Bile Of Gunn Rats Research Articles

In vitro formation of glutathione conjugates of the dimethylester of bilirubin

Rat hepatic microsomes catalyzed the formation of two distinct glutathione conjugates of bilirubin dimethylester (DMB). The two conjugates were identical to those isolated from the bile of Gunn rats infused with DMB. The microsomal reaction was dependent on NADPH, oxygen and glutathione and was inhibited by nitrogen and the cytochrome P450 inhibitors metyrapone, 1-benzylimidazole, and α-napthoflavone. Conjugate formation was inducible with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) but not phenobarbital pretreatment. The rate of formation of conjugates was not affected by washings of the microsomal pellet or by the presence of superoxide dismutase and/or catalase. Cation fast atom bombardment mass spectrometry (FAB/MS) of the conjugates indicated a molecular ion of 937 atomic mass units (amu). Fragmentation revealed a loss of 307 amu, consistent with glutathione, and a residual mass of 629 amu suggesting a hydroxylated derivative of DMB (612 amu). Cation FAB/MS/MS of conjugates formed in vitro under an atmosphere of oxygen-16 and oxygen-18 demonstrated the incorporation of molecular oxygen by a difference of 2 amu in the respective molecular ions. Our results suggest that DMB is oxidized by the cytochrome P450 IA gene family to an epoxide intermediate which is then subsequently conjugated with glutathione.

Urobilinogen-i is a major derivative of bilirubin in bile of homozygous Gunn rats

Gunn rats lack bilirubin UDP-glycosyltransferases, but diazo-negative derivatives of bilirubin have been described in their bile. In order to investigate this alternative disposal of bilirubin, crude bile samples from Gunn and Wistar rats were directly analysed by h.p.l.c. Besides bilirubin (in Gunn rats) or its glycosides (in Wistar rats), two major compounds were detected. A yellow one corresponded to the previously documented vitamin B-2 and was equally prominent in Gunn rats or Wistar-rat bile. The other compound was colourless, but on standing in contact with air it was spontaneously oxidized to a pinkish-yellow pigment. It was far more prominent in Gunn-rat bile. Analysis of bile obtained after intravenous injection of [14C]bilirubin to Gunn rats demonstrated that this compound was highly labelled. Freezing and thawing of the bile resulted in the formation of a series of diazo-negative derivatives, demonstrating that the original compound was quite labile. Spectral (adsorption and fluorescent) and chromatographic (h.p.l.c., t.l.c. and paper chromatography) analysis of the oxidized form of the labelled compound allowed its identification as urobilin-i. The colourless compound secreted in bile was urobilinogen-i. Administration of neomycin and bacitracin to Gunn rats or gut resection suppressed the biliary excretion of urobilinogen and thus confirmed its intestinal origin. Urobilinogen seems thus to represent the major bilirubin derivative present in Gunn-rat bile. Its breakdown products might represent the so-far-unidentified diazo-negative polar bilirubin derivatives. Since only a small amount of bilirubin is present in Gunn-rat bile, the urobilinogen formed in the intestinal lumen seems to be derived from bilirubin reaching the gut via routes other than the biliary one.

Biliary Excretion of Iopanoic Acid in Gunn Rats

AbstractPatients with the Crigler-Najjar syndrome lack hepatic microsomal UDP-glucuronyl transferase activity which is necessary for conjugating bilirubin with glucuronic acid. The gallbladder of these patients opacify normally on oral cholecystography with iopanoic acid (IOP), a contrast agent which like bilirubin, is conjugated with glucuronic acid prior to transport into bile. Experiments were performed with normal (Sprague-Dawley) and Gunn rats (a strain which lacks UDP-glucuronyl transferase activity) to determine the effect of this enzyme deficiency on the hepatic disposition of IOP. Bilirubin excretion in Gunn rats was less than 1% of the Sprague-Dawley animals, while IOP excretion in bile was not statistically different between the two strains whether the contrast agent was infused as IOP or as IOP-glucuronide. HPLC analysis showed that IOP distribution in Gunn rat bile was 92% IOP-G and 8% IOP. The hepatic intracellular distribution of IOP was the same in the two strains. The results indicate tha...

Uridine diphosphate-glucuronic acid-independent conversion of bilirubin monoglucuronides to diglucuronide in presence of plasma membranes from rat liver is nonenzymic.

TWO ROUTES HAVE BEEN PROPOSED FOR CONVERSION OF BILIRUBIN MONOGLUCURONIDE TO THE DIGLUCURONIDE: glucuronyl transfer (a) from UDP-glucuronic acid to bilirubin monoglucuronide, catalyzed by a microsomal UDP-glucuronyltransferase, and (b) from one molecule of bilirubin monoglucuronide to another (transglucuronidation), catalyzed by an enzyme present in liver plasma membranes. The evidence regarding the role of the latter enzyme for in vivo formation of bilirubin diglucuronide is conflicting. We therefore decided to reexamine the transglucuronidation reaction in plasma membranes and to study the conversion of bilirubin monoglucuronide to diglucuronide in vivo. Purified bilirubin monoglucuronide was incubated with homogenates and plasma membrane-enriched fractions from liver of Wistar and Gunn rats. Stoichiometric formation of bilirubin and bilirubin diglucuronide out of 2 mol of bilirubin monoglucuronide was paralleled by an increase of the IIIalpha- and XIIIalpha-isomers of the bilirubin aglycone, thus showing that dipyrrole exchange, not transglucuronidation, is the underlying mechanism. Complete inhibition by ascorbic acid probably reflects intermediate formation of free radicals of dipyrrolic moieties. The reaction was nonenzymic because it proceeded independently of the protein concentration and heat denaturation of the plasma membranes did not result in decreased conversion rates. Collectively, these findings show spontaneous, nonenzymic dipyrrole exchange when bilirubin monoglucuronide is incubated in the presence of rat liver plasma membranes. Because bilirubin glucuronides present in biological fluids contain exclusively the bilirubin-IXalpha aglycone, formation of the diglucuronide from the monoglucuronide by dipyrrole exchange does not occur in vivo. Rapid excretion of unchanged bilirubin monoglucuronide in Gunn rat bile after injection of the pigment provides confirmatory evidence for the absence of a UDP-glucuronic acid-independent process.

