of the bosentan trials for cholestatic liver injury, determined the cholestatic potency of bosentan in the rat, and studied the effects of bosentan and its metabolites on Bsep-mediated taurocholate transport in vitro. RESULTS: Bosentan caused dose-dependent and reversible liver injury in 2% to 18% of patients and caused a significant increase of serum bile salt levels (P RESULTS: Bosentan caused dose-dependent and reversible liver injury in 2% to 18% of patients and caused a significant increase of serum bile salt levels (P >.01). Concomitant administration of glyburide (INN, glibenclamide) enhanced the cholestatic potency of bosentan. Similar effects were seen in rats, in which serum bile salt levels were increased by glyburide less than by bosentan, which increased the levels less than a combination of bosentan and glyburide. In vitro, Bsep-mediated taurocholate transport was inhibited by bosentan (inhibition constant, approximately 12 micromol/L) and metabolites (inhibition constant, approximately 8.5 micromol/L for metabolite Ro 47-8634). CONCLUSIONS: These results indicate that bosentan-induced liver injury is mediated, at least in part, by inhibition of Bsep/BSEP-causing intracellular accumulation of cytotoxic bile salts and bile salt induced liver cell damage. The data further emphasize the pathophysiologic importance of drug-Bsep interactions in acquired forms of cholestatic liver injury.

Several portal hypertensive animal models are available and frequently used for haemodynamic studies. The portal venous inflows, measured with microspheres in pentobarbital anaesthetised rats, are compared here. The partial portal vein ligation model is characterised by a high portal venous inflow, together with extensive portal systemic shunting, at the cost of portal sinusoidal flow. In carbon tetrachloride-induced micronodular cirrhosis, portal sinusoidal flow, which reaches liver parenchyma, is high, and this is more pronounced in the presence of ascites. In bile duct ligation and excision-induced cirrhosis, an increase in liver weight was not equally followed by an increase in portal sinusoidal flow, pointing to a relatively underperfused liver.

Background/Aims : Hepatic stellate cells (HSC) are involved in the pathogenesis of liver fibrosis; although ET-1 is increased in cirrhosis, its pathophysiological role in fibrogenesis and portal hypertension remains controversial. The aim of this study was to investigate splanchnic hemodynamics and to correlate them with changes in ET-1 expression and HSC activation in bile duct ligated (BDL) rats. Methods/Results : Expression of the ET-1 gene was increased early as measured by quantitative reverse transcriptase-polymerase chain reaction (6-fold 3 days after BDL) whereas ET-1 peptide measured by RIA increased significantly only in the late phase (30-fold at 28 days). There was a linear correlation between portal pressure and the amount of ET-1 in the portal vein ( r =0.66; P =0.003), as well as between ET-1 and the volume fraction of myofibroblasts ( r =0.80, P −7 ) as assessed by morphometry and immunohistochemical staining using α-smooth muscle actin. Conclusions : During chronic liver injury activation of HSCs and of preproET-1 mRNA is accentuated in the early phase after BDL. The late increase in ET-1 peptide may indicate that this peptide is only secondarily involved in HSC activation. The correlation between ET-1 in portal vein and portal pressure suggests that ET-1 may play an important role in the development of portal hypertension.

Increased intrahepatic vascular tone can be pharmacologically manipulated in isolated cirrhotic livers. Intrahepatic endothelial dysfunction may lead to a decreased production of the potent endogenous vasodilator nitric oxide in cirrhotic livers. The aims of the study were to determine whether portal pressure can be lowered in vivo by injecting nitric oxide donors glyceryl trinitrate or nitroprusside directly in the portal vein and whether this is related to a decrease in intrahepatic resistance.In anaesthetised CCl4 cirrhotic rats, intraportal doses of glyceryl trinitrate 0.5, 1 or 5 microg/kg/ min or nitroprusside 1, 5 or 10 microg/kg/min did not decrease portal pressure but only arterial pressure. Systemic and splanchnic haemodynamics were measured before and during 15 min intraportal infusion of glyceryl trinitrate 10 microg/kg/min or nitroprusside 20 microg/kg/min.Glyceryl trinitrate decreased portal pressure from 14.0+/-1.1 to 11.8+/-1.4 mm Hg, splanchnic perfusion pressure from 102+/-10 to 74+/-5 mm Hg and portal sinusoidal flow from 2.11+/-0.38 to 1.70+/-0.35 ml/min/g liver (all p0.05). Nitroprusside did not decrease portal pressure significantly but led to a reduction of the splanchnic perfusion pressure (104+/-9 to 66+/-7 mm Hg) and the portal sinusoidal flow (2.39+/-0.50 to 1.77+/-0.31 ml/min/g liver; all p0.05). Portal sinusoidal resistance was not altered by either drug.Intraportal infusion of nitric oxide donors decreased arterial pressure more than portal pressure. Portal sinusoidal resistance remained unaffected, but the liver parenchyma became less perfused with high doses. The systemic effects of nitric oxide donating drugs prevailed.

Whereas early acute cellular rejection, even if successfully treated, seems to have an impact on late function and survival of kidney and heart transplants, little quantitative data are available on its effect(s) on liver transplants. Routine liver function tests, the functioning liver cell mass (galactose elimination capacity) and microsomal metabolic capacity (aminopyrine breath test) were determined prospectively in 37 consecutive patients 1 year after liver transplantation. Of these, 19 (7 females and 12 males, 32-69 years of age) had previously required treatment for at least one biopsy proven acute cellular rejection episode occuring a median 7 days after grafting, while 18 (6 females and 12 males, 30-67 years of age) had not. The functioning liver cell mass and microsomal metabolic capacity were both within normal limits for the majority of patients and did not differ significantly between patients with and without previous acute cellular rejection episodes. In contrast to other solid organ transplants, early acute cellular rejection episodes do not affect late function of liver allografts in man.

