The formation and distribution of bilirubin monoglucuronide and diglucuronide in rat liver slices.
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ABSTRACT:
SUBMITTER: Campbell MT
PROVIDER: S-EPMC1186652 | biostudies-other | 1979 Jun
REPOSITORIES: biostudies-other
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Project description:Highly purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver, when reconstituted with Gunn-rat liver microsomes (microsomal fraction), was able to catalyse the conversion of unesterified bilirubin into both bilirubin monoglucuronide and diglucuronide. Under zero-order kinetic conditions for monoglucuronide formation, the fraction of bilirubin diglucuronide formed by incubation of bilirubin with the reconstituted highly purified transferase accounted for 18% of total bilirubin glucuronides, which was only slightly lower than the fraction of diglucuronides (23% of total bilirubin glucuronides) formed by incubation with hepatic microsomes in the presence of UDP-N-acetylglucosamine or Lubrol. The reconstituted purified enzyme also catalysed the UDP-glucuronic acid-dependent conversion of bilirubin monoglucuronide into diglucuronide and, when bilirubin was incubated with UDP-glucose or UDP-xylose, the formation of bilirubin glucosides and xylosides respectively. These results suggest that a single microsomal bilirubin UDP-glycosyltransferase may be responsible for the formation of bilirubin mono- and di-glycosides.
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Project description:Menthofuran is a monoterpene present in mint plants that is oxidized by mammalian cytochrome P450 (CYP) to hepatotoxic metabolites. Evidence has been presented that p-cresol and other unusual oxidative products are metabolites of menthofuran in rats and that p-cresol may be responsible in part for the hepatotoxicity caused by menthofuran [ Madyastha, K. M. and Raj, C. P. (1992) Drug Metab. Dispos. 20, 295 - 301]. In the present study, several oxidative metabolites of menthofuran were characterized in rat and human liver microsomes and in rat liver slices exposed to cytotoxic concentrations of menthofuran. Metabolites that were identified were monohydroxylation products of the furanyl and cyclohexyl groups, mintlactones and hydroxymintlactones, a reactive γ-ketoenal, and a glutathione conjugate. A similar spectrum of metabolites was found in urine 24 h after the administration of hepatotoxic doses of menthofuran to rats. In no case was p-cresol (or any of the other reported unusual oxidative metabolites of menthofuran) detected above background concentrations that were well below concentrations of p-cresol that cause cytotoxicity in rat liver slices. Thus, the major metabolites responsible for the hepatotoxic effects of menthofuran appear to be a γ-ketoenal and/or epoxides formed by oxidation of the furan ring.
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Project description:Two important signaling pathways in liver fibrosis are the PDGF- and TGFβ pathway and compounds inhibiting these pathways are currently developed as antifibrotic drugs. Testing antifibrotic drugs requires large numbers of animal experiments with high discomfort. Therefore, a method to study these drugs ex vivo was developed using precision-cut liver slices from fibrotic rat livers (fPCLS), representing an ex vivo model with a multicellular fibrotic environment. We characterized the fibrotic process in fPCLS from rat livers after 3 weeks of bile duct ligation (BDL) during incubation and tested compounds predominantly inhibiting the TGFβ pathway (perindopril, valproic acid, rosmarinic acid, tetrandrine and pirfenidone) and PDGF pathway (imatinib, sorafenib and sunitinib). Gene expression of heat shock protein 47 (Hsp47), α smooth muscle actin (αSma) and pro-collagen 1A1 (Pcol1A1) and protein expression of collagens were determined. During 48 hours of incubation, the fibrosis process continued in control fPCLS as judged by the increased gene expression of the three fibrosis markers, and the protein expression of collagen 1, mature fibrillar collagen and total collagen. Most PDGF-inhibitors and TGFβ-inhibitors significantly inhibited the increase in gene expression of Hsp47, αSma and Pcol1A1. Protein expression of collagen 1 was significantly reduced by all PDGF-inhibitors and TGFβ-inhibitors, while total collagen was decreased by rosmarinic acid and tetrandrine only. However, fibrillar collagen expression was not changed by any of the drugs. In conclusion, rat fPCLS can be used as a functional ex vivo model of established liver fibrosis to test antifibrotic compounds inhibiting the PDGF- and TGFβ signalling pathway.
| S-EPMC3995767 | biostudies-literature
Project description:A radioassay for specific determination of the rates of UDP-glucuronic acid-dependent conversion of bilirubin into the two isomeric (C-8, C-12) bilirubin monoglucuronides and bilirubin diglucuronide is described and illustrated by its application to rat liver microsomes. The method is based on measurement of the relative amounts of radiolabel in unesterified bilirubin and its mono- and di-esterified reaction products after incubation with [14C]bilirubin as substrate. This analysis is performed by the alkaline-methanolysis procedure, combined with one of two t.l.c. systems developed in order to enhance the sensitivity, accuracy and precision of the radioassay. Results for rates of total bilirubin glucuronide formation obtained with the new assay and the standard enzyme assay based on the ethyl anthranilate diazo-method were identical. However, the sensitivity of the latter technique is approx. 10-fold lower than that of the radioassay.
| S-EPMC1144318 | biostudies-other
Project description: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.
| S-EPMC1146892 | biostudies-other
Project description:1. Reconstitution of purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver into Gunn-rat liver microsomes provides a better environment than phosphatidylcholine liposomes, such that the final specific activity of the Wistar-rat liver enzyme was increased up to 85 units/mg of protein. 2. Gunn- and Wistar-rat liver microsomes were equally effective for reconstitution of the purified enzyme. 3. The transferase activity does not appear to be fully expressed in the more rigid environment of foetal Wistar-rat liver microsomes. 4. These reconstitution experiments reveal a final specific activity for the purified bilirubin UDP-glucuronyltransferase consistent with the capacity of the whole rat liver to glucuronidate bilirubin and indicate that the absence of this enzyme activity in Gunn-rat liver microsomes is not due to an abnormal microenvironment.
