Initial studies on the N-glucosylation of phenobarbital by mouse liver microsomes using a radiochemical high-performance liquid chromatographic (HPLC) method.
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.
Soine
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WH
Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond 23298-0540.
Safi
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Westkaemper
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D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D015394:Molecular Structure; D010634:Phenobarbital / Q000378:metabolism*; D013237:Stereoisomerism; D014316:Tritium
D014316:Tritium; D010634:Phenobarbital