Background/Aims: In vivo studies have shown that arterial vasodilation induced by synthetic openers of ATP-sensitive K + (K ATP ) channels is decreased in rats with cirrhosis. Since vasodilation induced by these substances is mediated by membrane potential hyperpolarization in arterial smooth muscle cells, membrane potential hyperpolarization in response to K ATP channel openers may be altered in cirrhotic smooth muscle cells. The aim of the present study was to investigate the effects of K ATP channel modulators (i.e. openers and blockers of these channels) on the membrane potential in smooth muscle cells in isolated aortae from cirrhotic and normal rats. The influence of endothelin-1 production by endothelial cells on smooth muscle cells membrane potential responses to K ATP channel modulators was also studied. Methods: Cells were impaled in situ (in intact and endothelium-denuded aortae) with a microelectrode that was used to measure membrane potentials. K ATP channel openers were diazoxide or cromakalim; blockers were glibenclamide or tolbutamide. Bosentan (a mixed endothelin receptor antagonist) and exogenous endothelin-1 were also used. Preproendothelin-1 mRNA was assayed in aortae by RNase protection assay. Aortic wall endothelin-1 concentration was measured by double antibody radioimmunoassay technique. Results: As expected, in smooth muscle cells in intact normal aortae, K ATP channel openers induced membrane potential hyperpolarization and K ATP channel blockers membrane potential depolarization. In smooth muscle cells in intact cirrhotic aortae, K ATP channel openers and blockers did not significantly change the membrane potential. Endothelium removal or exposure of intact aortae to bosentan restored normal membrane potential responses to K ATP channel modulators in cirrhotic smooth muscle cells and did not alter the effects of these substances in normal smooth muscle cells. In endothelium-denuded aortae, exposure to exogenous endothelin-1 suppressed membrane potential responses to K ATP channel modulators. In intact aortae, the abundance of preproendothelin-1 mRNA and endothelin-1 did not significantly differ between normal and cirrhotic rats. Conclusions: K ATP channel opener-induced membrane hyperpolarization and K ATP channel blocker-elicited membrane depolarization are blunted in smooth muscle cells in intact cirrhotic aortae. This blunting is due to the activation of the endothelin-1 pathway in the aortic wall, downstream to the endothelial production of endothelin-1.

Most authors reporting on hepatectomy stress the importance of preoperative assessment of liver function and functional reserve to minimize the surgical risk, especially in patients with liver cirrhosis, jaundice or those undergoing prolonged chemotherapy [1] since it is these patients who have a markedly increased risk of developing liver failure [2–5]. According to a recent review, liver failure is the major cause of death after hepatectomy followed in importance by intraoperative and postoperative hemorrhage and sepsis accounting for 38, 29 and 21% of deaths, respectively [6]. For this reason it is of paramount importance to estimate liver function preoperatively and to predict postoperative remaining functional liver parenchymal mass and its reserve.

Naproxen covalently linked to human serum albumin (NAP-HSA) is efficiently targeted to endothelial and Kupffer cells of the liver and may offer a new therapeutic approach in the treatment of liver disease associated with inflammatory processes. In the present investigation we explored the pharmacokinetic behaviour of targeted and non-targeted naproxen as well as the pharmacokinetic properties of the active metabolite, Naproxen lysine (Nap lysine), in rats rendered fibrotic by bile duct ligation (BDL) for 4 weeks. Furthermore, we studied the effect of endotoxemia, experimentally induced by intravenous injection of 800 microg/kg lipopolysaccaride (LPS) upon the pharmacokinetics of these agents in order to investigate the feasibility of targeting naproxen to non-parenchymal cells in the inflamed and fibrotic liver. Our studies demonstrate that liver disease altered the pharmacokinetic behaviour of the different naproxen compounds. Thus, initial plasma concentrations of NAP HSA and naproxen were markedly lower in BDL rats accompanied by an increase of the volume of distribution during the terminal elimination phase (Vd(beta) BDL vs control 114 +/- 63 vs 50 +/- 7 and 202 +/- 24 vs 115 +/- 11 ml/kg for naproxen and NAP-HSA, respectively). After injection of LPS, no significant change in the pharmacokinetics of NAP-HSA was found whereas the naproxen treated control animals showed an increase in the terminal volume of distribution (176 +/- 34 vs 115 +/- 11 ml/kg) as well as an elevation of the plasma half-life (171 +/- 27 vs 116 +/- 14 min). The feasibility of targeting naproxen to the chronically diseased liver could be clearly demonstrated: 15 min after administration of the conjugate 46% and 55% of the administered dose was found in the liver of CTR and BDL rats, whereas after injection of free naproxen only 5% and 12% of the dose was detected in liver tissue, respectively. We conclude that targeting albumin-linked naproxen to non-parenchymal cells in the liver is still feasible under the pathological conditions induced in the present study. Liver fibrosis induced significant alterations in the pharmacokinetic behaviour of the studied compounds.

Bosentan has lately been described as a successful therapeutic agent for portopulmonary hypertension consecutive to child A cirrhosis. This is the first report of the effect of this substance with advanced liver cirrhosis (child C) and renal insufficiency. Low doses of bosentan (initially twice 31.25 mg/day and then 62.5 mg/day) increased cardiac output and allowed correction of renal insufficiency; it allowed one to stop the requirement of oxygen and not only improved the 6-min walking test by more than 400 m, but also decreased the severity of the liver cirrhosis to child B stadium. This suggests that patients, who would be excluded from a liver transplantation program because of their portopulmonary hypertension, could profit from a careful therapy with bosentan.

Rationale and Objectives The authors investigated the usefulness of dynamic phosphorus-31 magnetic resonance (MR) spectroscopy in the assessment of hepatic function by studying the effect of a fructose load on a rat model of liver cirrhosis. Methods In vivo P-31 MR liver spectra of eight rats with bile duct ligature and 10 control rats were obtained every 4.6 minutes before and after intraperitoneal fructose load (10 mmol per kilogram of body weight). Results In the basal spectra of the experimental group, the phosphomonoester peak was higher than in the control group (P = .026). After the fructose load, the phosphomonoester peak increase and the inorganic phosphate peak decrease were significantly less marked in the experimental group (P = .003). There was a linear correlation between the serum level of bilirubin and the phosphomonoester increase (r = .61, P Conclusion Dynamic P-31 MR spectroscopy may be useful in the assessment of hepatic function in chronic liver disease.

In vivo 31Phosphorus magnetic resonance spectroscopy (31P-MRS) permits evaluation of dynamic changes of individual phosphorus-containing metabolites in the liver parenchyma, such as phosphomonoester (PME), adenosine triphosphate, and inorganic phosphate (Pi). Intravenous fructose load alters phosphorus metabolites and allows assessment of liver function by 31P-MRS. 31P-MRS data obtained in alcoholic liver disease are however inconclusive. To study the hypothesis that fructose load can be used to investigate metabolic effects of ethanol ingestion, the interaction of different metabolites--i.e., fructose and ethanol--were followed in vivo. Using a 1.5 Tesla magnetic resonance system, six healthy volunteers were examined in three sessions each: a session after administration of (a) fructose only (250 mg/kg) was compared with (b) fructose load after ethanol ingestion (0.8 g/kg). A control experiment (c) was done after ethanol only. Spectra were acquired using one-dimensional chemical shift imaging with a temporal resolution of 5 min. Following a fructose load, the concomitant uptake of ethanol showed drastic changes of individual metabolic steps of the hepatic metabolism (averages +/- standard deviation). While the velocity of the net formation of PME (relative increase 0.46 +/- 0.11 without ethanol vs. 0.61 +/- 0.25 with ethanol) and the use of adenosine triphosphate (-0.13 +/- 0.03 vs. -0.16 +/- 0.03) and Pi (-0.022 +/- 0.009 vs. -0.021 +/- 0.004) were not significantly affected by ethanol uptake, a significant (p < 0.01) reduction of PME degradation (31.3 +/- 9.4 vs. 61.9 +/- 16.9 relative total area) and absence of an overshoot for Pi (10.5 +/- 4.9 vs. -7.1 +/- 5.3 relative area 13 min to 43 min) was observed after ethanol administration. Dynamic 31P-MRS allows the observation of individual steps of hepatic metabolism in situ; fructose metabolism in the human liver is slowed down by concomitant ethanol ingestion after the phosphorylation step of fructose. This could be explained by inhibition of aldolase rather than ethanol-induced changes of the hepatic redox state. Fructose load can be used to study effects of alcohol ingestion and might therefore be useful in patients with alcoholic liver disease.

