Determination of progesterone and some of its neuroactive ring A-reduced metabolites in human serum

A method for the separation and assay of some ring A-reduced metabolites of progesterone (pregnanediones and pregnanolones) is described. Serum was extracted with an organic solvent, and the extract chromatographed using high performance liquid chromatography (HPLC). A total of 50 fractions was collected for each sample and split using a stream splitter so that 30% was collected in counting vials for recovery while 70% was collected in test tubes which were assayed by radioimmunoassay. An antiserum raised in our laboratory to progesterone-3-CMO-BSA cross-reacted with five of these compounds (5 alpha- and 5 beta -dihydroprogesterone, 3 alpha- and 3 beta -5 alpha -tetrahydroprogesterone, and 3 beta ,5 beta -tetrahydroprogesterone). Since pregnenolone eluted with 5 alpha ,3 beta -tetrahydroprogesterone, pregnenolone was assayed separately and its effect subtracted. Using this method it was shown that picogram to nanogram/ml amounts of these metabolites are present in all human sera. Levels in men were comparable to those of women in the follicular phase of the menstrual cycle. 5 alpha -Dihydroprogesterone and 3 alpha ,5 alpha -tetrahydroprogesterone rose substantially in the luteal phase of the menstrual cycle and all lose considerably during pregnancy. (C) 2000 Elsevier Science Ltd. All rights reserved.