Emilio A. Rodríguez Garay

Separation and properties of conjugated biliverdin

Abstract 1. 1. Reversed-phase partition chromatography and paper chromatography have been used to separate free biliverdin from conjugated biliverdin. 2. 2. The colour of green post-mortem bile appears to be associated with both biliverdin monoglucuronide and biliverdin diglucuronide. 3. 3. Like pure biliverdin, the conjugated pigments have characteristic absorption spectra, and give a red fluorescence with zinc salts after iodine oxidation. Their chromatographic behaviour and solubility, however, differ markedly from that of unconjugated biliverdin. 4. 4. The paper-chromatographic technique described also separates bilirubin monoglucuronide and bilirubin diglucuronide from bilirubin and the biliverdins.

Alterations in renal function induced by aflatoxin B1 in the rat.

The effects of aflatoxin B1 (AFB1) on renal function were determined in adult male Wistar rats by in vivo and in vitro studies. In vivo studies demonstrated that AFB1 decreased glomerular filtration rate, tubular reabsorption of glucose, and tubular transport for p-amino-hippurate. Furthermore, AFB1 pretreatment increased urinary excretion of sodium and potassium and urinary gamma-glutamyl transferase content. In vitro studies showed that slices of renal cortical tissue obtained from AFB1-treated rats (100 microgram/kg) exhibited a diminished capacity to accumulate p-aminohippurate. On the other hand, total water content of tissue, amount of extracellular water, and intracellular sodium were increased. Intracellular potassium was diminished by treatment with AFB1. Although the molecular species responsible for the effects is not known, these results indicated that AFB1 is nephrotoxic in the rat following a single low dose (100 microgram/kg body wt, ip).

Preventive effect of silymarin against taurolithocholate-induced cholestasis in the rat

Increased amounts of monohydroxylated bile salts (BS) have been found in neonatal cholestasis, parenteral nutrition-induced cholestasis and Bylers disease, among others. We analyzed whether the hepatoprotector silymarin (SIL), administered i.p. at the dose of 100mg/kg/day for 5 days, prevents the cholestatic effect induced by a single injection of the model monohydroxylated BS taurolithocholate (TLC, 30 micromol/kg, i.v.) in male Wistar rats. TLC, administered alone, reduced bile flow, total BS output, and biliary output of glutathione and HCO(3)(-) during the peak of cholestasis (-75, -67, -81, and -80%, respectively, P<0.05). SIL prevented partially these alterations, so that the drops of these parameters induced by TLC were of only -41, -25, -60, and -64%, respectively (P<0.05 vs. TLC alone); these differences between control and SIL-treated animals were maintained throughout the whole (120 min) experimental period. Pharmacokinetic studies showed that TLC decreased the intrinsic fractional constant rate for the canalicular transport of both sulfobromophthalein and the radioactive BS [14C]taurocholate by 60 and 68%, respectively (P<0.05), and these decreases were fully and partially prevented by SIL, respectively. SIL increased the hepatic capability to clear out exogenously administered TLC by improving its own biliary excretion (+104%, P<0.01), and by accelerating the formation of its non-cholestatic metabolite, tauromurideoxycholate (+70%, P<0.05). We conclude that SIL counteracts TLC-induced cholestasis by preventing the impairment in both the BS-dependent and -independent fractions of the bile flow. The possible mechanism/s involved in this beneficial effect will be discussed.

Effect of silymarin on biliary bile salt secretion in the rat.

The effect of the hepatoprotector silymarin on bile secretion, with particular regard to bile salt secretion, was studied in Wistar rats. Silymarin (25, 50, 100, and 150 mg/kg/day, i.p., for 5 days) induced a dose-dependent increase in bile flow and bile salt secretion, the maximal effect being reached at a dose of 100 mg/kg/day (+17 and +49%, for bile flow and bile salt output, respectively; P < 0.05). Assessment of bile salt composition in bile revealed that stimulation of the bile salt secretion was accounted for mainly by an increase in the biliary secretion of beta-muricholate and, to a lesser extent, of alpha-muricholate, chenodeoxycholate, ursodeoxycholate, and deoxycholate. The maximum secretory rate (T(m)) of bile salts, as assessed by infusing the non-hepatotoxic bile salt tauroursodeoxycholate i.v. at stepwise-increasing rates, was not influenced by silymarin. The flavonolignan also increased the endogenous bile salt pool size (+53%, P < 0.05) and biliary bile acid excretion after bile acid pool depletion (+54%, P < 0.05), a measure of de novo bile salt synthesis. These results suggest that silymarin increases the biliary excretion and the endogenous pool of bile salts by stimulating the synthesis, among others, of hepatoprotective bile salts, such as beta-muricholate and ursodeoxycholate.

