Biosynthesis of [3H]7 alpha-hydroxy-, 7 beta-hydroxy-, and 7-oxo-dehydroepiandrosterone using pig liver microsomal fractions.
Current research on dehydroepiandrosterone (DHEA) is limited due to lack of radiolabeled metabolites. We utilized pig liver microsomal (PLM) fractions to prepare [(3)H]-labeled 7 alpha-hydroxy-DHEA (7 alpha-OH-DHEA), 7 beta-hydroxy-DHEA (7 beta-OH-DHEA), and 7-oxo-DHEA substrates from 50 microM [1,2,6,7-(3)H]DHEA (specific radioactivity 60-80 mCi/mmol). The metabolites were separated by preparative thin-layer chromatography (TLC) using ethyl acetate:hexane:glacial acetic acid (18:8:3 v:v:v) as the mobile phase, extracted with ethyl acetate, and dried under a stream of nitrogen. Metabolites assayed by TLC and gas chromatography-mass spectrometry were observed to be pure. In the presence of an reduced nicotinamide adenine dinucleotide phosphate (NADPH)-regenerating system initiated with 1 mM NADPH alone, 1 mg/ml PLM produced 7 alpha-OH-DHEA with minor amounts of 7-oxo-DHEA (68 and 14 nmol/2h/2 ml, respectively; 82% conversion), while in the presence of 1mM NADPH and 1 mM oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), more 7-oxo-DHEA than 7 alpha-OH-DHEA (58 and 11 nmol/2 ml/120 min, respectively; 69% conversion) was formed. When longer reaction times were used with NADPH and NADP(+), a mixture of 7 alpha-OH-DHEA, 7 beta-OH-DHEA, and 7-oxo-DHEA was produced (19,14, and 35 nmol/180 min/2 ml, respectively; 62% conversion). Using pig liver microsomes, the radiolabeled metabolites of DHEA can be prepared in stable, pure form at 10mM concentrations and >0.5 mCi/mmol levels of radioactivity for biochemical studies.
Robinzon
Boaz
B
Department of Animal Science, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O.B. 12, Rehovot 76100, Israel. robinzon@agri.huji.ac.il
Miller
Kristy K Michael
KK
Prough
Russell A
RA
D000818:Animals; D002855:Chromatography, Thin Layer; D003687:Dehydroepiandrosterone / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D006900:Hydroxylation; D007553:Isotope Labeling / Q000379:methods*; D008862:Microsomes, Liver / Q000737:chemistry* / Q000378:metabolism; D009249:NADP / Q000737:chemistry; D013552:Swine; D014316:Tritium / Q000737:chemistry
D014316:Tritium; D003687:Dehydroepiandrosterone; D009249:NADP