EVIDENCE FOR HUMAN PLACENTAL SYNTHESIS OF 24,25-DIHYDROXYVITAMIN-D(3)AND 23,25-DIHYDROXYVITAMIN-D(3)

The two principal dihydroxylated metabolites of the vitamin D prohormo ne 25-hydroxyvitamin D3 25(OH)D3! are 1alpha,25-dihydroxyvitamin D31 ,25(OH)2D3, the active hormone! and 24R,25-dihydroxyvitamin D3 24,25( OH)2D3, a putative regulator of developmental bone formation!. Althoug h several studies have demonstrated placental synthesis of 1,25(OH)2D3 from 25(OH)D3, placental production of 24,25(OH)2D3 has not been thor oughly investigated. Therefore, we studied 25(OH)D3 metabolism in term human placenta using a villous explant model and cultures of isolated trophoblast and villous mesenchymal cells. We determined that both vi tamin D-replete and vitamin D-deficient trophoblast metabolize 25(OH)D 3 predominantly via 24-hydroxylation. Placental 24,25(OH)2D3 was ident ified by cochromatography with authentic standard on four different HP LC systems, scanning UV spectrophotometry profile of the metabolite, s ensitivity to periodate cleavage, and mass spectrometry of the putativ e placental 24,25(OH)2D3 and its periodate cleavage product. We also i dentified for the first time placental synthesis of 23,25(OH)2D3 using cochromatography with authentic standard on two different HPLC system s, scanning UV spectrophotometry, resistance to periodate cleavage, an d mass spectrometry. When trophoblast was incubated for up to 4 h with physiologic concentrations of H-3!25(OH)D3 (6 nM) significant amount s of H-3!24,25(OH)2D3 were produced, but H-3!1,25(OH)2D3 could not b e consistently detected. In contrast, when we incubated trophoblast wi th supraphysiologic concentrations of 25(OH)D3 (6-10 muM), both 24,25( OH)2D3 and 1,25(OH)2D3 were synthesized. These results provide unequiv ocal evidence for placental synthesis of both 24,25(OH)2D3 and 23,25(O H)2D3. These findings also suggest that supraphysiologic substrate con centrations saturate the placental 24-hydroxylase and may permit accum ulation of placental 1,25(OH)2D3 by preventing its further metabolism. Consequently, the identification of this high basal 24-hydroxylase ac tivity in trophoblast may explain inconsistencies among previous repor ts regarding placental 1,25(OH)2D3 production. We speculate that activ e placental 24-hydroxylation may serve important functions in perinata l vitamin D metabolism.