Taurolithocholate-induced inhibition of biliary lipid and protein excretion in the rat

Taurolithocholate (TLC), a natural bile salt, induces selective impairment on canalicular membrane of the hepatocyte, which seems to be a major determinant of its cholestatic effect in experimental animals. In order to extend existing studies about the effects of TLC on bile secretion, we examined in TLC-treated rats the biliary excretion of compounds that are transported to canalicular membrane via vesicles, such as lipids and proteins. The single intravenous injection of TLC (3 mumol/100 g body wt.) inhibited transiently the biliary bile salt excretion, while the biliary excretion of lipids (i.e., cholesterol and phospholipids) and proteins remained inhibited even though the biliary excretion and composition of bile salts were normalized. Under such a condition, TLC also inhibited the transcellular vesicular pathway to the exogenous protein horseradish peroxidase entry into bile, without altering the paracellular biliary access of the protein. The hepatic uptake of horseradish peroxidase was unaffected by TLC-treatment. The results indicate that TLC can inhibit the biliary excretion of compounds that reach the canaliculus via a vesicular pathway, such as lipids and proteins, by a mechanism not related to a defective bile salt excretion. Possible explanations for these findings are discussed.

Inhibitory effect of unconjugated bilirubin on p-aminohippurate transport in rat kidney cortex slices.

The effects of unconjugated bilirubin on the accumulation of p-aminohippurate, kinetics of p-aminohippurate uptake, the efflux of pre-accumulated p-aminohippurate and water and electrolyte distribution were investigated in the rat kidney cortical slice. (2) The addition of unconjugated bilirubin to the incubation medium decreased the 60 min slice-to-medium concentration ratio of p-aminohippurate. (3) The decrease in p-aminohippurate accumulation by unconjugated bilirubin was found to be more pronounced by increasing the concentration of pigment in the medium. (4) The rate of uptake of p-aminohippurate as a function of p-aminohippurate concentration differed in aerobiosis and anaerobiosis, and unconjugated bilirubin decreased only the uptake of p-aminohippurate in aerobic conditions. (5) The efflux of pre-accumulated p-aminohippurate decreased when unconjugated bilirubin concentration in the medium was low (10-20 microM) but the efflux increased when the concentration of pigment was much higher (100 microM). (6) The addition of unconjugated bilirubin to the medium (40-100 microM) increased intracellular sodium and total tissue water content, and decreased intracellular potassium and oxygen consumption of tissue. However the slices incubated with low concentration of pigment (20 microM) did not exhibit significative changes in cellular functional parameters. (7) These findings suggest that unconjugated bilirubin impairs p-aminohippurate transport by a complex mechanism that might involve binding of pigment to sites necessary for anion transport, although effects related to pigment toxicity or to its oxidative decomposition are not excluded.

