Endocrinology, Vol 125, 1224-1230, Copyright © 1989 by Endocrine Society

ARTICLES

In vitro metabolism of 25-hydroxyvitamin D3 by human trophoblastic homogenates, mitochondria, and microsomes: lack of evidence for the presence of 25-hydroxyvitamin D3-1 alpha- and 24R-hydroxylases

BW Hollis, VN Iskersky and MK Chang
Department of Pediatrics, Medical University of South Carolina, Charleston 29245-2248.

In vitro studies were performed to assess the ability of term human trophoblastic tissue to metabolize 25-hydroxyvitamin D3 (25OHD3) and to compare this metabolism to that occurring in porcine renal mitochondria and microsomes. Human trophoblastic homogenates, containing a NADPH- generating system, were able to produce 1,25-dihydroxyvitamin D3 [1,25- (OH)2D3] at a rate of 225 pg/mg protein.h, but did not produce detectable quantities (less than 20 pg/mg protein.h) of 24,25- dihydroxyvitamin D3 [24,25-(OH)2D3]. Similarly, mitochondria and microsomes isolated from term human trophoblastic tissue produced 1,25- (OH)2D3 [249 +/- 156 and 199 +/- 82 (mean +/- SD) pg/mg protein.h, respectively] in the presence of an NADPH-generating system, but failed to produce detectable quantities (less than 200 pg/mg protein.h) of 24,25-(OH)2D3. The production of 1,25-(OH)2D3 from the trophoblastic mitochondria and microsomes could be increased by adding 140,000 x g trophoblastic cytosol to the subcellular incubation tubes. This treatment had no effect on the production of 24,25-(OH)2D3. The component(s) present in trophoblastic cytosol responsible for the increased 1,25-(OH)2D3 production by trophoblastic mitochondria and microsomes was shown to be heat labile, trypsin resistant, and less than 1000 mol wt in size. Comparing characteristics of the porcine renal 1 alpha- and 24R-hydroxylase systems with those of the human trophoblastic system revealed that 1) 1,2-dianilinoethane and EDTA totally blocked synthesis of 1,25-(OH)2D3 in trophoblastic mitochondria and microsomes, but had no effect on the synthesis of 1,25-(OH)2D3 by renal mitochondria; and 2) ketoconazole greatly inhibited the synthesis of 1,25-(OH)2D3 and 24,25-(OH)2D3 by renal mitochondria, but had no effect on the production of 1,25-(OH)2D3 by trophoblastic mitochondria or microsomes. Finally, production of 24,25-(OH)2D3 could not be demonstrated in trophoblastic homogenates, mitochondria, or microsomes, while the production of this compound was readily evident in renal mitochondria, but not microsomes. The results of this study question the existence of the 25-hydroxyvitamin D3-1 alpha- and 24R-hydroxylase systems in the trophoblastic portion of the human placenta. This study also suggests that 1,25-(OH)2D3 can be produced in vitro by a mechanism other than enzymatic 1 alpha-hydroxylation. The possibility exists that the mechanism involves the insertion of oxygen at the 1 position of 25- (OH)D3 by free radical chemistry.

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