Background/Aim: Interferon has become the mainstay of treatment of chronic hepatitis C; however, duration of treatment and dose remain unresolved questions. The present study aimed to compare standard dose interferon with a titrated dose regimen carried out for 1 year. Methods: This was a randomized, controlled multicenter trial. Patients with chronic hepatitis C were randomly allocated to a control group ( n =30), to a fixed dose group ( n =31) where interferon-α 2b 3 MU thrice weekly was given for 1 year or a titrated group ( n =34) where interferon was titrated starting at 5 MU thrice weekly to the lowest dose keeping the patient in remission as assessed by a normal ALT value. Liver biopsies were obtained before and at the end of treatment; in addition, galactose elimination capacity was measured as a measure of cytosolic function. Results: In the control, fixed an titrated groups a complete response was achieved in 229, 1028 and 1531, respectively ( p p =n.s. for the two treatments). The corresponding figure for sustained response was 129, 528 and 631 ( p =n.s.). In the titrated group, a complete (sustained) response was achieved with 5 MU in 2 (2), with 4 MU in 1 (0), with 3 MU in 4 (0), with 2 MU in 3 (0) and with 1 MU in 5 (4). Liver biopsy score and galactose elimination capacity improved significantly in responders but not in treatment failures. Conclusions: Both fixed and titrated dosing of interferon given for 1 year induced virus clearance in only a minority of treated patients. However, in a small number of patients, a complete and sustained response can be achieved with low doses of interferon. Dose titration could be an interesting approach to decreasing the cost and side effects in the treatment of chronic hepatitis C.

We here show the application of mRNA differential display to investigate changes in gene expression in rat liver cirrhosis and address problems inherent in the technique when applied to this complex disease model. A number of differentially expressed mRNA species could be identified and two were analyzed in more detail here. One was found to derive from a new gene while the other corresponded to fetuin, a 41 kDa N-glycoprotein that specifically inhibits tyrosine kinase activity of the insulin receptor when phosphorylated. Fetuin expression was reduced by 45% in liver cirrhosis induced by bile duct ligation, but not in cirrhosis induced by carbon tetrachloride/Phenobarbital, as compared to controls. Our results raise the possibility that fetuin plays a regulatory role in the proliferation of parenchymal liver cells.

Albumin and fibrinogen synthesis rates were measured in 15 subjects with different clinical stages of postviral cirrhosis and compared with galactose elimination capacity and aminopyrin breath test. Forty-three mg per kg body weight [ 2 H 5 ring]phenylalanine with an isotopic enrichment of 10 atom% were intravenously injected. [ 2 H 5 ring]phenylalanine enrichments in the plasma-free phenylalanine and the albumin and fibrinogen isolates were measured by gas chromatography-mass spectrometry. Fractional synthesis rates of albumin were normal in Child A cirrhosis (7.6 ± 2.2%d), but were lower in both Child B (3.5 ± 0.8%d) and C (4.5 ± 2.8%d). Absolute rates of albumin synthesis were (103 ± 30 mg/kg/d) in the child A group and substantially lower in the Child B (50 ± 3 mg/kg/d) and C (36 ± 20 mg/kg/d) group. The average fractional synthesis rate of fibrinogen was 16.7 ± 7.5%d and the absolute synthesis rate 11.6 ± 6.4 mg/kg/d. The values of the galactose elimination capacity and the aminopyrin breath test were below the normal range in all patients, gradually decreasing with an increase in the severity of the clinical stage of cirrhosis. Albumin synthesis rates significantly correlated with the Child scores, the galactose elimination capacity, and the aminopyrin breath test, whereas fibrinogen synthesis rates showed no such correlations. (Hepatology 1996 Jul;24(1):53-9)

Chronic bile duct obstruction in the rat leads to biliary cirrhosis but maintained hepatocellular mass. We have previously demonstrated translocation of epidermal growth factor receptor to nuclei. It remained unclear, however, whether this was due to hepatocyte proliferation and/or altered handling of epidermal growth factor receptor. Therefore, in the present investigation we stereologically estimated expression of proliferating cell nuclear antigen, a marker of the S phase of teh cell cycle at 1, 2, 3, 7, 14, 21 and 28 days after bile duct ligation. Proliferating cell nuclear antigen positive hepatocytes averaged 2.1 +/- 3.6% in sham-operated control animals. This increased to 20.7 +/- 6.4, 26.8 +/- 18.7, 31.3 +/- 23.9, 42.3 +/- 16.6 and 24.7 +/- 28.0% 3, 7, 14, 21 and 28 days after bile duct ligation, respectively (p0.005 by ANOVA). This was correlated with the number of epidermal growth factor receptor positive nuclei (rs = 0.737) and inversely with the maximal binding capacity of epidermal growth factor to a crude plasma membrane fraction (rs = 0.697) reported previously. We conclude that bile duct ligation in the rat induces a significant hepatocellular proliferation as assessed by proliferating cell nuclear antigen expression and that this process could, at least in part, be related to increased nuclear expression of the epidermal growth factor receptor.

