Bilirubin, a physiological antioxidant, can improve cryopreservation of human hepatocytes.

The availability of cryopreserved hepatocytes is required for a more widespread use of hepatocyte transplantation, but human hepatocytes are easily damaged during freezing-thawing. Here, preincubation with unconjugated bilirubin, a physiological antioxidant, resulted in increased viability and function of hepatocytes (as determined by trypan blue exclusion, mitochondrial succinate dehydrogenases activity, urea synthesis, and cytochrome P450 1A/2) compared with cells incubated without the pigment. These findings suggest that unconjugated bilirubin may be used as cryoprotectant in clinical hepatocyte transplantation.

Gekkonid lizards: average ages derived from tail-loss data.

Information on the rate of tail loss by autotomy, obtained from mark and recapture data, was used to estimate the average ages of adult individuals of the geckos Gehyra variegata and Heteronota binoei; the ages calculated were 4.4 and 1.9 years, respectively.

Hepatocyte transplantation followed by auxiliary liver transplantation--a novel treatment for ornithine transcarbamylase deficiency.

We report the first successful use of hepatocyte transplantation as a bridge to subsequent auxiliary partial orthotopic liver transplantation (APOLT) in a child antenatally diagnosed with severe ornithine transcarbamylase (OTC) deficiency. A total of 1.74 x 10(9) fresh and cryopreserved hepatocytes were administered intraportally into the liver over a period of 6 months. Immunosuppression was with tacrolimus and prednisolone. A sustained decrease in ammonia levels and a gradual increase in serum urea were observed except during episodes of sepsis in the first 6 months of life. The patient was able to tolerate a normal protein intake and presented a normal growth and neurological development. APOLT was successfully performed at 7 months of age. We conclude that hepatocyte transplantation can be used in conjunction with APOLT as an effective treatment for severe OTC-deficient patients, improving neurodevelopmental outcomes.

Effects of serum from patients with acute liver failure due to paracetamol overdose on human hepatocytes in vitro.

OBJECTIVE: Our goal was to investigate the effects of serum from patients with acute liver failure due to paracetamol (acetaminophen) overdose on the function of human hepatocytes in vitro. METHODS: Freshly isolated human hepatocytes plated on collagen-coated culture plates were, incubated (24 hours 37 degrees C) in medium containing pooled human sera (0%-80%) obtained from normal individuals or from patients with acute liver failure due to paracetamol overdose. The effects of the sera on cell function were assessed using MTT, [14C]-leucine incorporation, and cytochrome P450 (CYP1A1/2) activity assays. RESULTS: The overall cellular metabolic activity was significantly greater at all concentrations after exposure to acute liver failure serum compared to normal serum. There were no significant differences in the decreases produced by pooled acute liver failure and normal sera at concentrations up to 80% on the [14C]-leucine incorporation or CYP1A1/2 activity. CONCLUSION: The overall cell function/activity of human hepatocytes was not impaired in vitro on exposure to serum from patients with acute liver failure due to paracetamol overdose.

Serum F protein estimation in liver allograft recipients with graft dysfunction.

Serum levels of F protein, a 44 kD cytoplasmic protein mainly found in hepatocytes, became elevated during episodes of graft dysfunction following orthotopic liver transplantation. In a study of 27 liver transplant recipients, the rise in F protein did not precede rises in the other conventional biochemical indices of hepatic dysfunction. Serum F protein concentration significantly correlated with serum levels of aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, and bilirubin (all P less than 0.001) and also with the prothrombin time (P = 0.048). Despite its high concentration in liver cells, this marker does not provide any additional benefit in the diagnosis of graft dysfunction or in monitoring liver allograft function following transplantation.

Increased factor VIII complex in fulminant hepatic failure.

