Interaction of bilirubin and biliverdin with reactive nitrogen species

Bilirubin (BR) and biliverdin (BV), two metabolites produced during haem degradation by haem oxygenase, possess strong antioxidant activities toward peroxyl radical, hydroxyl radical and hydrogen peroxide. Considering the importance attributed to nitric oxide (NO) and its congeners in the control of physiological and pathophysiological processes, we examined the interaction of BR and BV with NO and NO-related species in vitro. Exposure of BR and BV to agents that release NO or nitroxyl resulted in a concentration- and time-dependent loss of BR and BV, as assessed by high performance liquid chromatography. Peroxynitrite, a strong oxidant derived from the reaction of NO with superoxide anion, also showed high reactivity toward BR and BV. The extent of BR and BV consumption largely depended on the NO species being analysed and on the half-lives of the pharmacological compounds considered. Of major importance, BR and BV decomposition occurred also in the presence of pure NO under anaerobic conditions, confirming the ability of bile pigments to scavenge the gaseous free radical. Increasing concentrations of thiols prevented BR consumption by nitroxyl, indicating that bile pigments and thiol groups can compete and/or synergise the cellular defence against NO-related species. In view of the high inducibility of haem oxygenase-1 by NO-releasing agents in different cell types, the present findings highlight novel anti-nitrosative characteristics of BR and BV suggesting a potential function for bile pigments against the damaging effects of uncontrolled NO production.     

Effect of biliverdin and bilirubin on the hepatic excretion of bile pigments in the rabbit

1. A study has been carried out on the effect of i.v. infusion of biliverdin and bilirubin at four different dose strengths in sodium pentobarbital-anaesthetized rabbits. 2. Both pigments show similar values for the maximum excretion rate in bile. 3. At doses of 0.45 and 0.60 mg/kg/min, the infusion of biliverdin does not affect bile flow in the resting state, contrary to the negative effect on flow induced by bilirubin. 4. This negative effect induced by bilirubin on bile flow is explained on the basis of its inhibitory action on the bile salt independent fraction (BSIF).     

Role of the hepatocyte microtubular system in the excretion of bile salts and biliary lipid: implications for intracellular vesicular transport

The role of the hepatocyte microtubular system in the transport and excretion of bile salts and biliary lipid has not been defined. In this study the effects of microtubule inhibition on biliary excretion of micelle- and non-micelle-forming bile salts and associated lipid were examined in rats. Low-dose colchicine pretreatment had no effect on the baseline excretion of biliary bile salts and phospholipid in animals studied 1 hr after surgery (basal animals), but slightly retarded the excretion of tracer [14C]taurocholate relative to that of lumicolchicine-pretreated (control) rats. However, colchicine pretreatment resulted in a marked reduction in the excretion of 2 mumol/100 g doses of a series of four micelle-forming bile salts of differing hydrophilicity, but had no significant effect on the excretion of the non-micelle-forming bile salt, taurodehydrocholate. Continuous infusion of 0.2 mumol of taurocholate/(100 g.min) following 24 hr of biliary drainage (depleted/reinfused animals) resulted in physiologic bile flow with biliary excretion rates of bile salts, phospholipid, and cholesterol that were markedly inhibited (mean 33, 39, and 42%, respectively) by colchicine or vinblastine pretreatment. Excretion of tracer [14C]taurocholate also was markedly delayed by colchicine in these bile salt-depleted/reinfused animals. In contrast, colchicine did not inhibit bile salt excretion in response to reinfusion of taurodehydrocholate. Thus, under basal conditions, the microtubular system appears to play a minor role in hepatic transport and excretion of bile salts and biliary lipid. However, biliary excretion of micelle-forming bile salts and associated phospholipid and cholesterol becomes increasingly dependent on microtubular integrity as the transcellular flux and biliary excretion of bile salts increases, in both bile salt-depleted and basal animals. We postulate that cotransport of micelle-forming bile salts and lipids destined for biliary excretion, via an intracellular vesicular pathway, forms the basis for this microtubule dependence.     