Lipid peroxidation and antioxidative mechanisms were investigated in liver mitochondria from bile duct ligated rats (BDL rats) and correlated with the activity of enzyme complexes of the electron transport chain. In comparison to pair-fed control rats, BDL rats had increased concentrations of thiobarbituric acid reacting substances (TBARS) per gram of liver and per milligram of mitochondrial protein 3, 7, 14, and 28 days after surgery. The hepatic glutathione (GSH) content was decreased in BDL rats 28 days after surgery when expressed per gram of liver but equal between BDL and control rats when expressed per liver. The mitochondrial GSH content was decreased in BDL rats by 20% to 33% from day 7 after surgery. The concentrations of ubiquinone-9 and ubiquinone-10, substances involved in electron transport and efficient antioxidants, were both decreased in BDL rats 14 and 28 days after surgery per gram of liver and per milligram of mitochondrial protein. When expressed per liver, ubiquinone-9 was decreased in BDL rats from day 7 after surgery. In comparison with controls, the decrease in total mitochondrial ubiquinone content in BDL rats averaged 52% 14 days and 38% 28 days after surgery. The activity of the succinate:ferricytochrome c oxidoreductase (complexes II and III of the electron transport chain) was decreased in BDL rats at days 7, 14, and 28 after surgery, and the activity of the ferrocytochrome c:oxygen oxidoreductase (complex IV) was reduced at 14 and 28 days after surgery. The mitochondrial concentration of TBARS showed a negative and the concentrations of GSH and ubiquinone a positive correlation with the activity of the succinate:ferricytochrome c oxidoreductase. We conclude that the mitochondrial concentration of antioxidants such as GSH and ubiquinone decreases in BDL rats, whereas the concentration of lipid peroxidation products increases. Hepatic mitochondrial dysfunction in BDL rats may at least partially result from oxidative damage to mitochondrial lipids and/or proteins.

To determine sirolimus steady-state pharmacokinetics, and to assess the relationship between time-normalized trough sirolimus concentration (C(min,TN)) and evidence of efficacy (rejection and death) and adverse reactions (stomatitis and pneumonia) in liver allograft patients.Dense sampling of sirolimus was performed over a single daily-dosing interval in 11 hepatic allograft recipients on day 28 and at 3 months after start of treatment. Serial trough concentration sampling was performed in 380 hepatic allograft recipients on days 1, 7, 14, 28, 42, 60, 90, 180, 270 and 360 after start of treatment. Occurrence of stomatitis, pneumonia, rejection, and death were collected for 360 days after start of treatment. Noncompartmental pharmacokinetic parameters were analyzed in the 11 densely sampled patients; C(min,TN) was determined in the 380 patients.Mean maximum concentration (C(max)), time to C(max) (t(max)), area under the curve for the given dose interval (AUC(tau)), and whole blood oral clearance at 3 months were 20.8 ± 7.6 ng/mL, 3 ± 1 h, 338 ± 144 ng·h/mL, and 10.0 ± 5.6 L/hr, respectively. In the 11 densely sampled patients, linear regression showed that C(min,TN) was highly predictive of AUC(tau) (r² = 0.77, P0.0001) at each analysis time point. Logistic regression showed a relationship between C(min,TN) in the 380 patients and pneumonia occurrence, but not between C(min,TN) and stomatitis, rejection, or death.In this study, the pharmacokinetic profile of sirolimus in hepatic allograft patients was consistent with that of renal transplantation recipients. With the exception of pneumonia, no correlation was observed between C(min,TN) and the occurrence of adverse events of interest.

Hepatic metabolism of fatty acids is impaired in experimental animals with long-term bile duct ligation. To characterize the underlying defects, fatty acid metabolism was investigated in isolated hepatocytes and isolated liver mitochondria from rats subjected to long-term bile duct ligation or sham surgery. After starvation for 24 hr, the plasma β-hydroxy-butyrate concentration was decreased in rats with bile duct ligation as compared with control rats. Production of β-hydroxybutyrate from butyrate, octanoate and palmitate by hepatocytes isolated from rats subjected to bile duct ligation was also decreased. Liver mitochondria from rats subjected to bile duct ligation showed decreased state 3 oxidation rates for L-glutamate, succinate, duroquinone, and fatty acids but not for ascorbate as substrate. State 3u oxidation rates (uncoupling with dinitrophenol) and activities of mitochondrial oxidascs were also decreased in mitochondria from rats subjected to bile duct ligation. Direct assessment of the activities of the subunits of the electron transport chain revealed reduced activities of complex I, complex II and complex III in mitochondria from rats subjected to bile duct ligation. Activities of the β-oxidation enzymes specific for short-chain fatty acids were all reduced in rats subjected to bile duct ligation. Mitochondrial protein content per hepatocyte was increased by 32% in rats subjected to bile duct ligation compared with control rats. Thus the studies directly demonstrate mitochondrial defects in fatty acid oxidation in rats subjected to bile duct ligation, which explain decreased ketosis during starvation. (HEPATOLOGY 1994;19:1272–1281.)

Cirrhosis is characterized by fibrogenesis, hepatocyte necrosis and the formation of regenerative nodules. Modulation of the epidermal growth factor receptor is an early event during regeneration. We have recently demonstrated alterations in the epidermal growth factor receptor during the development of biliary cirrhosis. The aim of the present study was to compare epidermal growth factor receptor distribution, expression and binding in biliary cirrhosis to that occurring in micronodular cirrhosis induced by phenobarbital/CCl4 exposition. Biliary cirrhosis and micronodular cirrhosis had similar functional impairment as assessed by the aminopyrine breath test. Epidermal growth factor receptor binding capacity was reduced in both models (control vs micronodular cirrhosis vs biliary cirrhosis: (mean +/- 1 SD) 60 +/- 22 vs 16 +/- 12 vs 27 +/- 9 fmol/mg protein, p0.05), while the binding constant was increased in biliary cirrhosis only. The receptor mass in plasma membrane, determined by Western blotting, was not changed. Distribution of epidermal growth factor receptor was assessed immunohistochemically on tissue sections. In both models, cytoplasmic staining was decreased and basolateral plasma membrane labeling was maintained. Nuclear localization was found in biliary cirrhosis only. In conclusion, in both models, cirrhosis induces an alteration in the binding properties, but not in the number of epidermal growth factor receptors in the plasma membrane. The loss of cytoplasmic epidermal growth factor receptor could reflect alterations in expression and/or in intracellular trafficking. This is supported by the reduced mRNA steady state levels for epidermal growth factor receptor which were found in both models, presumably representing down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Fifty-six patients with biopsy-proven, chronic active hepatitis B were included in a multi-center, randomized trial comparing steroid withdrawal followed by 1.5 MU recombinant interferon alpha 2b (Intron ® ) with placebo withdrawal followed by either 1.5 or 5 MU interferon. The patients were equally distributed between the treatment groups with respect to biochemical and histologic activity as well as with respect to DNA levels and quantitative liver function tests. One patient was lost to follow up. After 1 year of treatment, 10/18, 13/19 and 11/18 patients had lost hepatitis B virus DNA in the three groups, respectively (non-significant). Transaminase levels were normal in 27/34 of the responders but in only 4/21 of the non-responders ( p

Secondary biliary cirrhosis in the rat can be induced by bile duct ligation; the aim of the present study was to investigate whether susceptibility to this injury depends on development. Rats aged 4, 7, 14 and 22 weeks were bile-duct ligated or sham operated. Four weeks later, stereologic analysis of the liver was performed and the volume fraction of parenchyma, bile ducts and connective tissue was determined. Microsomal function was assessed in vivo by the aminopyrine breath test and in vitro by determining the microsomal cytochrome P450 content and microsomal lipid composition. In addition, portal pressure was measured. The volume fraction of parenchyma decreased in an age-dependent fashion in bile-duct ligated rats from 64.0 +/- 11.2% in the youngest to 46.4 +/- 8.4% in the oldest age group. This decrease was compensated by an age-dependent increase in both ductular proliferation and fibrosis. Microsomal function both in vivo and in vitro showed an age-dependent deterioration. Microsomal cholesterol and some individual phospholipids showed age-dependent changes. Portal hypertension developed in all bile-duct ligated groups, but portal pressure was significantly lower in the oldest bile-duct ligated groups (16.0 +/- 2.6 cmH2O) compared with other bile-duct ligated groups (around 21 cmH2O). We conclude that susceptibility to the sequelae of chronic cholestasis depends on the stage of development in rats. In experiments using this model, the age of the rats should be explicitly stated.

