Glucuronic acid conjugates of bilirubin-IXα in normal bile compared with post-obstructive bile. Transformation of the 1-O-acylglucuronide into 2-, 3-, and 4-O-acylglucuronides

Abstract

Structures have been determined for bilirubin-IXalpha conjugates in freshly collected bile of normal rats, dogs and man and in post-obstructive bile of man and rats. The originally secreted conjugate has been characterized as azopigment (I), i.e. a 1-O-acyl-beta-d-glucopyranuronic acid glycoside. Conversion of the acetylated methyl ester of azopigment (I) into methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-beta-d-glucopyranuronate (V) indicates the pyranose ring structure for the carbohydrate and a C-1 attachment for the bilirubin-IXalpha acyl group. Alternative procedures for deconjugation of azopigment (I) and its derivatives are also described. In post-obstructive bile, the 1-O-acylglucuronide is converted into 2-, 3- and 4-O-acylglucuronides via sequential intramolecular migrations of the bilirubin acyl group. The following approach was utilized. (1) The tetrapyrrole conjugates were cleaved to dipyrrolic aniline and ethyl anthranilate azopigments, and the azopigments were separated as the acids or methyl esters. (2) The isomeric methyl esters were characterized by mass spectral analysis of the acetates and silyl ethers. (3) The free glycosidic function was demonstrated by 1-oxime and 1-methoxime derivative formation. (4) The position of the dipyrrolic O-acyl group was determined for the methyl esters by protecting the free hydroxyl groups of the glucuronic acid moieties as the acetals formed with ethyl vinyl ether and by further conversion of the carbohydrates into partially methylated alditol acetates. These were analysed by using g.l.c.-mass spectrometry. The relevance of the present results with regard to previous reports on disaccharidic conjugates is discussed. Details of procedures for the formation of chemical derivatives for g.l.c. and mass spectrometry have been deposited as Supplementary Publication SUP 50081 (15 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978), 169, 5.

Unconjugated bilirubin induces apoptosis in colon cancer cells by triggering mitochondrial depolarization

“…1), it appears that it is the unconjugated form of bilirubin that specifically modulates tumorigenesis. Bilirubin is secreted into bile as the 1-O-acylglucuronide conjugate, 28,82 which partially undergoes acyl migration in the gallbladder and intestine to form 2-, 3-and 4-O-acyl isomers. 83,84 Deconjugation of bilirubin 1-O-acylglucuronide, but not the other positional isomers, 84 occurs within the intestinal lumen both by spontaneous nonenzymic hydrolysis 29 and through the action of mucosal and bacterial ␤-glucuronidases 30,31 present primarily in the distal small bowel and colon, where luminal bilirubin levels are the highest.…”

Mechanism of Bilirubin Diglucuronide Formation in Intact Rats

“…It is well established that formation of bilirubin monoglucuronide (BMG) is catalyzed by microsomal uridine diphosphate (UDP)-glucuronosyltransferase [UDP-glucuronate /3-glucuronosyltransferase (acceptor unspecific), EC 2.4.1.17], with UDP-glucuronic acid (UDPGlcUA) serving as sugar donor (5). It remained unexplained, however, whv on incubation of liver homogenate or microsomiies frolm humans, rats, dogs, and cats with UDPGlcUA, the reaction product was largely BMG (6), while in these species bile contains predominantly bilirubin diglucuronide (BDG) (2,3,(7)(8)(9). Because of this apparent inconsistenicy between the findings in vivo and in vitro, it has been postulated that conversion of B.MG to BDG is catalyzed by a separate enzyme that differs fromii bilirubin UDP-glucuronosyltransferase.…”

“…Collectively, these findings establish that a transglucuronidation mechanism is not operational in vivo and support the concept that bilirubin diglucuronide is formed by a microsomal UDP-glucuronosyltransferase system. INTRODUCTION Bilirubin,' the principal degradation product of heme in mammals (1), is excreted in bile largely in the form of polar derivatives in which one or both propionic acid ,8-substituents of the pigment are esterified with a sugar, predominantly glucuronic acid (2,3). This conjugation is thought to disrupt the multiple intramolecular hydrogen bonds responsible for the nonpolar character of bilirubin and thereby renders the pigment excretable in bile (4).…”

Uridine Diphosphate-Glucuronic Acid-independent Conversion of Bilirubin Monoglucuronides to Diglucuronide in Presence of Plasma Membranes from Rat Liver is Nonenzymic

“…1) and, predominantly, as the diglucuronide (BDG) (1)(2)(3)(4)(5). The first metabolic conversion step involves transfer of a glucuronyl residue from UDP-glucuronic acid to bilirubin, catalyzed by a microsomal UDP-glucuronyltransferase (EC 2.4.1.17) (6)(7)(8)(9).…”