In 30 patients with fulminant hepatic failure (FHF) all three components of the factor VIII complex were significantly increased (VIIIC = 6.43 +/- 1.12 u/ml; VIIIRAg = 3.91 +/- 0.25 u/ml; VIIIvWF = 3.89 +/- 0.29 u/ml). There was good correlation between all three parameters in control subjects, but only between VIIIRAg and VIIIvWF (r = 0.67; p less than 0.001) in patients with FHF. VIIIC was significantly higher than VIIIRAg and VIIIvWF. These results suggest that VIIIC and VIIIRAg are increased by different mechanisms in FHF. These processes may include endothelial cell damage, reduced reticuloendothelial system function and lack of production of inactivating substances by the damaged liver. Platelet adhesion to glass beads was increased in FHF (36.4 +/- 5.9% compared to 16.6 +/- 2.1%, p less than 0.01). There was no significant correlation between platelet adhesion and any of the parameters of the factor VIII complex. Thus the increase in platelet adhesion cannot be due to the increase in VIIIvWF in FHF.

Heparin response and clearance in acute and chronic liver disease.

Patients with liver disease are at risk of bleeding due to abnormalities of the clotting system although they must be anticoagulated if they require haemodialysis or haemoperfusion. The anticoagulant of choice is heparin. In this study we have investigated heparin kinetics in patients with fulminant hepatic failure (FHF) after a single intravenous dose of heparin (2,500 units) and found there was an increased clearance of heparin whether measured by its anti-Xa effect (t 1/2 = 27.8 +/- 2.9 min compared to t 1/2 = 50.2 +/- 2.7 min in normal controls p less than 0.001) or by the whole blood activated clotting time (t 1/2 = 23.7 +/- 2.2 min compared to t 1/2 = 37.0 +/- 2.0 min p less than 0.001). There was a decreased peak level of heparin measured by anti-Xa effect (peak level in FHF = 0.48 +/- 0.05 u/ml and in controls = 0.69 +/- 0.04 u/ml, p less than 0.02), but an increased sensitivity to heparin (sensitivity in FHF = 0.072 +/- 0.011 sec/unit, in controls 0.033 +/- 0.003 sec/unit, p less than 0.001). Patients with FHF had very low levels of antithrombin III (AT III), but there was no correlation between this and any parameters of heparin effect or clearance. In a group of patients with chronic liver disease heparin kinetics did not differ from controls despite low levels of AT III. The changes in heparin kinetics in FHF are likely to be complex with the balance between the proteins that act as cofactors, (e.g. AT III) and the proteins that have heparin neutralising activity, controlling the response of added heparin.

Clearance and tissue distribution of staphylococcal enterotoxin A in the rat and potential use of adsorbents for removal from plasma.

Many of the profound effects of staphylococcal septicaemia are thought to be the result of entry of enterotoxins into the systemic circulation. The aim of this study was to investigate the disposition of staphylococcal enterotoxin A (SEA) in the rat and its possible removal from blood. SEA labelled with 125I was administered intravenously (250 micrograms/kg) to rats. The blood clearance of SEA showed a biphasic pattern; an initial fast disappearance (half-life c. 3 min) was followed by a slower one (half-life c. 2 h). Thirty minutes after injection of 125I-labelled SEA, most of the radioactivity was concentrated in the kidneys, indicating that renal excretion was the main route of elimination of SEA. The adsorption capacities of polymer-coated activated charcoal (DHP-1 and Adsorba 150C), uncharged resin (Amberlite XAD-7), anion exchange resin (Dowex-1) and polymyxin B matrix were assessed by measurement of the equilibrium adsorption isotherms for SEA. DHP-1 charcoal, Amberlite XAD-7 resin and Dowex-1 resin adsorbed similar amounts of SEA in human plasma. Plasma perfusion experiments were performed in vitro with small columns containing either charcoal or resin adsorbents. Over 4 h perfusion, DHP-1 charcoal removed 50% of the initial amount of 125I-SEA, Adsorba 150C charcoal 8.1% of SEA and Amberlite XAD-7 resin 32.5% of SEA. These results suggest that it may be feasible to develop the adsorbent columns for direct removal of SEA from the plasma of patients with staphylococcal septicaemia.