Increased levels of natural benzodiazepine receptor agonists, produced in the body (endogenous) or ingested with food (exogenous) have been proposed as one of the factors causing hepatic encephalopathy in both experimental animals and human subjects. However, the divergent response of hepatic encephalopathy to benzodiazepine antagonists sheds doubt on this attractive hypothesis. Acute liver failure was induced in male Sprague-Dawley rats (n = 17) with intraperitoneal thioacetamide (600 mg/kg/day for 3 days) while 14 control rats received vehicle only. Acute liver failure developed in all treated rats (AST: 1,898 ± 1,359 IU/L vs. controls, 45 ± 5 IU/L, p < 0.005; bilirubin: 36 ± 27 μmol/L vs. controls, 1.5 ± 0.5 μmol/L, p < 0.005; centrizonal necrosis) and grade 3 or 4 hepatic encephalopathy (neurologic assessment and activity monitoring). However, benzodiazepine receptor ligand activity, measured in the supernatant of whole-brain homogenates with a [3H]fiumazenil binding competition assay, was clearly increased in only 1 of 17 rats with acute liver failure compared with controls (52.7 ± 34.1 vs. 44.3 ± 18.9 ng diazepam equivalents/gm; NS). To evaluate whether the reported increase in benzodiazepine receptor ligand activity could be due to prolonged residence of exogenous benzodiazepine-like substances, additional rats with acute liver failure and controls were treated with diazepam (five doses of 0.5 mg/kg at 12-hr intervals by gavage). Benzodiazepine receptor ligand activity was greater in animals with acute liver failure than in controls (223 ± 65 vs. 103 ± 23 ng diazepam equivalents/gm; p < 0.002) 1 to 3 hr after the last diazepam dose. Increased tissue levels of benzodiazepine receptor ligand activity are therefore neither necessary nor sufficient to explain the pathogenesis of hepatic encephalopathy in acute liver failure. We conclude that increased tissue levels of benzodiazepine receptor agonists in hepatic encephalopathy could be due to prolonged residence of exogenous benzodiazepine-like compounds of pharmaceutical or natural origin. (HEPATOLOGY 1993;18:1459–1464.)

The pharmacokinetics and haemodynamic effects of orally administered spirapril, a novel angiotensin-converting enzyme (ACE) inhibitor, have been investigated in patients with liver cirrhosis (n = 10), in patients with chronic, non-cirrhotic liver disease (n = 8) and in a control group of healthy subjects (n = 16). The absorption and elimination of spirapril did not differ between patients with liver disease and control subjects. In contrast, the bioavailability of spiraprilat, the metabolite responsible for the pharmacological action of spirapril, was significantly reduced in patients (AUC 820 micrograms.h.l-1, 923 micrograms.h.l-1 and 1300 micrograms.h.l-1 in patients with cirrhosis, patients with non-cirrhotic liver disease and in healthy subjects, respectively. Compared to healthy subjects, cirrhotic patients had a reduced rate constant of spiraprilat formation (1.10 h-1 in patients vs. 2.00 h-1 in control subjects) while the elimination half-life of spiraprilat was not different. The effect of spirapril on diastolic blood pressure was decreased in patients with chronic liver disease as compared to the controls. Thus, the pharmacokinetics of spirapril was unchanged in patients with different types of liver disease, including cirrhosis. However, the bioavailability of spiraprilat and hypotensive effect of spirapril were reduced in patients.

Secondary biliary cirrhosis in the rat is an attractive model since unlike other models it does not rely on exogenous toxic compounds to induce cirrhosis. However, because little is known about the microcirculatory abnormalities of this model, this study investigated hemodynamics in rats with predefined functional impairment and related them to different aspects of stereologically quantified structure. All animals with at least 50% reduction in microsomal function, assessed by the aminopyrine breath test, had portal hypertension. The sinusoidal space, as assessed by multiple indicator dilution in the perfused liver, was reduced whereas large vessel space was increased. This reduction in sinusoidal space could contribute to increased portal resistance. The degree of intrahepatic shunting varied as assessed by a microsphere technique (13.9 vs. 0.5% in controls). These alterations were confirmed by stereological analysis. Numerically, there was excellent agreement between functional indicator dilution data and anatomic quantitation. Microvascular exchange was impaired as in other models of cirrhosis as shown by a reduced extravascular albumin space (4.5 vs. 2.2%, p N -demethylase activity), the smooth endoplasmic reticulum was maintained (4.3 vs. 3.5 m 2 /ml cytosol, n.s.), which demonstrates that microsomal function in this model is reduced per unit hepatocyte. This suggests that the sick-cell hypothesis applies to secondary biliary cirrhosis in the rat.

In this study we investigated whether the retention of compounds which are excreted into the bile could contribute to portal hypertension in secondary biliary cirrhosis. Choledochovenous fistulas were grown in rats for 4 weeks. 6/13 of the animals had biochemical evidence of partial obstruction. Microsomal function, as measured by the aminopyrine breath test, was decreased in all animals with biliary retention while microsomal cytochrome P-450 content was decreased only in rats with evidence of obstruction. All animals with biliary retention with or without partial obstruction had portal hypertension. Animals with biliary retention and partial obstruction had hypercholeresis but decreased bile salt excretion. All animals with a chronic catheter in the biliary tree had a loss of the negative permselectivity of the sinusoidal-canalicular barrier and decreased maximal bile secretory pressure. Only animals with biochemical evidence of obstruction had moderate fibrosis and ductular proliferation as determined by stereological techniques. Unexpectedly, morphometric analysis also revealed an increase in hepatocyte mass induced by biliary retention. We conclude that bile contains a compound(s) which induces portal hypertension. This putative substance is neither bilirubin nor a bile acid since portal hypertension was also observed in animals with biliary retention without obstructive signs.