The biliary excretion of sulfbromophthalein in the pig

The characteristics of the hepatic metabolism of Sulfbromophthalein (BSP) have not been described previously for the pig. This is an important deficiency, since the pig is particularly suitable for studies of hepatic physiology and pharmacology which might apply to man. The aim of these experiments was to establish the pattern of serum clearance and biliary excretion of BSP and to determine that dose which would produce a maximal concentration in bile. A dose response and pattern of biliary excretion of BSP was studied at three dose levels administered either as a single bolus of a continuous infusion. All experiments were performed in conscious, conditioned pigs. The patterns of serum clearance and biliary excretion were found to be similar to other laboratory animals and to man. Maximary biliary concentration of BSP was achieved by a single bolus of 5-9 mumol/kg or a constant infusion of 0-59 mumol/kg/min. At these dose levels no significant alteration in bile flow was demonstrated nor was there any correlation between bile flow and BSP excretion. Supra-maximal doses produced a significant increase in bile flow and with these doses there was a significant positive correlation between bile flow and BSP excretion.     

Intrabiliary glutathione hydrolysis. A source of glutamate in bile

High concentrations of glutathione (GSH) and two of its constituent amino acids, glutamate and glycine, are normally found in rat bile. To examine the role of intrabiliary GSH hydrolysis as a source of these amino acids, as well as of cystine in bile, the biliary excretion of GSH and free amino acids was measured in normal male Sprague-Dawley rats; in animals given either phenol 3,6-dibromphthalein disulfonate or diethyl maleate, inhibitors of GSH secretion into bile; and after a retrograde intrabiliary infusion of (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), an irreversible inhibitor of gamma-glutamyl transferase activity. Total concentration of amino acids in normal rat bile ranged from 4 to 7 mM and was more than double the concentration in plasma (2-3 mM). Although most amino acids were detected in bile, glutamate and glycine were the most prevalent (1.2 and 1.0 mM, respectively), followed by the branched chain amino acids valine and leucine. The administration of phenol 3,6-dibromphthalein disulfonate (180 mumol/kg, intravenous), or of diethyl maleate (1 mmol/kg, intraperitoneal), resulted in a marked decrease in the biliary excretion of GSH, as well as a decrease in the excretion of glutamate, cystine, and glycine; however, the effects of these agents were not specific for the amino acid constituents of GSH. Following retrograde intrabiliary infusion of AT-125 (10 mumol/kg), there was an immediate and sustained doubling in the rate of biliary excretion of both GSH and glutathione disulfide and a marked decrease in the rate of excretion of glutamate. Varying the dose of AT-125 (0-20 mumol/kg) resulted in an inverse linear relation between hepatic gamma-glutamyl transferase activity and the biliary excretion of intact GSH. These findings suggest that most, if not all, of the free glutamate in excreted bile is formed from the intrabiliary hydrolysis of GSH. Prior to hydrolysis within the biliary tree, substantial concentrations of GSH must be transported from liver cells into bile; minimal canalicular concentrations of this tripeptide are estimated at 5 mM.     

Bile flow and electrolyte composition of bile associated with maximum bilirubin excretion in sheep.

Changes in the composition of bile accompanying the maximum biliary excretion (Emax) of bilirubin were investigated in sheep. Sheep fitted with chronic 'T-tubes' in the common bile duct were infused with taurocholate and bilirubin at various rates. Bile collected during both pre- and post-bilirubin steady-state periods was analyzed for the biliary concentration of electrolytes, bile salts, and bilirubin. Bilirubin Emax was 24.6 mumol/min while bile salt excretion during this period was 103 mumol/min. At Emax bilirubin entry into bile reached a concentration of 16.1 mumol/mL, increased the biliary concentration of sodium, did not change osmolarity of bile, and did not increase bile flow. The data suggest that bilirubin either interacts with mixed micelles in bile or forms molecular aggregates.     

