A 15N GC/MS study of in vivo glutamine synthesis in liver failure rats.

To clarify the nature of nitrogen metabolism between branched chain amino acid (BCAA) and glutamine (Gln) in liver failure, we measured arterial plasma concentrations of Gln and 15N uptake to amino-N and amide-N of Gln in normal and D-galactosamine-induced fulminant hepatic failure (FHF) rats after 15N-leucine (Leu) injection. Fifteen, 30 and 60 min after Leu injection, the arterial plasma concentrations of Gln were significantly higher in FHF rats than in controls. The concentrations of amino-15N Gln were also significantly higher in FHF rats than in controls at 5, 15, 30 and 60 min after injection. The concentrations of amide-15N Gln did not significantly differ between FHF and controls at 5, 15 and 30 min. However, at 60 min, the concentration was significantly higher in the FHF rats. The higher uptake of 15N to amino-N of Gln in FHF rats suggests the presence of an enhanced ability to synthesize Gln from Leu in FHF rats. The higher uptake of 15N to amide-N of Gln in FHF rats at 60 min after injection suggests that excessive administration of BCAA to patients with severely impaired urea-cycle capacity suffering with hepatic failure may lead to greater levels of hyperammonemia.

Ca2+ influx initiates death of hepatocytes injured by activation of complement.

To clarify the role of cytosolic Ca2+ in hepatocellular death, we exposed cultured hepatocytes to human serum and a monoclonal antibody directed against rat liver plasma membranes to produce complement-mediated cell injury. The change in cytosolic Ca2+ concentration was measured by fura2 and fluo3 fluorescence. With the addition of monoclonal antibody, an increase in cytosolic Ca2+ was observed, followed by cell death. Both the increase in intracellular Ca2+ and cell death were prevented by intracellular Ca2+ chelation or removal of extracellular Ca2+. We conclude that an increase in cytosolic Ca2+ plays a major role in hepatocellular injury induced by exposure of the cell membrane to monoclonal antibody.

Evaluation of serum uric acid to creatinine ratio in fulminant hepatitis.

Of the eight patients with fulminant hepatitis placed under total parenteral nutrition with an amino acid solution rich in branched chain amino acids and treated by plasma exchange, four survived and four died from hepatic failure. Serum uric acid levels in the non-survived group were significantly lower on days 1-6 compared with the survived group. The concentration ratios of uric acid to creatinine and prothrombin time were significantly lower on days 5-8 and days 3-8, respectively, in a similar comparison. Thus, the uric acid to creatinine ratio, which corrects for the possible renal dysfunction associated with acute hepatic failure, may serve as a clinically useful prognostic indicator for patients with fulminant hepatitis.

A multicenter study on the prognosis of fulminant viral hepatitis: early prediction for liver transplantation.

To determine the risk of death at an early stage of fulminant viral hepatitis, we created severity indexes drawn from clinical data on the day of development of encephalopathy in 128 patients with fulminant hepatitis B and 103 with fulminant hepatitis non-A, non-B. In fulminant hepatitis B, the risk score was 2.75 x BL + 2.75 x BR + 2.7 x AG + 2.3 x WB + 1.67 x CD + 1.56 x AL - 0.098 x PR - 0.88, where BL is 1 if total bilirubin is higher than 20 mg/dl, BR is 1 if the ratio of total to direct bilirubin exceeds 2.2, AG is 1 if age is above 40 yr, WB is 1 if white blood cell count is less than 4,000 cells/mm3 or more than 18,000 cells/mm3, CD is 1 if a hazardous disease coexists and AL is 1 if ALT is less than 100 times the upper limit of normal (otherwise all are 0), and PR is prothrombin time (percentage of normal value). Using a cutoff score of 0, we found the positive predictive value, negative predictive value and predictive accuracy to be 0.90, 0.86 and 0.89, respectively. Sensitivity and specificity were 0.94 and 0.77, respectively. In fulminant non-A, non-B hepatitis, the risk score was 2.66 x BR + 2.25 x BL + 2.24 x DI + 2.05 x AL +/- 1.38 x AG + 0.00021 x WB - 6.33.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration of a branched-chain amino acid preparation during hepatic failure: a study emphasizing ammonia metabolism.

We administered a branched-chain amino acid (BCAA) infusion to 16 patients with hepatic failure and two healthy subjects, and then evaluated its effects on ammonia metabolism and amino acid metabolic pool. Immediately after the BCAA infusion, the venous blood ammonia concentration increased in 12 of 15 patients with hepatic failure and in both two healthy subjects. Glutamine (Gln) also rose in all cases following the BCAA infusion, and this rise was particularly marked in the hepatic failure group. The increase in Gln due to the BCAA infusion and the arteriovenous difference in the pre-administration ammonia concentration showed a good correlation. These results suggest an increase in glutamine cycle capacity in patients with hepatic failure.