To investigate the potential role of lysosomes in cirrhosis, we analyzed the activity of lysosomal enzymes in rats exposed long-term to phenobarbital and carbon tetrachloride. The activity of lysosomal enzymes was markedly increased in the homogenate of cirrhotic livers (e.g., arylsulfatase 9 +/- S.D.2 vs. 16 +/- 6 nmoles.min-1.mg-1 in control rats and cirrhotic rats, respectively; p less than 0.001). The corresponding plasma levels were also increased (7 +/- 1 vs. 12 +/- 3 nmoles.min-1.mg-1; p less than 0.01), whereas biliary excretion was diminished (16 +/- 7 vs. 7 +/- 2 pmol.min-1.gm liver-1; p less than 0.05) in cirrhotic rats. Stereological quantification of lysosomes visualized cytochemically revealed an increase of pericanalicular lysosomes averaging 1.5 +/- 0.4 around a canaliculus in controls and 3.7 +/- 1.0 in cirrhotic rats (p less than 0.01). Because this suggested a defect in the transcellular vesicular pathway, we investigated the biliary excretion of horseradish peroxidase and epidermal growth factor in perfused livers. Bile flow and total horseradish peroxidase excretion were similar in control rats and cirrhotic rats. However, the early peak of biliary horseradish peroxidase excretion--usually taken as evidence of paracellular transport--was increased in cirrhotic rats (13 +/- 7 vs. 57 +/- 22%; p less than 0.01), whereas the second peak--reflecting the transcellular vesicular pathway(s)--was markedly reduced (87 +/- 7 vs. 43 +/- 22%; p less than 0.001). A similar reduction in the biliary excretion of intact epidermal growth factor and of its degradation products was found. These results demonstrate an increased number of lysosomes in hepatocytes of cirrhotic livers; this appears to be the result of accumulation rather than proliferation, in view of the reduced transcellular vesicular movement of different markers into bile.

Biliary cirrhosis with portal hypertension and hepatocellular failure is a well-known complication of extrahepatic obstruction. It is unclear to what extent these changes are reversible by biliodigestive anastomosis. Therefore a rat model of relief of biliary obstruction was developed by performing Roux-en-Y choledochojejunostomy in rats after bile duct obstruction. Patency of the biliodigestive anastomosis was documented by biliary scintigraphy. Microsomal function was assessed in vivo by the aminopyrine breath test and portal hypertension by spleen pulp pressure. Microsomal function was markedly impaired in obstructed animals but recovered after biliodigestive anastomosis. Microsomal cytochrome P450 content paralleled these changes. Similarly, portal hypertension was reversed after successful relief of obstruction. Stereologic analysis showed that biliodigestive anastomosis partially reversed bile ductular proliferation and fibrosis. Studying the time course of recovery showed that restoration of microsomal function was achieved after 2 weeks whereas recovery from portal hypertension required 4 weeks of biliary drainage. Recovery of microsomal function was paralleled by normalization of microsomal lipid composition while resolution of portal hypertension occurred parallel to resolution of the histologic abnormalities.

Mitochondrial metabolism was studied in liver mitochondria isolated from rats with secondary biliary cirrhosis induced by bile duct ligation for 5 wk. State 3 oxidation rates were decreased in mitochondrial preparations from bile duct-ligated rats as compared with sham-operated control rats by 63% and 42% using beta-hydroxybutyrate and succinate as substrates, respectively. In contrast, when the substrate was ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine state 3 oxidation rates were not affected by bile duct ligation. Oxidation rates after uncoupling with dinitrophenol were decreased for both beta-hydroxybutyrate and succinate as substrates in mitochondria from bile duct-ligated rats. The phosphate potential was reduced in mitochondria from bile duct-ligated rats (12.5 +/- 0.5 vs. 13.6 +/- 0.2 kcal in control and bile duct-ligated rats, respectively; p less than 0.05). The inner mitochondrial membrane of liver mitochondria from rats with secondary biliary cirrhosis contained three times more cholesterol as compared with control rats, whereas the phospholipid composition was essentially unchanged. Mitochondrial protein content expressed per liver (calculated on the basis of activities of mitochondrial enzymes determined in liver homogenate and in isolated mitochondria) was increased by 50% in bile duct-ligated rats as compared with control rats. In conclusion, the function of the electron transport chain in liver mitochondria isolated from rats with secondary biliary cirrhosis is impaired. This decrease could be related to altered lipid composition of the inner mitochondrial membrane.

Mitochondrial and cytosolic functions were studied in vivo and in perfused livers from rats with secondary biliary cirrhosis induced by bile duct ligation for 5 wk and in sham-operated controls. The livers were stereologically analyzed, and mitochondrial and cytosolic functions were related to liver structure. Oxygen consumption by perfused livers expressed per stereologically determined mitochondrial volume was decreased by 49% in bile duct-ligated rats compared with control rats. Glucose production (expressed per mitochondrial volume) was reduced by more than 90% in bile duct ligation, whereas urea production was not affected. Lactate production, a cytosolic function, was increased fivefold in bile duct ligation, and both the lactate/pyruvate and the beta-hydroxybutyrate/aceto-acetate ratios were increased in the liver perfusate of bile duct-ligated rats. In comparison with control rats, the stereologically determined mitochondrial volume fraction per hepatocyte was increased by 28% in bile duct-ligated rats. Activities of mitochondrial enzymes expressed per area of mitochondrial membrane or per mitochondrial volume were either unchanged (ATPase, cytochrome c oxidase and glutamate dehydrogenase) or decreased (monoamine oxidase) in bile duct ligation. Thus in comparison with control rats, mitochondrial metabolism is impaired in perfused livers from bile duct-ligated rats; increased mitochondrial volume per hepatocyte may represent a strategy to maintain hepatic energy metabolism in rats with secondary biliary cirrhosis.

Quantitative liver function tests are based on the clearance concept and measure the plasma disappearance of a test compound such as galactose. Metabolism is inferred to be predominantly hepatic, and usually no knowledge is obtained of the true time course of metabolite formation. Dynamic 31phosphorus magnetic resonance spectroscopy after intravenous administration of fructose directly measures hepatic sugar metabolism. To determine the feasibility and the utility of 31P magnetic resonance spectroscopy, we studied the responses of six healthy subjects and nine patients with nonalcoholic cirrhosis to a fructose load. Results were related to the impairment of hepatic function assessed by the galactose-elimination capacity test. Liver spectra were acquired in a 1.5 T whole-body nuclear magnetic resonance unit with a surface coil (9-cm diameter) placed ventrally on the liver; the one-dimensional chemical-shift imaging technique was used to obtain spectra from tissue slices parallel to the surface coil. After a basal spectrum had been obtained, fructose (250 mg/kg) was injected intravenously, and further spectra were collected sequentially every 6 min for 1 hr. Formation of monophosphate esters (9% +/- 5% vs. 20% +/- 8% of total area; p less than 0.01) and utilization of inorganic phosphate (5% +/- 4% vs. 11% +/- 3% of total area; p less than 0.005) were markedly decreased in cirrhotic patients. These measures correlated with the severity of the impairment of liver function measured by the galactose-elimination capacity (r = 0.53 to 0.69; p less than 0.05). We conclude that dynamic 31P magnetic resonance spectroscopy is a safe, clinically feasible test that allows detailed insights into biochemical events in liver disease.

