The effect of 3-sulphation and taurine conjugation on the uptake of chenodeoxycholic acid by rat hepatocytes.

The hepatic uptake of chenodeoxycholic acid, taurochenodeoxycholic acid, chenodeoxycholic acid 3-sulphate and taurochenodeoxycholate acid 3-sulphate by isolated rat hepatocytes was examined. Taurochenodeoxycholic acid, taurochenodeoxycholic acid 3-sulphate and chenodeoxycholic acid 3-sulphate uptake occurred by a saturable, energy-dependent process while chenodeoxycholic acid uptake was predominantly non-saturable, possibly simple diffusion. Apparent Km (mumol/l) and Vmax (nmol/mg protein per min) values (mean +/- S.D.), respectively, were: chenodeoxycholic acid (saturable component), 33 +/- 6.4 and 4.8 +/- 0.6; taurochenodeoxycholic acid, 11.1 +/- 2.0 and 3.1 +/- 0.5; chenodeoxycholic acid 3-sulphate, 6.1 +/- 0.9 and 2.3 +/- 0.4; and taurochenodeoxycholic acid 3-sulphate, 5.0 +/- 0.7 and 0.9 +/- 0.15. Both conjugation with taurine and sulphation at the 3 position resulted in a reduction in the values of Km and Vmax. Uptake of each of the bile acids taurochenodeoxycholic acid, taurochenodeoxycholic acid 3-sulphate and chenodeoxycholic acid 3-sulphate was competitively inhibited by the other two, with taurochenodeoxycholic acid a potent inhibitor of both taurochenodeoxycholic acid 3-sulphate and chenodeoxycholic acid 3-sulphate uptake. Other bile acids also inhibited. Uptake was inhibited by albumin in the order chenodeoxycholic acid 3-sulphate greater than taurochenodeoxycholic acid 3-sulphate greater than taurochenodeoxycholic acid and was dependent on the extent of bile acid binding to albumin.

Investigation of the role of reactive oxygen species in bilirubin metabolism in the Gunn rat.

It has been previously established that the attenuation of hepatic lipid peroxidation by a fat-free diet is accompanied by a marked rise in plasma bilirubin in Gunn rats. Present in vitro studies confirmed that microsomal lipid peroxidation caused the concurrent degradation of added bilirubin but failed to show that microsomal superoxide, hydroxyl radical or hydrogen peroxide would degrade bilirubin. Moreover, although injection of vitamin E completely inhibited microsomal lipid peroxidation and bilirubin degradation it had no effect on plasma bilirubin. No evidence has therefore been obtained that in Gunn rats, in the absence of bilirubin glucuronidation, that reactive oxygen species provide a significant physiological pathway of bilirubin disposal.

A fluorimetric and enzymatic method for the estimation of serum total bile acids.

The enzymatic technique using 3alpha-hydroxysteroid dehydrogenase for determining bile acids in blood has been modified by measuring the reduced nicotinamide adenine dinucleotide fluorimetrically. The increased sensitivity attained enables the concentration of total bile acids in serum to be estimated using 3 ml for normal subjects and 1 ml for jaundiced patients. The range of normal values in serum was found to be 0-4.7 mumol/litre for males and 1.0-8.2 mumol/litre for females.

A direct fluoroimmunoassay for conjugated chenodeoxycholic acid using antibody coupling to magnetisable particles.

A novel solid phase fluoroimmunoassay for conjugated chenodeoxycholic acid has been developed employing an antiserum coupled to magnetisable cellulose particles and a chenodeoxycholyl glycinefluorescein thiocarbamyl ethylene diamine conjugate as the label. The data obtained from serum samples from 25 patients correlated closely (r = 0.99) with those obtained by radioimmunoassay. The assay is rapid (30 minutes), simple, and ideal for routine clinical purposes.

A distinctive pattern of serum bile acid and bilirubin concentrations in benign recurrent intrahepatic cholestasis.