In vivo and in vitro studies on the protective effects of 9 beta-methylcarbacyclin, a stable prostacyclin analogue, in galactosamine-induced hepatocellular damage.

Early treatment with prostacyclin (PGI2) was previously shown to reduce mortality in the galactosamine model of acute hepatic failure in the rat, with a decreased release of hepatic cytosolic and lysosomal enzymes. In this study, 9 beta-methylcarbacyclin, a chemically stable analogue of PGI2, had similar protective effects to PGI2 in vivo but required approximately 100-fold higher concentrations (2 mg kg-1). These effects were only obtained when 9 beta-methylcarbacyclin was given early (0 to 6 h post-galactosamine) but not later (24 to 30 h). In isolated rat hepatocytes in vitro galactosamine up to a concentration of 100 mM caused a dose-dependent inhibition of L-[U-14C] leucine incorporation into protein and increase in the release of the cytoplasmic enzyme lactate dehydrogenase. Studies on the short-term effects of 9 beta-methylcarbacyclin using isolated hepatocytes treated with galactosamine (5 mM) showed that this agent, at an optimum concentration of 30 ng ml-1, was capable of significantly reducing the inhibition of protein synthesis caused by galactosamine but did not alter the rate of release of lactate dehydrogenase. The results demonstrate that the protective effects of 9 beta-methylcarbacyclin occur early in the time course of galactosamine action, and include direct effects on the hepatocytes.

Increased elastase-alpha 1-antitrypsin complex in fulminant hepatic failure: relationship to bacterial infection and activation of coagulation.

To study the effect of infection, a frequent complication of fulminant hepatic failure (FHF), on the release of elastase from polymorphonuclear leucocytes and its inhibition in circulation we have measured the concentrations of alpha 1-antitrypsin, which binds and inhibits elastase in the circulation, and of elastase-alpha 1-antitrypsin complex, in 30 patients with FHF. Elastase-alpha 1-antitrypsin complex was significantly increased in FHF as compared to controls (303 +/- 51 micrograms/l compared to 37 +/- 5 micrograms/l; n = 10; P less than 0.001) demonstrating activation of leucocytes in FHF. Infection caused greater release of leucocyte elastase, complex levels were significantly greater in patients who were infected when compared to those who were not (463 +/- 84 micrograms/l; n = 13 compared to 180 +/- 46 micrograms/l; n = 17; P less than 0.01). Also patients who survived had significantly lower complex levels than those who did not (212 +/- 49 micrograms/l; n = 18 compared to 440 +/- 94 micrograms/l; n = 12; P less than 0.02). alpha 1-Antitrypsin activity was not significantly different from control subjects (0.99 +/- 0.06 U/ml compared to 0.97 +/- 0.05 U/ml). However alpha 1-antitrypsin activity was significantly higher in patients who survived (1.17 +/- 0.05 U/ml; n = 18) compared to those who did not (0.71 +/- 0.03 U/ml; n = 12; P less than 0.001) and patients who died had significantly lower levels than control subjects (P less than 0.01) indicating the importance of maintenance of normal inhibitor levels in patients with FHF. The leucocyte activation and release of elastase in FHF was linked to activation of the coagulation system; elastase--alpha 1-antitrypsin complex levels correlated significantly with thrombin-antithrombin III complex levels (r = 0.68; P less than 0.001) and inversely with fibrinogen (r = -0.71; P less than 0.001).

Fibrinolytic abnormalities following liver transplantation in patients with fulminant hepatic failure.