Oxidative drug metabolism is impaired in liver cirrhosis; it is unclear, however, whether this depends on the etiology of cirrhosis. Therefore, we studied the metabolism of dextromethorphan in two rat models: biliary cirrhosis induced by bile duct ligation and micronodular cirrhosis induced by chronic exposure to CCl4/phenobarbital. Results were compared with aminopyrine N-demethylation assessed by a breath test in vivo; the latter was reduced to a similar extent in biliary (-41%) and micronodular (-37%) cirrhosis compared to controls. In contrast, clearance of dextromethorphan was significantly (P less than 0.001) reduced in biliary (25.4 +/- 5.3 mL/min/kg) but not in micronodular cirrhosis (48.6 +/- 15.6) as compared to controls (62.2 +/- 16.2). Intrinsic clearance of dextromethorphan in vitro was reduced by 95% and 63% in biliary and micronodular cirrhosis, respectively (P less than 0.001 vs controls). It correlated with dextromethorphan clearance in vivo (r = 0.68, P less than 0.001) whereas correlation with aminopyrine N-demethylation was weak (r = 0.42, P less than 0.05). Our results demonstrate a differential effect of biliary and micronodular cirrhosis on isoenzymes responsible for aminopyrine and dextromethorphan demethylation.

The renal response to atrial natriuretic factor is blunted in cirrhosis with ascites. This might be due to alterations of renal receptors for atrial natriuretic factor. Therefore density and affinity of glomerular atrial natriuretic factor binding sites of bile duct-ligated rats with ascites (n = 10) and of sham-operated controls (n = 10) were determined. Glomerular atrial natriuretic factor binding sites were identified to be of the B-(biologically active) and C-(clearance) receptor type. Discrimination and quantitative determination of B and C receptors for atrial natriuretic factor were achieved by displacement experiments with atrial natriuretic factor(99-126) or des(18-22)atrial natriuretic factor(4-23), an analogue binding to C receptors only. Density of total glomerular atrial natriuretic factor binding sites was significantly increased in bile duct-ligated rats (3,518 ± 864 vs. 1,648 ± 358 fmol/mg protein; p < 0.05). This was due to a significant increase of C-receptor density (3,460 ± 866 vs. 1,486 ± 363 fmol/mg protein; p < 0.05), whereas density of B receptors was not significantly different in bile duct-ligated rats (58 ± 11 vs. 162 ± 63 fmol/mg protein). Affinity of atrial natriuretic factor to its glomerular binding sites did not differ significantly between both groups. These data suggest that an altered glomerular atrial natriuretic factor receptor density could be involved in the renal resistance to atrial natriuretic factor in cirrhosis with ascites.

To investigate whether the impairment of mitochondrial function in cirrhosis is due to a reduction in liver cell mass or whether mitochondrial function is altered specifically, we analyzed mitochondrial volume and surface density of mitochondrial membranes in control and cirrhotic rats by stereological means. Cirrhosis was induced by long-term exposure to phenobarbital and CCl4. Hepatocellular and mitochondrial volumes were reduced to a similar extent, by 39% and 40%, respectively, in cirrhotic animals (p less than 0.01). Thus the fraction of hepatocytes occupied by mitochondria did not differ between the two groups. Both total outer (31 +/- 3 vs. 19 +/- 6 m2; p less than 0.01) and inner (87 +/- 24 vs. 45 +/- 12 m2; p less than 0.01) mitochondrial membranes were significantly reduced. Membrane surface was normal per unit of mitochondrial volume, however, suggesting intact mitochondrial structure. Matrix and outer membrane enzyme activities expressed per compartment did not differ between control and cirrhotic animals. Inner membrane, in contrast, had an increased enzyme content per unit area both for cytochrome oxidase (10.3 +/- 2.9 vs. 13.0 +/- 1.6; p less than 0.05) and ATPase (13.7 +/- 1.4 vs. 21.2 +/- 2.9; p less than 0.01). Basal oxygen consumption measured in the perfused liver in situ was significantly reduced in cirrhotic livers (1.6 +/- 0.1 vs. 1.1 +/- 0.4 mumol/min-1/gm-1) but was unchanged when expressed per square meter of inner membrane. Our results demonstrate that impaired mitochondrial function is mainly due to loss of hepatocellular mass. Increased enzyme activity per unit surface area of inner mitochondrial membrane may be important to maintain mitochondrial function of the cirrhotic liver.

Antipyrine metabolism depends on at least three isoenzymes of cytochrome P450 forming the main metabolites 3-OH-, 4-OH- and norantipyrine. We investigated to which extent antipyrine clearance and metabolite formation in vivo correlate with metabolite formation by microsomal fractions in vitro. The influence of sex was investigated in two rat strains. Antipyrine clearance in saliva was determined in 10-month-old Sprague-Dawley and Dark Agouti rats of either sex. Antipyrine and its metabolites in urine and microsomes were measured by a new HPLC method after solid phase or liquid extraction. Antipyrine clearance was 46% higher in males than in female rats. This was associated with a 40% higher urinary excretion of 3-OH-antipyrine in the male rats, the other metabolites being excreted to a similar extent. This higher production of 3-OH-antipyrine in vivo was paralleled by a higher intrinsic clearance in vitro while no sex difference in intrinsic clearance for the formation of the other metabolites was seen. The correlation between in vivo and in vitro metabolic clearance for 3-OH-antipyrine was good (r = 0.75) but unconvincing for 4-OH- (r = 0.49) and norantipyrine (r = 0.01). This could be due to further metabolism of 4-OH- and norantipyrine.

The pharmacokinetics of albendazole and its main metabolite, albendazole sulphoxide, have been examined after giving a single oral dose of 200 mg albendazole to 19 patients with either Echinococcus multilocularis or E. granulosus, 5 of whom had significant extrahepatic obstruction due to the underlying disease. The AUC of albendazole sulphoxide was increased in the latter patients (mean 122 μmol · h · l−1 compared to 17 μmol · h · l−1 in the non-obstructed group). Obstructed patients had delayed absorption, ka averaging 0.39 compared to 1.41 h−1 in non-obstructed patients. The corresponding elimination rate constant, ke was also prolonged, averaging 0.041 and 0.13 h−1 in the two groups, respectively. Four patients were restudied after complete or partial resolution of the cholestasis. The pharmacokinetic parameters in them had returned towards values comparable to those in the non-obstructed patients.