Evaluation of antibody against AR142, a synthetic peptide derived from the core sequence of hepatitis C virus, in the diagnosis of non-A, non-B chronic liver disease.

We evaluated serum anti-hepatitis C virus (HCV) using a synthetic peptide (AR142) which includes an epitope in the core region of HCV. The incidence of anti-AR142 in 98 patients with type non-A, non-B chronic liver diseases (NANB-CLD) was 89.8%, while all the 28 patients with non-type C chronic liver diseases were negative for anti-AR142. Among 98 NANB-CLD patients, 74 were positive for both anti-AR142 and anti-C100-3, 23 showed discordant results, and one was positive for neither. Eighty-one NANB-CLD patients underwent reverse transcription-polymerase chain reaction assay to detect viremia and 76 (93.8%) had a detectable level of HCV-RNA. Titers of anti-AR142 were not different among groups of different disease activities, genotypes of HCV, nor amount of serum HCV-RNA. These observations suggest that anti-AR142 could be a useful marker for chronic HCV infection.

The role of Kupffer cells in complement activation in D-Galactosamine/lipopolysaccharide-induced hepatic injury of rats.

To investigate the role of Kupffer cells in complement activation, we used a rat model of acute hepatic injury induced by D-Galactosamine (GalN) and lipopolysaccharide (LPS). In in vivo study, minimal histological changes were observed after i.p. GalN (200 mg/kg) single administration. Complement hemolytic activity (CH 50) decreased to 70% of its initial value 2-3 h after i.p. LPS (1.5 mg/kg) single administration. Massive hepatic necrosis was induced by simultaneous administration of GalN and LPS. After 2-3 h, CH 50 decreased to 70% of its initial value, and deposition of C3 fluorescence (C3) was observed in Kupffer cells. After 4 h, GPT was greatly increased (1286 +/- 240 IU/l), CH 50 was further reduced, and C3 was observed on hepatocyte membranes and in the cytosol. In in vitro study, we used hepatocyte cultures and co-cultures of hepatocytes and Kupffer cells to investigate the participation of GalN, LPS, complement, and Kupffer cells in hepatic cell necrosis. We found no increase of LDH (% leakage) when LPS and complement were added to the medium (22.7 +/- 5.7%). A moderate increase was observed with the addition of GalN (33.2 +/- 2.6%). A remarkable increase was observed only with the addition of GalN, LPS, and complement to the co-culture (50.0 +/- 8.8%). These results suggest that Kupffer cells activated by LPS are very important in promoting acute hepatic injury by complement.

[Amino acid metabolism in liver cirrhosis].

In patients with liver cirrhosis the fasting plasma alpha-amino nitrogen concentration is high as the rule, due to reduced clearance of total alpha-amino nitrogen. The urea cycle is diminished of its capacity in cirrhotic patients than in the control subject, and to compensate for this, the extrahepatic glutamine cycle capacity is enlarged in the patients. The following important topics were taken up in this mini review: some problems concerning Fischer ratio, amino acids metabolism and pH regulation in the liver, and the supplementation therapy with branched chain amino acids under the condition of organ relationship.

Immunotherapy using Freund's adjuvant and recombinant interleukin-2 combined with transarterial chemoembolization for hepatocellular carcinoma.

A new immunotherapy for hepatocellular carcinoma (HCC) using Freund's adjuvant and recombinant interleukin-2 (IL-2) combined with conventional transarterial chemoembolization therapy was performed. In 16 patients with HCC and one patient with metastatic liver cancer receiving this therapy, decrease and suppression of reelevation of alpha-fetoprotein after therapy was observed. Disappearance of tumor thrombi of HCC in the main portal vein was observed in a patient, and decrease of carcinoembryonic antigen was also observed in a patient with metastatic liver cancer. The present therapy using Freund's adjuvant and IL-2 is likely to open a new avenue for the treatment of patients with advanced liver cancer.

Dynamic relationship between urea and glutamine synthesis in the mechanism of ammonia detoxication: a tracer study using 15NH4Cl in fulminant hepatic failure rats.

