Elevation of the serum bilirubin diconjugate fraction provides an early marker for cholestasis in the rat.

AIMS/METHODS: During cholestasis, components normally excreted into bile, e.g. bilirubin, accumulate in liver cells and biliary passages. In order to assess the conjugation of bilirubin retained in the hepatocyte during cholestasis, we analyzed the pattern of bilirubin pigments in rat serum and bile, using reversed phase alkaline methanolysis-HPLC. Cholestasis was induced by bile flow interruption for 1 to 2 h. RESULTS: One hour after initiation of cholestasis, the serum concentration of total bilirubin rose 2-fold due to increases in bilirubin di- (BDC) and monoconjugate (BMC), while unconjugated bilirubin (UCB) decreased by 33%. As a result, the BDC/BMC ratio increased to 1.67+/-0.20 vs 0.60+/-0.10 in controls (p<0.01) and the BMC/UCB ratio to 1.0+/-0.2 vs 0.1+/-0.1 (p<0.01). After relief of biliary obstruction, biliary output rose to 8.0+/-0.5 vs 5.5+/-0.3 micromol x min(-1) x kg (p<0.01), and the biliary BDC/BMC ratio to 4.0+/-0.3 vs 1.5+/-0.2 (p<0.01). In contrast, the biliary BMC/UCB ratio remained unchanged throughout. Increasing the duration of obstruction to 2 h led to a further increase in the serum BMC/UCB ratio to 2.2+/-0.3 (p<0.01), but not in the BDC/BMC ratio. Serum aminotransferase activity and the concentration of total bile acids increased 3- and 100-fold above their respective control values. Alkaline phosphatase activity remained unaltered, and electron microscopical features of cholestasis became apparent only after 2 h of biliary obstruction. CONCLUSIONS: We suggest that one of the initial events of cholestasis is a more efficient conjugation of bilirubin retained in the hepatocyte. This results in a shift of the equilibrium among bilirubin pigments towards BDC, the end-product of conjugation. Such a shift provides an early marker for cholestasis.

Increase in the relative amount of bilirubin diconjugates in rat bile and serum under infusion of phthaleins and indocyanine green.

The effect of organic anions on bilirubin metabolism and excretion was investigated in rats. Biliary excretion of bromosulfophthalein, bromcresol green and indocyanine green led to a significant decrease in excretion of bilirubin pigments and to an increase of their concentration in serum. This was concomitant with a marked increase in the ratio of bilirubin diconjugates to monoconjugates in bile and serum. These changes were unrelated to either bile flow, biliary lipid output, or hepatic activity of bilirubin UDP-glucuronyl transferase. Following bolus injection of [14C]bilirubin, peak excretory rate of radioactivity was markedly delayed in rats infused with bromosulfophthalein, as compared to controls. It is concluded that administration of the organic anions increased the ratio of bilirubin diconjugates to monoconjugates in bile and serum, by slowing down the intrahepatic transit of bilirubin pigments. This, in turn, allowed more efficient enzyme-catalyzed formation of bilirubin diconjugates from the intermediate bilirubin monoconjugates.

The maximal biliary excretory rate (Tm) of ioglycamide in the rat. Effect of taurocholate.

Biliary excretion of ioglycamide was studied in Wistar and Gunn rats. A hepatic transport-maximum (Tm) was observed. Higher Tm-values were found in Gunn rats, which have a greater bile flow compared to the parent Wistar rats, in spite of having a similar bile acid output. This suggests that the Tm is related to the bile acid-independent bile flow. In bile acid-depleted Wistar rats, bile acid output was 30% of control values whereas bile flow and ioglycamide-Tm had only decreased by approximately 15%. Ioglycamide excretion could not be increased by taurocholate infusion. An additional 22.0 ml of bile was excreted per mmol of biliary ioglycamide. Loads of the contrast agent markedly exceeding the Tm resulted in a decrease of its own biliary excretion and its choleretic properties. These presumed 'toxic' effects were counteracted by near-physiological amounts of taurocholate. Thus, the effect of taurocholate varies greatly depending upon the amounts of the contrast agent and the taurocholate administered.