Cholestasis induced by perfusion of the liver with hypocalcemic media has been ascribed to several defects in bile secretion including increased biliary permeability. To investigate this model of cholestasis further, livers perfused with hypo- and normocalcemic media were examined stereologically using thin sections and freeze-fracture replicas. Organization of tight junctions was not altered by hypocalcemia; neither the number of strands nor the junctional depth were significantly affected. By contrast, the volume of hepatocytes decreased by 11% ( p p p + efflux of 62 μmol/g liver was observed corresponding to approx. 8% of the hepatocellular water space. Our results suggest that hypocalcemia-induced cholestasis is due, at least in part, to a disturbance of the osmotic equilibrium, possibly caused by impairment of an ion transport system involved in hepatocellular volume control.

Rapamycin is an immunosuppressant with antiproliferative properties. We investigated whether rapamycin treatment of bile duct-ligated (BDL) rats is capable of inhibiting liver fibrosis and thereby affecting hemodynamics. Following BDL, rats were treated for 28 days with rapamycin (BDL SIR). BDL animals without drug treatment (BDL CTR) and sham-operated animals served as controls. After 28 days, hemodynamics were measured, and livers were harvested for histology/immunohistochemistry. Liver mRNA levels of transforming growth factor (TGF)-beta1, connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF)-beta, cyclin-dependent kinase inhibitor p27(kip) (p27), and cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21) were quantified by real-time polymerase chain reaction. Liver protein levels of p27, p21, p70 S6 kinase (p70(s6k)), phosphorylated p70(s6k) (p-p70(s6k)), eukaryotic initiation factor 4E-binding protein (4E-BP1), p-4E-BP1 (Thr37/46), and p-4E-BP1 (Ser65/Thr70) were determined by Western blotting. Portal vein pressure was lower in BDL SIR than in BDL CTR animals. Volume fractions of connective tissue, bile duct epithelial, and desmin- and actin-positive cells were lower in BDL SIR than in BDL CTR rats. On the mRNA level, TGF-beta1, CTGF, and PDGF were decreased by rapamycin. p27 and p21 mRNA did not differ. On the protein level, rapamycin increased p27 and decreased p21 levels. Levels of nonphosphorylated p70(s6k) and 4E-BP1 did not vary between groups, but levels of p-p70(s6k) were decreased by rapamycin. Rapamycin had no effect on p-4E-BP1 (Thr37/46) and p-4E-BP1 (Ser65/Thr70) levels. In BDL rats, rapamycin inhibits liver fibrosis and ameliorates portal hypertension. This is paralleled by decreased levels of TGF-beta1, CTGF, and PDGF. Rapamycin influences the cell cycle by up-regulation of p27, down-regulation of p21, and inhibition of p70(s6k) phosphorylation.

Background: The role of endothelial nitric oxide synthase 3 (NOS-3) in the hyperdynamic circulation associated with cirrhosis is established but not that of the neuronal (NOS-1) isoform. We therefore investigated aortic NOS-1 levels in NOS-3 knock-out (KO) and wildtype (WT) mice and in hepatic arteries of patients. Methods: Mice rendered cirrhotic by bile duct ligation (BDL) were compared with sham-operated controls. Hepatic arteries of cirrhotic patients were collected during liver transplantation; donor vessels served as controls. mRNA levels were quantified by real-time PCR, protein levels by Western blotting and NO production by Nω-nitro-l-arginine methyl ester inhibitable arginine–citrulline assay. Results: Aortae of NOS-3 KO mice exhibited higher NOS-1mRNA (5.6-fold, P

Secondary complications of diabetes mellitus often involve gastrointestinal dysfunction. In the experimental Goto Kakizaki rat, a model of Type II diabetes, hyperglycaemia and reduced glucose clearance is associated with elevated plasma endothelin (ET)-1 levels and selective decreases in nitric oxide synthase in circular muscle, longitudinal muscle and neuronal elements of the gastrointestinal tract. Functionally, this is accompanied by decreased nitrergic relaxatory responses of jejunal longitudinal muscle to tetrodotoxin-sensitive electrical field stimulation. Long-term treatment with a selective ET A-type receptor antagonist, markedly reduced hyperglycaemia and restored plasma glucose clearance rates towards normal. This was associated with a restoration of NG-nitro-L-arginine methyl ester-sensitive relaxatory responses of jejunal longitudinal muscle to electrical field stimulation. The results indicate that beneficial effects of ETA receptor blockade on gastrointestinal function may result from an improvement in insulin sensitivity with concomitant reduction of the severity of hyperglycaemia. ETA receptor blockade may represent a new therapeutic principle for improving glucose tolerance in Type II diabetes and could be beneficial in alleviating or preventing hyperglycaemia-related secondary complications in this condition.

To analyze a single center's 6-year experience with 258 consecutive patients undergoing major hepatic resection for primary or secondary malignancy of the liver, and to examine the predictive value of preoperative liver function assessment.Despite the substantial improvements in diagnostic and surgical techniques that have made liver surgery a safer procedure, careful patient selection remains mandatory to achieve good results in patients with hepatic tumors.In this prospective study, 258 patients undergoing hepatic resection were enrolled: 111 for metastases, 78 for hepatocellular carcinoma (HCC), 21 for cholangiocellular carcinoma, and 48 for other primary hepatic tumors. One hundred fifty-eight patients underwent segment-oriented liver resection, including hemihepatectomies, and 100 had subsegmental resections. Thirty-two clinical and biochemical parameters were analyzed, including liver function assessment by the galactose elimination capacity (GEC) test, a measure of hepatic functional reserve, to predict postoperative (60-day) rates of death and complications and long-term survival. All variables were determined within 5 days before surgery. Data were subjected to univariate and multivariate analysis for two patient subgroups (HCC and non-HCC). The cutoffs for GEC in both groups were predefined. Long-term survival (60 days) was subjected to Kaplan-Meier analysis and the Cox proportional hazard model.In the entire group of 258 patients, a GEC less than 6 mg/min/kg was the only preoperative biochemical parameter that predicted postoperative complications and death by univariate and stepwise regression analysis. A GEC of more than 6 mg/min/kg was also significantly associated with longer survival. This predictive value could also be shown in the subgroup of 180 patients with tumors other than HCC. In the subgroup of 78 patients with HCC, a GEC less than 4 mg/min/kg predicted postoperative complications and death by univariate and stepwise regression analysis. Further, a GEC of more than 4 mg/min/kg was also associated with longer survival.This prospective study establishes the preoperative determination of the hepatic reserve by GEC as a strong independent and valuable predictor for short- and long-term outcome in patients with primary and secondary hepatic tumors undergoing resection.