Fulminant hepatic failure (FHF) was produced in rats with intraperitoneal injection of D-galactosamine. 15NH4Cl (50 mg/kg of body weight) was injected into the rats via the tail vein. Arterial blood was drawn before and 5, 15, 30, 60 min after the injection of 15NH4Cl. 15N-ammonia, -urea, and -glutamine (amide and amino) were determined by gas chromatography and mass spectrometry. The plasma 15N-ammonia level was higher but decreased more rapidly in the FHF rats than in the control rats. This suggests that in FHF rats, the systemic vascular pool of ammonia is enlarged and ammonia clearance from blood is increased. The incorporation of 15N into urea was significantly different between the two groups. In FHF rats, the plasma urea-15N level rose 5 min after the injection, decreased at 15 min and was elevated again up to 60 min after injection. This biphasic change suggests that in FHF rats the incorporation of 15N into the extrahepatic glutamine pool is accelerated up to 15 min and that after 30 min a larger volume of glutamine-amide-15N is transferred to the urea cycle.

Photodynamic therapy of rat liver cancer: protection of the normal liver by indocyanine green.

The application of photodynamic therapy (PDT) to hepatocellular carcinoma (HCC) has been difficult because hematoporphyrin derivatives (HpD) accumulate not only in cancer cells but also in normal hepatocytes and, hence, laser irradiation causes injuries in both tissues. Protection of the normal liver tissue from laser phototoxicity was demonstrated using indocyanine green (ICG) as a protective agent. In vitro, argon laser irradiation decolored the green tint of ICG much faster in solutions containing HpD than those without, suggesting that ICG captured singlet oxygen from HpD. Degeneration of Change hepatocytes induced by HpD and laser irradiation was prevented by an addition of ICG into the medium. In vivo, laser irradiation of the rat liver surface caused hyperemia when HpD was injected two days before, while the hyperemia was much milder in rats additionally receiving ICG injection 10 minutes before the irradiation. ICG injected into rat HCC accumulated only in the normal liver tissue. Laser irradiation of rat HCC preinjected with both HpD and ICG destroyed only the cancer tissue, while the surrounding liver tissue was preserved. Both in vitro and in vivo results suggest that ICG has a scavenger effect against excited oxygen and it might be used as a protective agent in PDT of HCC.

Increased urine level of amino-terminal peptide derivatives of type III procollagen in patients with liver diseases.

The amino-terminal peptides of type III procollagen (PIIIP) in the urine of 40 patients with various liver diseases were determined with a commercial radioimmunoassay kit. The level of urinary PIIIP (uPIIIP) was correlated well with serum PIIIP (sPIIIP) in 9 patients, the coefficient of correlation being r = 0.836 (p less than 0.01) and the regression line being y = 1.42x + 24. Urinary PIIIP consisted of at least 4 different molecular species with molecular weights of 49 k, 18 k, 10 k and 4.6 k as estimated by column chromatography on Sephadex G-100. Furthermore. uPIIIP was found to be significantly elevated in acute hepatitis, chronic hepatitis, liver cirrhosis, hepatocellular carcinoma and other liver diseases, in which the elevation of sPIIIP has been reported by others. The mean values +/- standard deviations of uPIIIP were 44.0 +/- 32.0, 60.4 +/- 32.0, 62.0 +/- 46.5, 53.0 +/- 27.1 and 48.1 +/- 22.8 ng/ml for the respective liver diseases, and 13.2 +/- 4.5 for the non-hepatic disease group.

Metabolism of branched-chain amino acids in rats with acute hepatic failure: a tracer study using 15N-leucine.

Fulminant hepatic failure (FHF) was produced in rats with intraperitoneal injection of D-galactosamine. Control rats received only physiological saline. 15N-leucine (200 mg/kg of body weight) was injected into the rats via the tail vein. Arterial blood was drawn before and 5, 15, 30 and 60 min after the injection of 15N-leucine. 15N-amino acids were determined quantitatively by gas chromatography and mass spectrometry. The plasma 15N-leucine level decreased logarithmically in the same manner in both groups. This result suggests that leucine is mainly metabolized in extrahepatic tissues. The incorporation of 15N into plasma isoleucine and valine was not significantly different between the groups. Plasma alanine and glutamine concentrations increased in controls and decreased in FHF rates after the injection. The incorporation of 15N into plasma alanine in rats with FHF was significantly later than in controls. This result may suggest that undergoing hyperammonemia causes to form more glutamine from glutamate in extrahepatic sites as the same manner as for chronic hepatic failure. Additionally, insulin levels increased temporarily after the injection of leucine in both groups. This increase may play a role in the decrease in plasma isoleucine and valine concentrations after injection of leucine.

Peritoneoscopy of alcoholic liver cirrhosis in comparison with non-alcoholic liver cirrhosis.

