Measurement of ring A-reduced progesterone metabolites by enzyme-linked immunoassay with colorimetric detection: baseline levels of six metabolites, including pregnanolone, in male rat plasma.

The performance of an antiserum to progesterone and pregnane neurosteroids was assessed in two competitive assay setups: radioimmunoassay and enzyme-linked immunoassay with colorimetric detection, both with the same limit of detection of 2 pg. The enzyme-linked immunoassay was less labor-intensive and had better precision of measurement and was used to measure progesterone and six of its ring A-reduced metabolites in rat plasma. The measured levels of allopregnanolone and progesterone were in agreement with those reported previously when measured by gas chromatography/mass spectrometry and high-performance liquid chromatography coupled with radioimmunoassay and substantially lower than those previously measured by radioimmunoassay without chromatographic separation. Both isomers of dihydroprogesterone and all four isomers of pregnanolone were detected in rat plasma, indicating that progesterone is metabolized by reduction at the C5 and C3 position of the A ring, in both alpha and beta configurations. In addition to 5beta-dihydroprogesterone and isopregnanolone, which have not been previously detected in the rat, we found considerable amounts of pregnanolone, which is neuroactive, with similar potency to that of allopregnanolone but was previously thought not to be produced in rats.