OBJECTIVE: To evaluate the fibrinolytic system after liver transplantation in patients with fulminant hepatic failure. DESIGN: Seven patients were studied prior to, and for 4 days after, liver transplantation. METHODS: Both activators and inhibitors of the fibrinolytic system were investigated in seven patients with fulminant hepatic failure who underwent liver transplantation. RESULTS: alpha 2-antiplasmin and C1-inhibitor levels increased rapidly after transplantation (81 and 53% of normal on day 1; 106 and 99% on day 2, respectively). Plasminogen levels remained low throughout the 4-day study period. Plasminogen activator inhibitor-1 was higher than normal before transplantation (21.0 compared with 7.4 U/ml) and increased further on the first day after operation (37.5 U/ml; P < 0.05 versus pre-transplantation). Tissue plasminogen activator levels remained normal (pre-operative, 7.0 IU/ml; Day 4, 0.2 IU/ml). D-dimer remained elevated during the postoperative period showing increased fibrinolytic activity. Thrombin-antithrombin III complex was also elevated during the study period. Antithrombin III was greatly reduced prior to transplantation (13.7% of normal) and plasma levels were less than 50% of normal values during the study. CONCLUSIONS: Measures of fibrinolytic activity are raised after liver transplantation in patients with fulminant hepatic failure. This is probably due to increased fibrin formation caused by a coexisting hypercoagulable state.

Use of extracorporeal liver assist device and auxiliary liver transplantation in fulminant hepatic failure.

The case history of a 14-year-old boy with fulminant hepatic failure secondary to non-A, non-B hepatitis who fulfilled selection criteria for orthotopic liver transplantation is described. Two forms of liver support were used (extracorporeal liver assist device and an auxiliary partial orthotopic liver transplantation) to provide additional time to allow spontaneous recovery to occur. During the 66 h of extracorporeal haemoperfusion through the device, haemodynamic stability was maintained along with improvements in serum bilirubin (555 to 381 mumol/l), and international normalized ratio (INR) (3.7 to 2.9). Deterioration in these parameters was observed following cessation of treatment and 10 h later, after a donor liver had become available, an auxiliary transplant was performed. Clinical recovery, though initially slow, was eventually complete, with histopathological and scintigraphic evidence of full liver regeneration at 3 months. Withdrawal of his immunosuppressive drugs began at 6 months and was complete by 14 months after auxiliary transplantation. He has since remained well with normal liver function tests. Temporary liver support may provide additional time for spontaneous recovery of the native liver to occur in selected cases of fulminant hepatic failure, even when criteria are fulfilled for orthotopic liver grafting.

Hepatocyte growth factor and plasminogen activation in fulminant hepatic failure.

Human hepatocyte growth factor (hHGF) has considerable sequence homology with plasminogen and both proteins can be activated by plasminogen activators. The aim of this study was to investigate the relationship between plasma hHGF and fibrinolysis in patients with fulminant hepatic failure (FHF), in whom proteases of coagulation are known to be activated and hHGF levels have been shown to be raised as a consequence of hepatic regeneration. Serum hHGF measured by ELISA was increased in FHF (median 6.67 ng/ml, range 1.2-62 ng/ml), but the values did not correlate with the decreased plasminogen level (median 9%., range 0.7-35.5%) or the level of t-PA which was normal. There was a significant correlation between serum hHGF and increased plasma D-dimer (median 2,163 microgram/l, range 39-7 311 microgram/l), produced by the action of plasmin on fibrin and increased plasma thrombin-antithrombin III complexes (TAT, median 31.7 microgram/l, range 3.7-105 microgram/l). These relationship could be indicative of an involvement of blood coagulation, possibly a specific serine protease, in hHGF activity. After liver transplantation, plasma hHGF was rapidly cleared to almost normal levels, whereas D-dimer and TAT continued to be at elevated levels.

9 beta-methylcarbacyclin, a stable prostacyclin analogue, in prevention of platelet losses during charcoal haemoperfusion.