| S-EPMC1163694 | biostudies-other
Project description:1. Glucuronide formation of bilirubin and p-nitrophenol in vitro with excess of UDP-glucuronic acid by UDP-glucuronyltransferase from livers of young and adult rabbits was studied. 2. The development of UDP-glucuronyltransferase for the two substrates followed a markedly different pattern during maturation of young rabbits, p-nitrophenol-conjugation ability being much higher at birth than that for bilirubin. 3. Mg(2+) increased bilirubin conjugation, but inhibited p-nitrophenyl glucuronide formation. 4. p-Nitrophenol acted as a potent non-competitive inhibitor for bilirubin conjugation but bilirubin did not affect p-nitrophenyl glucuronidation. 5. The enzyme for bilirubin conjugation was inactivated at pH9 during treatment with snake venom, whereas in the same preparation the activity of the corresponding enzyme for p-nitrophenol was enhanced. In addition, some solubilization of the latter enzyme could be achieved by this method. 6. The possibility of the existence of more than one enzyme system for the formation of O-glucuronides is discussed.
| S-EPMC1265020 | biostudies-other
Project description:1. Rat liver microsomal preparation can effect the transglucosylation from UDP-glucose to bilirubin in the presence of Mg(2+). 2. Other nucleotides, namely CDP-glucose, ADP-glucose and GDP-glucose, were not active as glucosyl donors. 3. Only trace amounts of galactose, galacturonic acid and N-acetylglucosamine were conjugated to bilirubin when their respective UDP derivatives were used in the reaction mixture. 4. The azobilirubin glucosides produced by coupling with p-diazobenzenesulphonic acid and diazotized ethyl anthranilic acid were separable from the corresponding azobilirubin glucuronides by t.l.c. 5. The glucoside was, however, hydrolysed by both beta-glucosidase and various preparations of beta-glucuronidase; azobilirubin and glucose were liberated in the process. 6. Kinetic studies showed that the effects of pH and Mg(2+) on the two conjugating systems were similar. 7. The specific activities of hepatic bilirubin UDP-glucosyltransferase, expressed as mug of bilirubin ;equivalents' conjugated/h per mg of protein, are respectively 1.7 and 2.4 for male and female rats. 8. The K(m) values for bilirubin and UDP-glucose are 5.7x10(-5)m and 1.6x10(-3)m respectively. 9. The glucoside and glucuronide conjugations of bilirubin are discussed in relation to the availability of the conjugating agents and aglycone in the liver.
| S-EPMC1178253 | biostudies-other
Project description:Fibrotic processes in the liver of non-alcoholic steatohepatitis (NASH) patients cause microcirculatory dysfunction in the organ which increases blood vessel resistance and causes portal hypertension. Assessing blood vessel function in the liver is challenging, necessitating the development of novel methods in normal and fibrotic tissue that allow for drug screening and translation toward pre-clinical settings. Cultures of precision cut liver slices (PCLS) from normal and fibrotic rat livers were used for blood vessel function analysis. Live recording of vessel diameter was used to assess the response to endothelin-1, serotonin and soluble guanylate cyclase (sGC) activation. A cascade of contraction and relaxation events in response to serotonin, endothelin-1, Ketanserin and sGC activity could be established using vessel diameter analysis of rat PCLS. Both the sGC activator BI 703704 and the sGC stimulator Riociguat prevented serotonin-induced contraction in PCLS from naive rats. By contrast, PCLS cultures from the rat CCl4 NASH model were only responsive to the sGC activator, thus establishing that the sGC enzyme is rendered non-responsive to nitric oxide under oxidative stress found in fibrotic livers. The role of the sGC pathway for vessel relaxation of fibrotic liver tissue was identified in our model. The obtained data shows that the inhibitory capacities on vessel contraction of sGC compounds can be translated to published preclinical data. Altogether, this novel ex vivo PCLS method allows for the differentiation of drug candidates and the translation of therapeutic approaches towards the clinical use.
| S-EPMC8135082 | biostudies-literature
Project description:Background/aimsMost prognostic prediction models for patients with liver cirrhosis include serum total bilirubin (TB) level as a component. This study investigated prognostic performance of serum direct bilirubin (DB) and developed new DB level-based prediction models for cirrhosis.MethodsA total of 983 hospitalized patients with liver cirrhosis were included. DB-Model for End-Stage Liver Disease (MELD) score was calculated using MELD score formula, with serum DB level replacing TB level.ResultsMean age of study population was 56.1 years. Alcoholic liver disease was the most frequent underlying condition (471 patients, 47.9%). Within 6 months, 144 patients (14.6%) died or received liver transplantation due to severe liver dysfunction. The area under the receiver operating characteristic curve (AUROC) for prediction of 6-month mortality with DB level was significantly higher than that with TB level (p<0.001). The AUROC of DB-MELD score for prediction of 6-month mortality was significantly higher than that of MELD score (p<0.001). Patients were randomly divided into training (n=492) and validation (n=491) cohorts. A new prognostic prediction model, "Direct Bilirubin, INR, and Creatinine" (DiBIC) score, was developed based on the most significant predictors of 6-month mortality. In training set, AUROC of DiBIC score for prediction of 6-month mortality was 0.892, which was significantly higher than that of the MELD score (0.875, p=0.017), but not different from that of DB-MELD score (0.886, p=0.272). Similar results were observed in validation set.ConclusionsNew prognostic models, DB-MELD and DiBIC scores, have good prognostic performance in liver cirrhosis patients, outperforming other currently available models.
| S-EPMC8283287 | biostudies-literature