Excretion of administered and endogenous photobilirubins in the bile of the jaundice gunn rat.

Radiolabeled photobilirubins, prepared in vitro by anaerobic illumination of [34C]bilirubin, were injected intravenously into homozygous jaundiced Gunn rats with an external bile fistula. With the animals kept in darkness, the labeled photobilirubins were excreted rapidly in bile. Photobilirubins IA and IB were excreted primarily as unconjugated bilirubin, whereas photobilirubin II was excreted primarily as photobilirubin II and not converted into bilirubin. Bile of Gunn rats given no exogenous pigments, but undergoing phototherapy, contained a large proportion of photobilirubin II and, if collected in liquid nitrogen, traces of photobilirubins I; neither was found in bile when these rats were kept in the dark. Because there is prior evidence that these rats were kept in the dark. Because there is prior evidence that these photobilirubins are isomers of bilirubin, these experiments indicate that the major mechanism of phototherapy is photoisomerization of bilirubin. Photobilirubin II is the unidentified major photoderivative described previously, whereas formation of photobilirubins IA and IB, and their reversion to bilirubin-IXalpha, account for the remarkably increased output of the parent pigment during phototherapy.

Evidence for conversion of bilirubin to dihydroxyl derivatives in the Gunn rat

Three bilirubin derivatives, isolated from the bile of the jaundiced Gunn rat, are chromatographically and spectrally identical with certain products generated by photo-oxidation of bilirubin in CHCl 3. The pure pigments exhibit the spectrochemical characteristics respectively of a rubin, rhodin, and purpurin tetrapyrrole chromophore. Mass spectroscopy is compatible with their formulation as dihydroxyl derivatives of bilirubin which could be formed by established photodegradation processes involving singlet oxygen. Excretion of these products in Gunn rat bile in the dark suggests that a bilirubin oxygenase(s) is involved in the alternate pathways of bilirubin metabolism in these animals which are congenitally deficient in bilirubin conjugation.

Photocatabolism of labeled bilirubin in the congenitally jaundiced (Gunn) rat.

To elucidate the mechanism by which phototherapy reduces serum bilirubin, studies were performed on the catabolism of labeled bilirubin in homozygous jaundiced Gunn rats before, during, and after a period of exposure to 1700 foot candles of daylight fluorescent light. Following equilibration with the body pool of an intravenously administered tracer dose of (3)H- or (14)C-bilirubin, radioactive and diazo reactive compounds were excreted in the bile at a slow, steady rate and plasma specific activity declined semilogarithmically. Subsequent exposure to light caused a marked increase in the biliary excretion of radioactive and diazoreactive compounds. Fecal and urinary radioactivity increased also but remained minor fractions of the total excreted radioactivity. After extinguishing the lights, these variables reverted gradually to control values. Spectral and chromotographic analysis of the excreted pigments and their azopigments demonstrated that the increased biliary radioactivity during phototherapy consisted of two roughly equal fractions: (a) unconjugated bilirubin, excreted at rates comparable to the output of conjugated bilirubin in the bile of normal nonjaundiced rats; and (b) water-soluble bilirubin derivatives, chromatographically identical with those found in Gunn rat bile under control lighting conditions but different from the products of photodecomposition of bilirubin in vitro. In some animals, phototherapy produced little decline in plasma bilirubin despite comparable acceleration of bilirubin catabolism. This was attributed tentatively to increased synthesis of early labeled bilirubin in these animals.

Differences between the biliary excretion of tri [14C] methyl-(3-hydroxy-phenyl) ammonium iodide in Wistar and Gunn rats.

1. The biliary excretion of [(14)C]trimophonium iodide [tri[(14)C]methyl(3-hydroxyphenyl)ammonium iodide] was studied in normal Wistar animals and in jaundiced homozygous Gunn rats. 2. In normal Wistar rats small amounts of radioactivity (approx. 3% of the dose in 4h) were excreted in bile as two glucuronide conjugates, i.e. [(14)C]trimophonium glucuronide [tri[(14)C]methyl-(3-oxyphenyl)ammonium glucuronide] (85%) and 3-di[(14)C]methylaminophenyl glucuronide (10-15%). Only minor amounts of the unchanged drug were detected in bile. 3. In the homozygous jaundiced Gunn rat large amounts of radioactivity (26% of the dose in 4h) were eliminated in bile as [(14)C]trimophonium glucuronide alone. The quantitative excretion of this metabolite in Gunn rat bile was about ten times that in normal animals. 4. It is proposed that the biochemical lesion in the homozygous Gunn rat may indirectly affect the biliary transport of exogenous glucuronides across the canalicular membrane.

Excretion of Unconjugated Bilirubin in the Bile of Gunn Rats

Conjugation is a generally accepted prerequisite for biliary excretion of bilirubin, but small amounts of unconjugated bilirubin regularly are identifiable in bile. This unconjugated pigment fraction usually is present in the bile together with much larger amounts of conjugated bilirubin suggesting a relationship in excretion between these two pigment types. Data are presented which suggest that the biliary excretion of unconjugated bilirubin is enhanced approximately 30-fold by the concomitant forced excretion of conjugated pigment in Gunn rats. Complex formation between the two pigment types (“bilirubin monoglucuronide”) is postulated to explain this phenomenon.