The effect of 9 beta-methylcarbacyclin, a stable analogue of prostacyclin, as a platelet protective agent, has been investigated during in-vitro charcoal haemoperfusion with fresh heparinized human blood, a model of progressive platelet damage. There were no platelet losses over 3 h perfusion after an initial bolus (1 microgram ml-1) of 9 beta-methylcarbacyclin (101.6 +/- s.e. 1.9% of initial value) whereas in the paired control circuit there was a significant reduction in the platelet level (73.5 +/- 4.1%, P less than 0.05). Prevention of rises in plasma thromboxane B2 by 9 beta-methylcarbacyclin confirmed the lack of platelet damage and a significant, but less marked, effect on white cell losses was observed. In a second series of experiments a bolus of 9 beta-methylcarbacyclin in one circuit was compared with prostacyclin infusion in the other which demonstrated that this prostaglandin analogue was as effective as prostacyclin and on the basis of these initial results it would merit clinical evaluation.

Screening of hepatoprotective plant components using a HepG2 cell cytotoxicity assay.

Identification of the active components of plants with hepatoprotective properties requires screening of large numbers of samples during fractionation and purification. A screening assay has been developed based on protection of human liver-derived HepG2 cells against toxic damage. Various hepatotoxins were incubated with HepG2 cells in 96-well microtitre plates (30,000 cells well-1) for 1 h and viability was determined by metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS). Bromobenzene (10 mM) and 2,6-dimethyl-N-acetyl-p-quinoneimine (2,6-diMeNAPQI, 200 mM) had greater toxic effects than tert-butyl hydroperoxide (1.8 mM) or galactosamine (10 mM), reducing mean viability to 44.6 +/- 1.2% (s.e.m.) and 56.1 +/- 2.1% of control, respectively. Protection against toxic damage by these agents was tested using a crude extract of a known hepatoprotective Sri Lankan plant, Osbeckia aspera, and two pure established hepatoprotective plant compounds, (+)-catechin and silymarin (1 mg mL-1). Viability was significantly improved by Osbeckia (by 37.7 +/- 2.4%, P < 0.05, and 36.5 +/- 2.1%, P < 0.05, for bromobenzene and 2,6-diMeNAPQI toxicity, respectively). Comparable values for (+)-catechin were 68.6 +/- 2.9% and 63.5 +/- 1.1%, and for silymarin 24.9 +/- 1.4% and 25.0 +/- 1.6%. This rapid and reproducible assay should prove useful for the isolation and identification of active hepatoprotective compounds in crude plant extracts.

Protective effects of Osbeckia octandra against galactosamine and tert-butyl hydroperoxide induced hepatocyte damage.

Ayurvedic and other 'traditional' medical practitioners in Sri Lanka use the mature leaves of the plant Osbeckia octandra for its hepatoprotective properties. In this study the effects of an aqueous extract of Osbeckia octandra against injury induced by D-galactosamine and tert-butyl hydroperoxide (TBH) were investigated in freshly isolated rat hepatocytes. The plant extract (500 micrograms/ml) significantly reduced the inhibition of protein synthesis (as assessed by the incorporation of 14C-leucine into protein) in hepatocytes incubated for 1 h with 10 mM galactosamine by a mean of 25.6 +/- 3.6% and decreased the release of cellular lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) enzyme activities into the medium by 55.3% and 32.8%, respectively. With TBH, the plant extract decreased lipid peroxidation (estimated from malondialdehyde formation) by a mean of 29.9 +/- 1.1% together with a 46.8% and 54.7% decrease in the release of LDH and AST, respectively into the incubation medium. Significant protection was also obtained when the Osbeckia extract was added to the incubation medium up to 30 min after pre-exposure of the hepatocytes to either galactosamine or, to a lesser extent, TBH. The results support the use of Osbeckia as a hepatoprotective agent.

Determination of the ketone body ratio in fulminant hepatic failure.