The pattern of serial serum bile acid and bilirubin concentrations in 3 patients with benign recurrent intrahepatic cholestasis (BRIC) was compared with those from patients with other liver diseases. In BRIC the peak bile acid concentration (260- 575 micromol/l was found at the onset of the cholestasis. The bilirubin concentration increased slowly so that maximum values (185-550 micromol/l) were attained between 33 and 51 days after the onset of symptoms. Both the serum bile acid and bilirubin concentrations returned to normal after 79 to 98 days. Percutaneous biliary drainage of extrahepatic biliary obstruction (3) caused a dramatic reduction in the serum bile acid level (mean 89% after 48 hours), but only a slight fall in serum bilirubin (mean 22%). In primary biliary cirrhosis (2) the bile acid and bilirubin concentrations changed in parallel until the onset of liver failure when serum bilirubin, but not bile acids, increased markedly. Serum bile acid and bilirubin concentrations changed in parallel throughout cholestatic viral hepatitis (2), chronic active hepatitis (2) and alcoholic hepatitis (1). The data indicates that a distinctive pattern is found in BRIC and this may be of diagnostic value.

Twenty-five years of progress in bilirubin metabolism (1952-77).

This review deals with the development of our understanding of the chemistry of bilirubin and its glucuronide derivatives during the years 1952-1977. It examines the relation between haem metabolism and bilirubin formation and our present knowledge of hepatic transport of bilirubin. The heterogeneity of familial hyperbilirubinaemia is discussed.

The use of Bond-Elut for the estimation of serum bile pigments bonded covalently to albumin.

A simple and precise method has been devised for the quantitation of biliprotein (delta-bilirubin or albumin bound bilirubin) in serum. In the presence of caffeine/benzoate, Bond-Elut (C8, 200 mg) extracts unconjugated and conjugated bilirubin but not pigments that are covalently bonded to albumin which pass through the column and can be quantitated by a standard diazo method. Following elution from the Bond-Elut column with methanol-acetonitrile (50:50, v/v) unconjugated and conjugated bilirubin can be quantitated either as total pigments or individually by HPLC.

Changes in the binding of radioactive conjugated bile salts to serum proteins in cholestatic jaundice.

1. A micro-partition centrifugal ultrafiltration technique has been used to monitor the percentage of [14C]glycocholate, [3H]glycochenodeoxycholate and [3H]glycochenodeoxycholate-3-sulphate bound to serum proteins of patients with cholestatic liver disease. 2. In comparison with normal individuals the percentage of binding of [14C]glycocholate and, to a lesser extent, of [3H]glycochenodeoxycholate and [3H]glycochenodeoxycholate-3-sulphate was reduced. 3. The binding of [14C]glycocholate was inversely related to the serum bile salt and bilirubin concentrations. In contrast, the binding of [3H]glycochenodeoxycholate and [3H]glycochenodeoxycholate-3-sulphate were not altered by the severity of the cholestasis. 4. Studies in vitro indicated that the reduction in the binding of [14C]glycocholate in cholestatic liver disease was not due to high concentrations of bile salts, unconjugated bilirubin or fatty acids.

The isolation and characterization of a bile ductule cell population from normal and bile-duct ligated rat livers.

The separation of bile ductule cells from Kupffer and sinusoidal endothelial cells in a suspension of non-parenchymal cells has been attempted. Bile duct ligation was performed so that a four-fold increase in the total number of non-parenchymal cells isolated from rat liver was attained and the proportions of both Kupffer and bile ductule cells were elevated. Rate zonal centrifugation, through a Ficoll gradient, partially separated the cells into two populations: (1) small cells (4-6 micrometer diameter) probably originating from the sinusoidal endothelium and (2) larger bile ductule and Kupffer cells (8-12 micrometer diameter). A more successful separation was achieved by isopycnic centrifugation through a linear metrizamide gradient. Bile duct ligation (14 days) altered the distribution of cells on the gradient and the peak containing bile ductule and Kupffer cells partially subdivided into the separate cell types. Antiserum raised against the bile ductule fraction was shown to be compatible with that raised against common bile duct tissue. gamma-glutamyl transpeptidase and leucine aminopeptidase activity were preferentially located in the rate zonal fraction containing bile ductule cells. Their specific activity increased after bile duct ligation as did that of beta-glucuronidase, which was raised in all cells.