Endogenous bilirubin excretion in Bolivian squirrel monkeys with a Gilbert's-like syndrome

Fasted Bolivian squirrel monkeys (BoSM) exhibit a marked hyperbilirubinemia when compared to fed BoSM. This fasting hyperbilirubinemia (FH) is similar to that in human patients with Gilbert's syndrome. Endogenous bilirubin (BR) excretion (production) into bile was elevated two-fold in BoSM upon fasting. The fraction of injected dose of 3 H-amino-levulinic acid (ALA) incorporated into biliary BR in fasted monkeys was of less magnitude than in fed monkeys and was associated with lower specific activities of 3 H-BR. Both the lower incorporation of ALA and lower specific activities of 3H-BR in fasted BoSM suggest that increased BR excreted may have arisen from pre-existing non-labeled pools of either heme or BR.     

Endotoxin-induced reduction in biliary indocyanine green excretion rate in a chronically catheterized rat model

Using a nonstressed chronically catheterized rat model in which the common bile duct was cannulated, we studied endotoxin-induced alterations in hepatic function by measuring changes in the maximal steady-state biliary excretion rate of the anionic dye indocyanine green (ICG). Biliary excretion of ICG was calculated from direct measurements of biliary ICG concentrations and the bile flow rate during a continuous vascular infusion of ICG. Despite significant elevations in mean peak serum tumor necrosis factor-alpha (TNF-alpha) concentrations (90.9 +/- 16.2 ng/ml), there was no effect on mean rates of bile flow or biliary ICG clearance after administration of 100 microg/kg endotoxin at 6 or 24 h. Significant differences from mean baseline rates of bile flow and biliary ICG excretion did occur after administration of 1,000 microg/kg endotoxin (mean peak TNF-alpha 129.6 +/- 24.4 ng/ml). Furthermore, when rats were treated with up to 16 microg/kg of recombinant TNF-alpha, there was no change in mean rates of bile flow or ICG biliary clearance compared with baseline values. These data suggest that the complex regulation of biliary excretion is not mediated solely by TNF-alpha.     

Hepatic organic anion transport kinetics and bile flow during short-term total parenteral nutrition in the rabbit

Plasma disappearance of sulfobromophthalein (BSP) after an intravenous bolus (5 mg/kg) was determined in six lab chow-fed (LCF) rabbits and in six rabbits maintained on total parenteral nutrition (TPN) for 5 days. A common bile duct cannula enabled measurements of bile flow and biliary BSP excretion. Compartmental analysis of the biexponential plasma disappearance curve yielded three fractional transfer rates, plasma to liver (hepatic uptake), liver to plasma (reflux), and liver to bile (canalicular excretion). The transfer rates for hepatic uptake were 0.253 +/- 0.061/min for LCF and 0.147 +/- 0.040/min for TPN (P less than 0.01) and for the canalicular excretion of BSP were 0.038 +/- 0.019/min for LCF and 0.019 +/- 0.002/min for TPN (P less than 0.05). Model-computed rates for BSP excretion in bile over 60 min were lower with TPN (61%) than with LCF (80%); the measured excretory rates were 53% for TPN rabbits and 75% of injected dose for LCF animals. Basal biliary flow was reduced by 50% in the TPN group. With a two-compartmental model, assuming two pools and three transfer rates, we have demonstrated for the first time significant decreases in hepatic uptake and canalicular excretion of the organic anion BSP during TPN. A decrease in hepatic blood flow due to the enteral fast of TPN could have contributed in part to the decreased hepatic uptake. But, because the second exponent of the biexponential curve is independent of hepatic blood flow, the decrease in liver to bile transfer rate is a true approximation of a diminished canalicular excretory capacity during TPN. It is concluded that the movement of organic anions along the hepatic BSP/bilirubin transport system is impaired early during TPN.     

