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 (5alpha- and 5beta-dihydroprogesterone, 3alpha- and 3beta-5alpha-tetrahydroprogesterone, and 3beta, 5beta-tetrahydroprogesterone). Since pregnenolone eluted with 5alpha, 3beta-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. 5alpha-Dihydroprogesterone and 3alpha,5alpha-tetrahydroprogesterone rose substantially in the luteal phase of the menstrual cycle and all rose considerably during pregnancy.