Bilirubin Uridine Diphospho-glucuronyltransferase in Rat Liver Microsomes: Genetic Variation and Maturation

Abstract

Extract: Optimal conditions for the in vitro assay of bilirubin uridine diphospho- (UDP) glu-curonyltransferase activity in rat liver microsomes are described. Solvent partitioning was used to separate the conjugated from nonconjugated bilirubin, thus avoiding dependency on the rate of coupling with diazotized sulfanilic acid for the distinction between bilirubin and its conjugated form. The inclusion of uridine diphospho-N-ace-tylglucosamine (UDPNAG) in the reaction mixture permitted the rate of conjugation of bilirubin by fresh rat liver homogenates and microsomes to occur at greater saturation of the available enzyme with the substrates bilirubin and UDP-glucuronic acid. Liver microsomes, isolated in 0.15 M KC1, increased their activity for bilirubin conjugation and decreased their dependency on UDPNAG during the first 10 days of storage at — 15°. Chromatographic separation of the azo pigments of the conjugated bilirubin gave evidence to suggest that bilirubin monoglucuronide was the initial product and bilirubin diglucuronide appeared in increasing amounts in more prolonged incubations. These results suggested that bilirubin monoglucuronide can be intermediate to the formation of bilirubin diglucuronide. Bilirubin UDP-glucuronyltrans-ferase activity in hepatic microsomes of adult homozygous Gunn rats was not demonstrable. In microsomes of heterozygous Gunn rats and normal Wistar and Sprague-Dawley rats bilirubin UDP-glucuronyltransferase activity was found to be 31.0 and 58.0 μg bilirubin conjugated/mg microsomal N/30 min, respectively. Measurements in developing rats indicated that the maturation in enzyme activity occurred by at least two distinct means: increase of specific activity of the microsomes, and an increase in the content of microsomes per gram of liver (Table IV). Speculation: A method for quantitative measurement of bilirubin UDP-glucuronyltransferase activity applicable to samples obtained by needle biopsy has been needed. The method described in this report meets this need and may permit more precise differential diagnosis of retention jaundice of infancy and childhood and especially the “physiologic” jaundice of the newborn.