Depression of biliary glutathione excretion by chronic ethanol feeding in the rat

The effects of chronic alcohol feeding on biliary glutathione excretion were studied in rats pair fed diets containing either ethanol (36% of total energy) or isocaloric carbohydrate for 4-6 weeks. An exteriorized biliary-duodenal fistula was established and total glutathione (GSH) and oxidized glutathione (GSSG) were measured. A significant decrease was observed in rats fed alcohol chronically compared to their pair fed controls in the biliary excretion of GSH (55.7 +/- 37.0 vs 243.1 +/- 29.0 micrograms/ml bile, p less than 0.025) as well as biliary GSSG (12.5 +/- 5.0 vs 49.9 +/- 8.0 micrograms/ml bile, p less than 0.05) and in bile flow (23.1 +/- 1.6 vs 29.2 +/- 1.3 micrograms/min, p less than 0.05). An acute dose of ethanol tended to exaggerate the decrease on biliary GSH and GSSG in the two groups of animals. The depression in biliary GSH could not be attributed to decreased GSH synthesis since S35-L-methionine incorporation into hepatic and biliary GSH was unchanged or even increased after chronic ethanol feeding.     

Sodium arsenite induced alterations in bilirubin excretion and heme metabolism

The acute administration of sodium arsenite (AsIII) to rats resulted in a biphasic alteration of the hepatic cytosolic "free" heme pool. The first stage was an increase in the cytosolic "free" heme without significant effects on the content of cytochrome P-450 or on bilirubin excretion. The second stage consisted of a continuous fall of the cytosolic "free" heme and of the content of cytochrome P-450. These changes were concurrent with an eight-fold increase in heme oxygenase activity and associated with marked elevations in the biliary excretion of bilirubin. The bile was collected from chronically cannulated rats to avoid artifacts related to anesthesia or post anesthetic effects. The rapid increase in biliary excretion of labeled heme degradation products indicated an increased breakdown of newly synthesized heme. Immunoelectrophoresis of bile proteins showed an altered pattern of bile protein excretion. The increased biliary haptoglobin suggested some hemolysis, while the reduction in the free immunoglobulin A (IgA) secretory component showed an AsIII-related decreased protein transport across hepatocytes to bile. Further research is required to assess the direct role of an increased heme degradation in the genesis of the hepatotoxic effects of AsIII.     

The effect of secretin and cholecystokinin-pancreozymin on the secretion of bile in the anaesthetized rabbit.

Effects of secretin and Cholecystokinin-Pancreozymin (CCK-PZ) on the secretion of bile in anaesthetized rabbits have been studied. Single injections of secretin (5.0 u.kg(-1) significantly increased the flow of bile irrespectively of whether the cystic duct was free or had been tied. A sustained increase in bile flow could be obtained by the continuous infusion of secretin. Cholecystokinin-Pancreozymin was effective in increasing the bile flow in doses of 1.0 u.kg(-1). Much of the effect could be attributed to contraction of the gallbladder but a significant increase in flow could still be elicited after ligation of the cystic duct. Our findings strongly suggest that the biliary secretion in rabbits is not as different from the general pattern as has previously been suggested.     

The effects of bromosulphophthalein, indocyanine green and bilirubin on the biliary excretion of methylmercury

The effects of three ligands for ligandin on the biliary excretion of methylmercury were investigated in male rats injected intravenously with 1.0 mg/kg Hg as Me203 HgCl. Bromosulphophthalein and indocyanine green inhibited the biliary excretion of methylmercury, while bilirubin had no such effect. None of the compounds tested which inhibited the biliary excretion of methylmercury decreased bile flow or changed the hepatic concentration of mercury of non-protein thiols. The possibility of the involvement of ligandin in the biliary excretion of methylmercury is discussed.     

Quantification of LDL cholesteryl ester contribution to biliary steroids in the rat

The proportion of LDL cholesteryl ester converted to biliary steroids was quantified in the rat. The pre-existing pool of bile was allowed to drain for 10-12 h through a bile duct cannula. A single intravenous pulse injection of LDL labelled with [3H]cholesterol linoleyl ester was made, followed by a constant infusion of the same material in order to maintain constant specific radioactivity in plasma. A new steady state was achieved within 6 h and bile samples were then collected hourly until 12 h. Although substantial amounts (53-61 micrograms/h) of cholesteryl ester were released into the liver during LDL catabolism, only a very small fraction (0.8-1.90 micrograms/h) was found in biliary steroids. The proportion of LDL cholesteryl esters contributing to biliary steroids was only 1-2%. These results perhaps explain why perturbations to accelerate bile acid excretion have no effect on plasma LDL cholesterol concentration in the rat.     