Taurocholate increases the biliary output of bilirubin pigments in the rat, due to haemolysis.

In order to investigate the effect of bile salts on the biliary transport of bilirubin, we infused rats with taurocholate at 1-3 mumol/min/kg body weight. When administered in saline, taurocholate induced haemolysis, and a dose-dependent increase in biliary excretion and serum concentration of all bilirubin pigments. The addition of human or rat serum albumin to the infusate, at an albumin:taurocholate molar ratio equal to or above 200, completely prevented haemolysis and the above-mentioned alterations in bilirubin transport, without any change in the bile acid output. In order to mimic a haemolytic condition, an erythrocyte haemolysate was further added to the taurocholate-albumin solution. This again resulted in an increased bilirubin output. We suggest that taurocholate treatment increases bilirubin output due to the increased load of the pigment on the liver, as a result of the bile acid-induced haemolysis.

Effects of ioglycamide on the hepatic transport of bilirubin and its mono- and diconjugates in the rat.

Bilirubin seems to share the biliary excretion pathway with other organic anions, but not with bile acids. We studied the effects of the organic anion ioglycamide, an iodinated contrast agent, on bilirubin metabolism in Wistar rats. This compound does not undergo conjugation and is characterized by a maximal biliary secretory rate (Tm). The results show that in spite of producing a 3-fold increase in bile flow, ioglycamide excretion under Tm conditions decreased the output of unconjugated bilirubin and its monoconjugate by approximately 90%. Diconjugated bilirubin decreased by only 50% and became by far the predominant pigment in bile (86.5 +/- 6.0% of total pigment vs. 61.0 +/- 4.0% in basal conditions, n = 12). Unconjugated and monoconjugated bilirubins changed in parallel suggesting that the former arises from the monoconjugates. In serum, diconjugated bilirubin augmented from trace amounts to 1.15 +/- 0.17 mumole per liter. Total conjugated pigments in serum increased from 5 to 85% of total bilirubin. Bile acid output remained unchanged. Pretreatment of rats with ioglycamide altered neither the activity of bilirubin UDP-glucuronyltransferase nor the ratio of diconjugate to monoconjugate formed at both low (25 microM) and high (164 microM) bilirubin concentrations. The observed biological effects of ioglycamide were dose-dependent and fully reversible. We suggest that ioglycamide interferes with the excretion of conjugated bilirubins ("bilirubinostasis"). The monoconjugates retained in the hepatocyte might then undergo more efficient transformation to diconjugates, the latter thus becoming the most important bile pigments in serum and bile.

Effect of chronic administration of cyclosporin A on hepatic uptake and biliary secretion of bromosulfophthalein in rat.

Cyclosporin A (CyA) decreases bile flow and bile salt secretion in the rat. The purpose of this study was to examine the influence of CyA on the hepatic transport of bromosulfophthalein (BSP). Male Sprague-Dawley rats were injected with CyA at the daily dose of 10 mg/kg (treated animals) or solvent (controls) during three weeks. Hepatic uptake of BSP (assessed by the plasma disappearance curve of the dye) and biliary secretion during infusions (95.5 and 178 nmol/min/100 g) were examined in both groups. Administration of CyA resulted in a decrease in both bile flow and BSP biliary secretion at the two infusion rates used. BSP plasma disappearance rate was significantly lower in treated animals than in controls. Conjugation of the dye was unaffected by CyA. There was no modification in ALT activity or in liver histology. These data show that chronic administration of CyA in rats decreases both hepatic uptake and biliary secretion of BSP. Thus, the inhibitory effect of CyA on biliary secretion is not limited to bile salts but also is observed with other cholephilic substances.