The ratio of acetoacetate to beta-hydroxybutyrate, the ketone body ratio, was measured in arterial blood from 28 patients with fulminant hepatic failure as an index of the hepatic energy charge. The ketone body ratio was significantly reduced in the total group of patients with fulminant hepatic failure as compared with control subjects (0.27 +/- 0.03 SE as compared with 0.48 +/- 0.03; p less than 0.001). Patients who survived had significantly less reduction of the ketone body ratio on admission than those who died (0.39 +/- 0.06, n = 10 as compared with 0.20 +/- 0.02, n = 19; p less than 0.02). In seven patients who died, in whom ketone body ratio was measured less than 12 hours before death there was a significant decrease in ketone body ratio as compared with that on admission (0.24 +/- 0.05 to 0.15 +/- 0.04; p less than 0.05). In contrast, in seven patients who survived there was no significant change in ketone body ratio when measured within 12 hours of regaining consciousness as compared with the figures on admission. Measurement of arterial ketone body ratio may give an indication of prognosis, and may be of use in testing the efficacy of treatments which aim to enhance hepatic regeneration or to remove toxic substances that may reduce the hepatic energy charge.

Effect of supernatants from Kupffer cells stimulated with galactosamine and endotoxin on the function of isolated rat hepatocytes.

Activated Kupffer cells may release substances that are involved in liver injury induced by galactosamine and endotoxin. In the present study Kupffer cells were isolated from rat livers, cultured for 24 hours and incubated with galactosamine, endotoxin (LPS) or tumor necrosis factor-alpha for 4 or 24 hours. The Kupffer cell-conditioned media were than added in separate experiments to freshly prepared isolated rat hepatocytes to determine their cytotoxic effect. No significant effects on the rate of protein synthesis, as assessed by the incorporation of 14C-leucine on lactate dehydrogenase enzyme release from hepatocytes during 1 h incubation was found as compared with conditioned media from control Kupffer cells. In further experiments, Kupffer cells incubated for 4 hours with LPS and galactosamine were shown to produce thromboxane B2 and also the potentially cytoprotective prostaglandins PGE2 and small amounts of prostacyclin measured as 6-keto-PGF1 alpha. It is concluded that under the conditions of the present experiments, factors secreted by cultured Kupffer cells have no cytotoxic effects on isolated rat hepatocytes during short-term incubation.

In vitro studies on the immunomodulatory effects of extracts of Osbeckia aspera.

Ayruvedic medical practitioners in Sri Lanka use aqueous extracts of the mature leaves of Osbeckia aspera to treat liver disease. The extract has been shown to have hepatoprotective effects in vitro and in vivo, and to have inhibitory effects on the complement system and on in vitro phagocytosis by polymorphonuclear cells. The aim of this study was to investigate the effect of an aqueous extract of Osbeckia on lymphocyte proliferation stimulated by mitogens and antigen. In control peripheral blood mononuclear cells (PBMC), high concentrations of the Osbeckia extract were inhibitory to proliferation stimulated by phytohaemagglutinin (PHA) and tuberculin purified protein derivative (PPD). On stimulation by phorbol myristate acetate and ionomycin (PMA+I) the extract showed stimulation of proliferation at low concentrations (<10 microg/ml) with inhibition at higher concentrations. A similar inhibitory pattern on mitogen/antigen stimulation was seen with PBMC from patients with chronic hepatitis C virus (HCV) infection. These results suggest that the inhibitory agent(s) in the aqueous extract of Osbeckia may have an effect on antigen-presenting cell function. The combined hepatoprotective and immunosuppressive effects of the extract are more likely to be beneficial in acute hepatitis rather than chronic hepatitis viral infection.

Review of methods to remove protein-bound substances in liver failure.

A wide range of toxic substances accumulates in the circulation of patients with liver failure, including more lipid-soluble substances, which bind to plasma proteins. Serum albumin is the most important binding protein for ligands such as bilirubin and bile acids, which are potentially toxic and can cause apoptosis in astrocytes and hepatocytes respectively in vitro. Resin haemoperfusion was originally investigated to remove these compounds, as well as inflammatory cytokines. Current effective methods for removal of protein-bound compounds in patients with liver failure include high volume plasmapheresis and different forms of albumin dialysis. Bioartificial liver support systems need adsorbent and/or dialysis modules to replace the lack of excretory function.