Hepatic protoporphyrin metabolism in patients with advanced protoporphyric liver disease.

Protoporphyria is a genetic disorder in which liver damage is caused by the toxic effect of protoporphyrin accumulation in the liver. In this study protoporphyrin was measured in the resected livers of 7 patients who had liver transplantation and an additional patient from whom liver tissue was obtained post mortem. Comparison of liver, erythrocyte and serum protoporphyrin levels demonstrated a marked gradient between these compartments: erythrocyte, 5781 +/- 655 micrograms/dl; serum, 384 +/- 102 micrograms/dl; liver 377,238 +/- 55,568 micrograms/100 gm wet weight, (mean +/- SE). Protoporphyrin levels in bile of 3 patients were 55,559, and 1,153 micrograms/dl, indicating a gradient between liver and bile as well. Examination of the livers by polarization microscopy and electron microscopy demonstrated protoporphyrin pigment crystals. In one patient who had recurrent liver disease after transplantation, the protoporphyrin concentration in the graft at the time of death was similar to that in the resected liver. These data indicate that liver protoporphyrin levels in patients with advanced protoporphyric liver disease are much higher than levels in blood and bile, in part because protoporphyrin forms crystalline deposits in liver tissue. Thus, progressive hepatic accumulation of protoporphyrin occurs in the face of impaired biliary excretion. An intrinsic defect in hepatic excretion of protoporphyrin is probably not necessary for this condition to develop because liver disease can occur in the graft following transplantation.     

The bile flow and biliary excretion of ursocholate in the rat

Several studies reported that ursodeoxycholate (but not its conjugates), when administered intravenously, increased the biliary bicarbonate concentration in the rat (1–3). At the same time, a complete dissociation between bile flow and the bile salt excretion rate was produced in the second hr of infusion (2). In order to examine whether this property was due to the 7β-hydroxy group in its molecular structure, the choleretic property of ursocholate (3α, 7β, 12α-trihydroxy-5β-cholanoic acid) was investigated in male Wistar rats. Immediately after the start of iv infusion of ursocholate at a rate of 1.2 μmole/min/100 g b. wt., both the bile flow and bile salt excretion rate began to increase. However, unlike with ursodeoxycholate, the bile salt excretion rate continued to be high in the second and third hr of infusion, while the bile flow rate gradually increased. Furthermore, the bicarbonate concentration in the bile fell slightly 10 min after the start of ursocholate infusion. Although the concentration tended to return to the baseline value before the bile salt infusion in the later period of observation, no significant increase in bicarbonate concentration was observed during the whole observation period. These properties were quite similar to those of cholate rather than those of ursodeoxycholate. However, a cholate infusion at the same rate of 1.2 μmole/min/100 g b.wt. caused a cholestasis as early as 20 to 30 min after the start of an infusion. These results suggest that the previously reported properties of ursodeoxycholate (that it causes a complete dissociation between the bile flow and bile salt excretion rate in the second hr and that it increases the biliary bicarbonate concentration) were not due to the 7β-hydroxy group in its steroidal structure, and that the choleretic property of ursocholate is similar to its 7α-hydroxy epimar, cholate. However, the much lower cytotoxicity of ursocholate compared to cholate appears to be due to the 7β-hydroxy group that ursocholate has.     

