Initial studies on the N-glucosylation of phenobarbital by mouse liver microsomes using a radiochemical high-performance liquid chromatographic (HPLC) method

• Published in: Pharmaceutical research 1992-05, Vol.9 (5), p.613-616
• Main Authors: Soine, W H, Safi, H, Westkaemper, R B
• Format: Article
• Language: English
• Subjects: Animals; Chromatography, High Pressure Liquid; Glycosylation; Male; Mice; Mice, Inbred ICR; Microsomes, Liver - enzymology; Microsomes, Liver - metabolism; Molecular Structure; Phenobarbital - metabolism; Stereoisomerism; Tritium
• ISSN: 0724-8741
• DOI: 10.1023/A:1015889707922

A method is described for the assay of phenobarbital N-glucosylation using UDP-D-[6-3H]glucose. The radioactive phenobarbital N-glucoside conjugates [(5R)-PBG, (5S)-PBG] formed during the incubations were resolved from each other and from uncharacterized radioactive products by semipreparative HPLC. The product ratio of the N-glucosides of (5R)-PBG/(5S)-PBG was 2.9 for the crude liver homogenate and 3.0 +/- 0.5 for the microsomes. Magnesium was necessary for optimal activity. The Km values for formation of (5R)-PBG, (5S)-PBG, and (5R + 5S)-PBG were 1.55 +/- 0.35, 1.27 +/- 0.14, and 1.47 +/- 0.21 mM, respectively. The Vmax values for formation of (5R)-PBG, (5S)-PBG, and (5R + 5S)-PBG were 1.34 +/- 0.05 x 10(-6), 0.43 +/- 0.01 x 10(-6), and 1.77 +/- 0.04 x 10(-6) mumol/min/mg microsomal protein, respectively. It was observed that at concentrations greater than 5 mM sodium phenobarbital, inhibition of formation of phenobarbital N-glucosides occurred. The product ratio of (5R)-PBG/(5S)-PBG is comparable to that observed in the urinary excretion studies with the mouse and opposite to that observed in urinary excretion studies in humans.