Bilirubin Mono- and diglucuronide formation by human liver In vitro : Assay by high-pressure liquid chromatography

Abstract

Bilirubin diglucuronide, the major pigment in human bile is formed in two steps. Bilirubin is converted to bilirubin monoglucuronide by transfer of the glucuronosyl moiety of uridine diphosphoglucuronic acid catalyzed by the microsomal enzyme, uridine diphosphoglucuronate glucuraonosyl transferase (UDP glucuronyl transferase, EC 2.4.1.17). Bilirubin monoglucuaronide is converted to bilirubin diglucuronide in vitro by two enzymatic mechanisms: (a) UDP glucuronyl transferase-mediated transfer of a second mole of glucuronic acid form UDP-glucuronic acid to bilirubin monoglucuronide; (b) dismutation of 2 moles of bilirubin monoglucuronide to 1 mole of bilirubin diglucuronide and 1 mole of unconjugated bilirubin, catalyzed by bilirubin monoglucuronide dismutase (bilirubin glucuronoside glucuronosyl transferase EC 2.4.1.95). Assay methods for the three enzymatic mechanisms in human liver homogenate by high-pressure liquid chromatography analysis of underivatized bilirubin tetrapyrroles have been developed. UDP glucuronyl transferase was activated in five human liver homogenates with digitonin, Triton X-100, or UDP-N-acetylglucosamine. Greatest activation was observed with Triton X-100. The pH optimum for conversion of bilirubin to bilirubin monoglucuronide was 7.4, and UDP glucuronyl transferase activity was 625 +/- 51 nmoles per 20 min per gm liver. At high initial bilirubin concentrations (342 microM), the product of UDP glucuronyl transferase assay with bilirubin as substrate was predominantly bilirubin monoglucuronide. At lower initial bilirubin concentrations (6.5 to 34 microM), up to 15% bilirubin diglucuronide was formed. Glucuronyl transferase-mediated UDP glucuronic acid-dependent conversion of bilirubin monoglucuronide to diglucuronide was assayed using UDP-14-C-glucuronic acid. The pH optimum was 7.4, and the rate was 21 +/- 7 nmoles per gm liver per 20 min. The rate of bilirubin diglucuronide formation by enzymatic dismutation of bilirubin monoglucuronide was 470 +/- 112 nmoles per gm liver per min. The pH optimum was 6.6. The products of enzymatic dismutation were of the IX alpha configuration.

UDP-Glucuronosyltransferase 1A1 Is the Principal Enzyme Responsible for Etoposide Glucuronidation in Human Liver and Intestinal Microsomes: Structural Characterization of Phenolic and Alcoholic Glucuronides of Etoposide and Estimation of Enzyme Kinetics

“…Bilirubin is putatively considered as the most specific substrate for UGT1A1 (Bosma et al, 1994;Burchell et al, 1995;King et al, 2000). However, simultaneous and accurate determination of bilirubin and its multiple glucuronidation isomers (including one di-glucuronide, and two cis/trans and two positional isomers of mono-glucuronides) is difficult because of the light sensitivity of bilirubin, and the instability or interconversion of mono-and di-glucuronide of bilirubin in vitro and in vivo (Jansen et al, 1977;Chowdhury et al, 1981;Gordon et al, 1984;Miners et al, 2004;Zhang et al, 2005). In addition, it has been reported that bilirubin may also inhibit UGT1A4 (Ghosal et al, 2004).…”

Long Term Correction of Bilirubin-UDP-glucuronosyltransferase Deficiency in Gunn Rats by Administration of a Recombinant Adenovirus during the Neonatal Period

“…Samples were collected at 30-min intervals, and the remaining bile was returned via the duodenal cannula to avoid bile salt depletion and dehydration. Bile samples were analyzed by high pressure liquid chromatography (HPLC) using a Bondapak C-18 column (Millipore-Waters, Milford, MA), as described previously (20). In brief, the HPLC column was equilibrated with 50 mM ammonium acetate/acetic acid (pH 4.5) and 50% methanol.…”

“…UGT Activity toward Bilirubin-Tissue homogenates were prepared from liver biopsy specimens from two different lobes of the liver and UGT activity toward bilirubin was determined by a sensitive HPLCbased method as described previously (1,20).…”

“…Bile Pigment Analysis-For definitive evaluation of bilirubin glucuronidation, selected rat were provided with a polyethylene bile duct cannula and pigments excreted in bile were analyzed by HPLC using a Bondapak C-18 column (Millipore-Waters, Milford, MA) as described (20).…”

A novel strategy for in vivo expansion of transplanted hepatocytes using preparative hepatic irradiation and FasL-induced hepatocellular apoptosis

“…Bile pigments were analyzed by reverse-phase HPLC using a m-bondapak C18 column (MilliporeWaters, Milford, CA, USA) as described. 35 Peaks were detected by absorbance at 436 nm, identified by retention time using authentic pigment standards, and quantitated by electronic integration of the peak areas.…”