The effect of secretin on bile flow and bile acid and bilirubin excretion following relief of prolonged bile duct obstruction in the rat.

Bile flow was re-established in rats whose bile ducts had been obstructed for 5, 10, 15 and 28 days (Groups I, II, III and IV, n = 5). The effect of i.v. secretin on bile flow in control rats, whose bile ducts had been cannulated, was minimal, but in cholestatic rats there was an immediate response which was related to the duration of the obstruction and the degree of bile duct proliferation. In 40 min the mean excess bile flow production amounted to 76, 258, 320 and 432 microliters/100 g body wt. in Groups I, II, III and IV, respectively. Choleresis was prolonged in the Group IV rats that had developed cirrhosis. Synthetic secretin had a minimal effect on bile acid and bilirubin excretion. It is postulated that the proliferating bile ductules are the site of secretin choleresis, although the possibility that reduced inactivation of the hormone plays a role cannot be excluded.

Enzymic oxidation of unconjugated bilirubin by rat liver.

The presence of the enzyme bilirubin oxidase, which degrades bilirubin in vitro, was demonstrated in the liver. Subcellular-fractionation experiments indicate that bilirubin oxidase is located in both the inner and outer membranes of the mitochondria. The mean rate of the reaction is 1.57 +/- 0.38 (S.D.) nmol of bilirubin degraded/min per mg of mitochondrial protein (munits/mg of protein). With respect to the overall breakdown of bilirubin, the enzyme has a Km' of 136 microM-bilirubin and a Vmax.' of 9.13 munits/mg of protein. Its activity is influenced by the ionic strength of the media and is inhibited by KCN, thiol reagents, NADH and albumin. The enzyme is aerobic, and between 1 and 1.5 mol of O2 are consumed per mol of bilirubin degraded. The products of the reaction include propentdyopents. The hepatic bilirubin oxidase activity of the jaundiced Gunn-rat liver is not significantly different from that of the Sprague-Dawley rat, and it is not induced by beta-naphthoflavone.

Hepatic transport of sulfated and nonsulfated bile acids in the rat following relief of bile duct obstruction.

The effect of bile duct ligation for 5 days on the hepatic transport of sulfated and nonsulfated bile acids was studied. Tracer doses of radioactive bile acids [3H]taurochenodeoxycholate-3-sulfate [3H]chenodeoxycholate-3-sulfate, [3H]taurochenodeoxycholic acid and [14C]taurocholic acid were injected 90 min after relief of obstruction when the plasma total bile acid concentration had reverted to normal. Plasma clearance and biliary excretion of sulfated bile acids were lower than those of nonsulfated bile acids, particularly in the cholestatic rats (p less than 0.02). For each bile acid, hepatic transport in the cholestatic rats was significantly reduced compared with the control rats. [3H]Chenodeoxycholate-3-sulfate and [3H]taurochenodeoxycholic acid were partially metabolized to [3H]taurochenodeoxycholate-3-sulfate prior to biliary excretion. This data suggests that the hepatic transport system for sulfated bile acids is less efficient and more easily impaired by cholestasis than that for nonsulfated bile acids.

Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat.

Hepatic morphological abnormalities were examined in rats whose bile ducts had been either cannulated and then obstructed or irreversibly ligated for 5, 10, 15 and 28 days or longer. Throughout the experiment most of the morphological changes observed in the cannulated group were comparable to those in the ligated group. Portal inflammation and marginal bile duct proliferation were noted with the same frequency in both groups. Biliary obstruction for 15 days or more led to cirrhosis. After 28 days obstruction, five out of six cannulated rats and four out of six ligated animals respectively developed cirrhosis. The development of cirrhosis was progressive and associated with ascites. It is concluded that in the rat the morphological sequelae of long term cholestasis induced by either cannulation and obstruction or ligation of bile ducts are similar and are accompanied by cirrhosis. The advantages of this experimental model for the study of human cirrhosis are discussed.

