RT Journal Article
SR Electronic
T1 A SENSITIVE RADIOACTIVE ASSAY FOR HEXOBARBITAL HYDROXYLASE IN HEPATIC MICROSOMES
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 760
OP 765
VO 1
IS 6
A1 D. KUPFER
A1 J. ROSENFELD
YR 1973
UL http://dmd.aspetjournals.org/content/1/6/760.abstract
AB The in vitro oxidation of hexobarbital by rat liver microsomes yields 3-hydroxyhexobarbital as the prime product. Therefore, the presently described method was developed to assay 3-OH-Hex.2 The assay involves the incubation of 2-14C-hexobarbital with rat liver microsomes, in the presence of an NADPH-generating system, at 37°C in an atmosphere of air. To terminate the reaction, 1 M citrate buffer, pH 5.5 (containing 15% NaCl), is added and the unmetabolized hexobarbital is extracted with 1-chlorobutane. The 3-OH-Hex in the aqueous phase is then extracted with ethyl acetate and an aliquot of the EtOAc is evaporated and counted in a scintillation spectrometer. Thin-layer chromatography demonstrated that the BuCl phase contained almost exclusively the unmetabolized hexobarbital, whereas the EtOAc contained 3-OH-Hex and occasionally a small amount of 3-ketohexobarbital. The simplicity of the assay permits a large number of determinations in a given time. The high sensitivity of the assay allows determinations when a small amount of the product, 3-OH-Hex (corresponding to conversion of less than 1% of hexobarbital) is formed; namely, when initial reaction rates are measured and when a small amount of tissue is available. Copyright © 1973 by The American Society for Pharmacology and Experimental Therapeutics