Hormonal regulation of biliary calcium excretion in rats: inhibition of calcitonin action by alpha 1-adrenergic stimulation

The effect of synthetic [Asu1,7] eel calcitonin (CT) and other hormones on biliary calcium excretion was investigated in rats cannulated bile duct. Administration of CT (80 mU/100 g body weight) produced a significant increase in liver calcium and a corresponding elevation of bile calcium content. The increase in bile calcium content was also caused by administration of insulin (0.1 U/100 g), epidermal growth factor (10 micrograms/100 g), glucagon (10 micrograms/100 g), epinephrine (10 micrograms/100 g), norepinephrine (10 micrograms/100 g), 4 beta-phorbol 12-myristate-13-acetate (10 micrograms/100 g) and ATP (1.0 mg/100 g), suggesting that this increase may be a receptors-mediated response. Of these hormones and drugs, norepinephrine, a alpha-receptor mediator, clearly prevented CT effect on biliary calcium excretion. Moreover, phenylephrine, a alpha 1-receptor agonist, caused an inhibition of the CT effect, while the agonist significantly increased biliary calcium excretion. The present study clearly demonstrates that biliary calcium excretion is stimulated by various hormones which increase calcium influx into liver cells, and suggests that the CT action may be inhibited by alpha 1-adrenergic stimulation.     

Effects of pancreatic polypeptide on biliary flow and bile acid secretion stimulated by secretin and cholecystokinin in the conscious pig

Fourteen castrated male Large White pigs, weighing 42.5 +/- 1.0 kg, were fitted with biliary and duodenal fistulae for biliary secretion studies. Furthermore, catheters were placed in a carotid artery for blood sampling and in a jugular vein for peptide infusion. Bile was automatically restituted to the animals and continuously sampled for analysis on experimental days. Following an 8 day recovery period, infusion studies were performed after an overnight fast. After a 30 min basal period, sustained biliary flow and bile acid output were obtained and maintained throughout the assay with secretin (36 pmol/kg/h) and CCK-8 (600 pmol/kg/h) infusion. Then, 200, 400, 600, 800 or 1200 pmol/kg/h of porcine pancreatic polypeptide (PP) were infused for 60 min. Secretin plus CCK infusion was continued for 1 h after PP infusion was stopped. Each dose of PP was given on a separate day. Biliary flow was not affected by PP except for the dose of 400 pmol/kg/h. On the contrary, bile acid concentration and output decreased with the lowest dose of PP (200 pmol/kg/h). As soon as the first dose of PP was infused, bile acid concentration and output fell to about 60% of values obtained with secretin plus CCK. Plasma levels of PP were below or similar to postprandial values for 200, 400 and 600 pmol/kg/h and they were significantly larger with 800 and 1200 pmol/kg/h. Bile acid concentration and output did not return to values obtained with secretin plus CCK infusion after cessation of PP infusion. In conclusion, porcine PP given in physiological doses to the pig decreases bile acid output whereas biliary flow remains unaffected.     

Influence of bile salts on the endogenous excretion of bile pigments

Effect of the infusion of glycodeoxycholate (GDC), taurocholate (TC) and dehydrocholate (DHC) on bile flow and on bile salt, biliary lipid and bile pigment secretion, has been studied in pentobarbital-anesthetized rabbits. GDC increased bile flow the most, while DHC increased it more than TC. The different choleretic actions of these bile salts cannot be explained by means of variations in their capacity to form micelles. Only GDC and TC were able to stimulate biliary lipid secretion, which suggests that both bile salts increase the formation of mixed micelles. GDC and TC to a lesser extent increased bile pigment excretion, DHC being without effect. These results favour the hypothesis that micellar binding could be an important factor responsible for the effect of bile acids on bile pigment excretion and should not be completely ruled out.     

Cholagogic effect of trimethylglycine in normal animals of different ages and in experimental atherosclerosis

Trimethylglycine at a dose of 1.5 g/kg was found to produce marked bile secretory effect in young and old rats. In rabbits with experimental atherosclerosis, trimethylglycine increased the content of biliary acids in the bile and normalized the indexes of lipid metabolism in the blood serum. Apparently, the effect on cholesterol transformation into biliary acids and its excretion with the bile is one of the mechanisms of anti-atherosclerotic action of trimethylglycine.