PT  - JOURNAL ARTICLE
AU  - D. KUPFER
AU  - J. ROSENFELD
TI  - A SENSITIVE RADIOACTIVE ASSAY FOR HEXOBARBITAL HYDROXYLASE IN HEPATIC MICROSOMES
DP  - 1973 Nov 01
TA  - Drug Metabolism and Disposition
PG  - 760--765
VI  - 1
IP  - 6
4099  - http://dmd.aspetjournals.org/content/1/6/760.short
4100  - http://dmd.aspetjournals.org/content/1/6/760.full
SO  - Drug Metab Dispos1973 Nov 01; 1
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