The role of dietary lipid in the regulation of unconjugated hyperbilirubinaemia in Gunn rats.

1. The purpose of this study was to characterize the mechanisms of diet-induced hyperbilirubinaemia in Gunn rats with emphasis on the role of lipids, and to examine their relationship with regard to fasting hyperbilirubinaemia. 2. A lipid-free normocaloric diet produced a threefold increase in plasma bilirubin concentration (baseline 109.4 mumol/l), which was maximal by 10 days and thereafter remained constant. The level of hyperbilirubinaemia attained was not influenced by fasting or phenobarbitone, and returned to baseline concentration within 10 days of resuming a normal diet. 3. Determination of hepatic bilirubin showed that the magnitude of the hepatic bilirubin pool was increased by the lipid-free diet but was unchanged by fasting. Hepatic ligandin concentrations were comparable in fasted Gunn rats and those fed normal or lipid-free diets, although total hepatic ligandin was reduced in the fasted animals. 4. The hyperbilirubinaemic effect of the lipid-free diet was largely reversed by the inclusion of 10% lipid in the diet and was affected to a lesser extent by 5% lipid. Similar reductions in plasma bilirubin concentration were observed with a variety of other lipids (10%), regardless of their fatty acid chain length or degree of saturation. 5. In fasting animals a direct correlation was observed between plasma bilirubin and free fatty acid concentrations and insulin levels were greatly depressed, whereas in those fed on the lipid-free diet no significant changes were evident in plasma concentrations of free fatty acids or insulin. 6. Plasma bilirubin concentration was unrelated to alterations in plasma triglycerides produced by the administration of clofibrate. However, an unexplained decrease in plasma bilirubin (40%) without a significant change in triglycerides was noted when clofibrate was added to the lipid-free diet. 7. Analysis of kinetic data obtained from [14C]bilirubin clearance studies revealed that hyperbilirubinaemia associated with the lipid-free diet reflected a marked reduction (60%) in plasma clearance with no change in bilirubin turnover. This was accompanied by a relative redistribution of bilirubin from the extravascular pool to the plasma pool. 8. Although these studies indicate that fasting and the withdrawal of dietary lipid have some similar effects on bilirubin metabolism, it seems likely that different mechanisms are responsible for the hyperbilirubinaemia.

Excretion of conjugated bilirubin in the isolated perfused rat kidney.

1. Renal mechanisms of conjugated bilirubin excretion have been studied in isolated rat kidneys perfused with a protein-free dextran medium, containing conjugated bilirubin isolated from human bile. 2. In nine perfused kidneys with a low glomerular filtration rate (GFR) (less than 0.5 ml/min) and depressed tubular function, there was a significant linear correlation between conjugated bilirubin clearance and GFR (r = 0.97). 3. In contrast, nine kidneys with a normal GFR (greater than 0.8 ml/min) and good tubular function exhibited substantial tubular reabsorption of filtered conjugated bilirubin (mean 74%). Reabsorption was proportional to the filtered conjugated bilirubin load and a tubular transport maximum was not observed even at high concentrations (144 mumol/1). 4. The fractional reabsorption of bilirubin was unchanged by the addition of sodium aminohippurate to the medium. Perfusion with an albumin medium (10 g/1) resulted in a tenfold reduction in conjugated bilirubin clearance. 5. These observations indicate that non-protein-bound conjugated bilirubin is freely filtered by the glomeruli and then largely reabsorbed in the tubules. Evidence of tubular secretion was not obtained. 6. Chromatographic separation of bilirubin conjugates showed that the proportion of di- to mono-conjugates in the urine was greater than in the perfusate. Whether this incicated further conjugation by the kidney of the monoconjugates or differential clearance of the conjugates was not established.