Peritoneoscopic findings of 39 patients with alcoholic liver cirrhosis (ALC) were compared with those of 95 patients with non-alcoholic liver cirrhosis (NALC). They were selected from 245 patients with liver cirrhosis subjected to peritoneoscopy in the 7 year period from 1975 to 1981. Out of the 95 NALC patients, 24 had hepatitis B surface antigen. The ALC patients had nodules which varied in size (61%), large depressions (69%), and a markedly rounded edge of the liver (33%) more often than NALC patients (18, 43 and 3%, respectively). Nodularity differed between the right and left lobes in ALC (41%) more often than in NALC (16%). Interstitial reddish markings and patchy nodules were, however, more frequent in NALC (51 and 28%, respectively) than in ALC (8 and 5%, respectively). Lymphatic vesicles were observed both in ALC (85%) and NALC (78%). In conclusion, the peritoneoscopic features which suggested ALC were the coexistence of nodules of various sizes, large depressions and a markedly dull edge of the liver. Interstitial reddish markings and patchy nodules were more indicative of NALC than ALC.

Comparison of estimation methods of liver maximum removal rate of indocyanine green.

Three linear plots by which the liver's maximum removal rate (Rmax) of indocyanine green (ICG) and the Michaelis constant (Km) can be calculated were compared in a microcomputer simulation study. The widely-used Lineweaver-Burk plot (1/V vs. 1/S; V, ICG initial removal rate (mg/kg/min); S, ICG loading dose (mg/kg] presented the greatest bias and variance. There was no remarkable difference in bias between the S/V vs. S plot and the V vs. V/S plot, but the latter possessed a smaller variance. Therefore, the V vs. V/S plot was considered the best for estimating Rmax. The best combination of three ICG loading doses was 0.5, 2, and 5 mg/kg. This combination was selected by comparison of the Rmax estimated from three points with that estimated from six points (0.5, 1, 2, 3, 4 and 5 mg/kg).

Comparative diagnosis of alcoholic liver diseases by multivariate and histological analysis.

Sixty-seven cases of alcoholic liver disease were histologically classified into 4 groups: alcoholic liver cirrhosis (ALC), alcoholic hepatitis (AH), alcoholic liver fibrosis (ALF) and alcoholic fatty liver (AFL). They were statistically reclassified by the likelihood method using age, total alcohol intake, hepatomegaly and 12 liver function tests. A score table for likely diagnosis was constructed from the incidences of each range. The cases were re-evaluated using the score table, with an overall correct diagnosis rate of 73%. The best combination of 5 parameters included the indocyanine green plasma disappearance rate, total alcohol intake, cholesterol, choline esterase and glutamic oxaloacetic transaminase/glutamic pyruvic transaminase ratio. A correct diagnosis rate of 75% was attained using these 5 parameters, and 94% of patients were correctly diagnosed by the first or the second likelihood diagnosis. Differential diagnosis of alcoholic liver diseases was easily and confidently obtained with the likelihood score table.

Effect of ammonia on plasma and cerebrospinal fluid amino acids in dogs with and without portacaval anastomoses.

To elucidate the possible connection between ammonia-induced changes of plasma and cerebrospinal fluid (CSF) amino acid levels and the development of hepatic encephalopathy in dogs, beagle dogs were given an ammonium acetate infusion both before and following portacaval shunt (PCS). During ammonia-induced coma and after recovery in the dogs prior to PCS the plasma and CSF concentrations of most amino acids were decreased. Following PCS the plasma and CSF concentrations of the aromatic amino acids (AAA), phenylalanine and tyrosine, increased and the levels of the branched chain amino acids (BCAA), valine, leucine, and isoleucine, decreased during ammonia-induced coma. The CSF/plasma molar ratio for the AAA exhibited a marked increase after recovery as compared to the value during coma in the Eck-fistula dogs. With respect to the AAA, no correlation was observed between signs of neurologic impairment in the animals and the following parameters: glutamine and methionine levels of CSF, and the plasma molar ratio (Formula: see text). The data obtained do not support the hypothesis that high concentrations of phenylalanine and tyrosine in the brain may be primarily responsible for altered neurotransmission leading to the development of hepatic encephalopathy.

Effects of sake and bourbon on liver histopathology and function in rats.

Sake or bourbon (8g ethanol/kg body weight) was intragastrically administered to rats for 12 days. An equal dose of ethanol in water or an isocaloric glucose solution was administered to control groups. Food was withheld, but water freely provided. Neither mortality nor liver and body weights were different between the alcohol-treated groups. Glutamic oxaloacetic transaminase and glutamic pyruvic transaminase were more elevated in the sake group than in the other groups. Additionally, liver fibrosis was more pronounced, and vacuole formation or steatosis was less in this group. These results suggest that sake is more fibrogenic. Some components other than ethanol, such as long-alkyl chain alcohols, may have been responsible for the differential histopathology.