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Prednisolone is metabolized in the perfused human placental lobule to prednisone, 20 alpha-dihydroprednisone, 20 beta-dihydroprednisone and 20 beta-dihydroprednisolone. The pathway of metabolite formation was defined in perfusions of placental lobules using prednisone and 20 beta-dihydroprednisone separately as substrates and with prednisolone co-perfused with glycyrrhetinic acid, a potent inhibitor of the 11-oxidase component of the 11 beta-hydroxysteroid dehydrogenase enzyme system. The pattern of metabolites identified from 6 h samples indicated a reversible formation of prednisone from prednisolone, the production of the 20 alpha- and 20 beta-dihydro metabolites of prednisone from prednisone, the formation of 20 beta-dihydroprednisolone from 20 beta-dihydroprednisone only and no direct formation of 20 beta-dihydroprednisolone from prednisolone. Kinetic analysis at two substrate concentrations confirmed that the formation of three of the four steroid metabolites followed first order kinetics. In perfusions with an initial prednisolone concentration of 1 microgram/ml (n = 4) or 100 ng/ml (n = 3), the rate constants obtained were (mean +/- SD, maternal compartment, h-1): prednisone, 1.97 +/- 0.49 and 2.25 +/- 0.15, P > 0.1; 20 alpha-dihydroprednisone, 0.0006 +/- 0.0004 and 0.0017 +/- 0.0006, P < 0.1; 20 beta-dihydroprednisone, 0.15 +/- 0.022 and 0.15 +/- 0.0077, P > 0.1. In contrast, the rate constant for formation of 20 beta-dihydroprednisolone at an initial prednisolone concentration of 100 ng/ml (0.083 +/- 0.0095 h-1) was significantly (P < 0.01) greater than the corresponding rate constant at the higher initial prednisolone concentration (0.039 +/- 0.015 h-1). A significant increase (P < 0.05) was observed for the formation of 20 beta-dihydroprednisolone at the end of 6 h perfusions at the lower initial substrate concentration (11.2 +/- 1.9%) compared with the 1 microgram/ml concentration (6.0 +/- 2.5%).

Previous studies of the metabolism of 11 beta-hydroxy corticosteroids by placental tissue have indicated that the only product is the C11-oxidized metabolite. In the present study we have re-examined the metabolism of prednisolone in the isolated, perfused, dual recirculating human placental lobule, using a perfusate based on tissue culture medium 199. Four metabolites were identified in both the maternal and fetal compartments in 6 h perfusions by comparison of relative retention times measured by HPLC and capillary gas chromatography (GC) and of mass spectra recorded by capillary gas chromatography-mass spectrometry (GC-MS) with those of authentic reference standards. The steroids were derivatized as the MO-TMS ethers for mass spectral measurements. Analysis of samples from five perfusion experiments resulted in the following percentage conversions after 6 h perfusion (mean +/- SD, maternal and fetal perfusate, respectively): prednisone (49.1 +/- 7.8, 49.1 +/- 6.6), 20 alpha-dihydroprednisone (0.84 +/- 0.29, 0.81 +/- 0.35), 20 beta-dihydroprednisone (39.1 +/- 6.7, 39.2 +/- 5.9), 20 beta-dihydroprednisolone (6.8 +/- 2.7, 6.3 +/- 1.6) and unmetabolized prednisolone (4.1 +/- 1.8, 4.6 +/- 2.1). No evidence was found for metabolites formed by 6 beta-hydroxylation or cleavage of the C17-C20 bond.

A reversed-phase liquid chromatographic assay was developed to quantitate prednisolone, prednisone and the 20 alpha-dihydro and 20 beta-dihydro reduced metabolites of both parent compounds in tissue culture media from in vitro perfusions of the human placental lobule. Steroids were extracted from perfusate, using reversed-phase cartridges, with average recoveries of 95.2% or greater. The internal standard for the analyses was 6 alpha-methylprednisolone. In this assay cortisol coelutes with prednisolone, however, no other significant interferences were found. Assay of each steroid was linear in the range 0-1 microgram/ml. Intra-assay coefficients of variation were measured at 10 and 750 ng/ml with ranges of 3.4% (20 alpha-dihydroprednisone) to 8.8% (20 beta-dihydroprednisolone) and 4.1% (20 beta-dihydroprednisone) to 8.8% (prednisone). The corresponding inter-assay coefficients of variation were 3.3% (20 alpha-dihydroprednisone) to 9.1% (20 beta-dihydroprednisolone) and 1.9% (prednisolone) to 3.5% (prednisone). The analyses utilized two C18 columns which were linked together and maintained at 40 degrees C.