Studies on the bilirubin sulphate conjugate excreted in human bile

Abstract 1. 1. Chemical and chromatographic studies of the polar bile pigment present in the bile, serum and urine of patients with obstructive jaundice have established it as a bilirubin conjugate containing sulphur. This pigment gives an alkali-stable azobilirubin derivative, azo C, from which inorganic sulphate is liberated by acid hydrolysis. The molar ratio of sulphate to azo pigment is approx. 1. 2. 2. The azodipyrrolyl derivative of natural bilirubin sulphate in ascending partition chromatography has been shown to be more polar than bilirubin glucronide. 3. 3. A simple densitometric paper chromatographic method is described for the determination of the percentage of bile pigments present as bilirubin disulphate in bile, serum and urine. 4. 4. The alkali-stable fraction of human bile contains 10–15% of bilirubin disulphate and 5–10% of another unknown conjugated pigment. 5. 5. Increased biliary excretion of bilirubin disulphate after intravenous injection of synthetic bilirubin sulphate and in vivo synthesis of sulphate conjugate following administration of radioactive sulphate, has been observed using rats with biliary fistula. Radioactivity is mainly associated with azo C after the formation of the azo derivative and with other nonbilirubinic compounds excreted in the bile. 6. 6. Similarities and dissimilarities between natural and synthetic bilirubin sulphate are discussed.

Hepatic transport of organic anions in taurolithocholate-induced cholestasis in rats.

The hepatic transport of organic anions was evaluated in taurolithocholate-induced cholestasis in rats. Taurolithocholate (3 mumol per 100 g body wt., i.v.) diminished bile flow by 61%, whereas biliary excretion of bile salts was normalized after 80 min. Tm studies of sulfobromophthalein revealed reduced biliary excretion (-58%) and increased hepatic content of the dye (+75%). Conjugation pattern in bile showed that free sulfobromophthalein was increased by 57%, suggesting that hepatic conjugation was also impaired. This finding, however, could not fully explain the reduced sulfobromophthalein excretion since Tm of its non-metabolizable analog phenol-3,6-dibromophthalein was also decreased (-41%). Compartmental analysis of plasma decay of both dyes revealed that, whereas hepatic uptake was unaltered, canalicular excretion was reduced and reflux from the liver into plasma was increased by the cholestatic agent. Studies on transport of phenol-3,6-dibromophthalein by isolated hepatocytes showed that while uptake was unaffected, the treatment reduced (-36%) the release from hepatocytes preloaded with the dye. Neither glutathione S-transferase activity nor binding of sulfobromophthalein to cytosolic proteins was altered when evaluated in vitro, suggesting that reduced conjugation and enhanced sinusoidal reflux were not due to an irreversible effect of taurolithocholate on this enzyme. In conclusion, taurolithocholate impairs the hepatic transport of organic anions by impairing canalicular excretion and intrahepatic conjugation, as well as by increasing transfer from the liver into the plasma.

Inhibition of rat liver microsomal bilirubin UDP-glucuronosyltransferase by ursodeoxycholic acid

Ursodeoxycholic acid and its endogenous metabolite tauroursodeoxycholic acid inhibited in vitro the microsomal bilirubin UDP-glucuronosyltransferase from rat liver. The magnitude of the inhibition correlated well with the loss of integrity of microsomal vesicles, suggesting that bile salts needed to reach the lumen to exert their inhibitory effects. The endogenous bile acids cholic acid, chenodeoxycholic acid and deoxycholic acid also exhibited inhibitory effects on bilirubin glucuronidation in digitonin-disrupted microsomes. Ursodeoxycholic acid inhibitory capacity was similar to that of chenodeoxycholic acid and deoxycholic acid but greater than that of cholic acid, the major endogenous bile salt. Kinetic studies, performed in detergent-activated preparations, showed that the inhibitions produced by ursodeoxycholic and tauroursodeoxycholic acids were competitive toward both bilirubin and UDP-glucuronic acid. The estimated Ki(app) for both substrates did not differ statistically between ursodeoxycholic and tauroursodeoxycholic acids. Both bile salts were weak inhibitors toward bilirubin but rather strong inhibitors toward UDP-glucuronic acid.

Biliverdin pigments in green biles

Abstract 1. 1. The nature of the green bile pigments in various animal species has been studied using reversed-phase partition chromatography and paper chromatography. 2. 2. The chromatographic behaviour of the pigments has been compared with that of synthetic biliverdin derivatives. 3. 3. In chicken, turkey, toad and snake bile, biliverdin behaved chromatographically like a bile acid complex of sodium biliverdinate. 4. 4. In toad bile-free biliverdin and its sodium salt were also found. 5. 5. In green human bile and the bile of rabbits and some varieties of snakes, the dominant green pigments were biliverdin glucuronides.