Abstract
Cholate-solubilized chick kidney mitochondria that 1-hydroxylated 25-hydroxyvitamin-D3 (25-OH-D3) upon reconstitution also produced 10-oxo-19-nor-25-OH-D3, which co-eluted with 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) on normal phase high performance liquid chromatography (HPLC) with hexane:propanol-2 (9:1), the traditional chromatographic system for isolating 1,25-(OH)2-D3. The 10-oxo derivative was separated from 1,25-(OH)2-D3 by normal phase HPLC with dichloromethane:propanol-2 (19:1) or by reverse phase HPLC with methanol:water (4:1). Unlike 1,25-(OH)2-D3 production, formation of 10-oxo-19-nor-25-OH-D3 did not require a source of reducing equivalents and was blocked by the antioxidants, diphenyl-rho-phenylenediamine, and butylated hydroxytoluene, implicating a free radical or peroxidative synthetic mechanism. Rat kidney mitochondria solubilized with cholate or with cholate and Emulgen 911 produced 10-oxo-19-nor-25-OH-D3 but no detectable 1 alpha,25-(OH)2-D3. These results stress the importance of careful identification of vitamin D metabolites produced in vitro and suggest the use of alternate chromatographic conditions for isolating 1,25-(OH)2-D3 or inclusion of antioxidants in the assay of solubilized 1 alpha-hydroxylase to eliminate contamination of 1,25-dihydroxyvitamin D3 with 10-oxo-19-nor-25-OH-D3.
Highlights
Tuted with NADPH, beef adrenal ferredoxin reductase, and upon reconstitution produced lO-oxo-19-nor-25- ferredoxin from beef adrenal (13) or chickenkidney(14)
OH-D3 did not requairesource of reducing equivalentfsromthose of pig, beefa,nd chicken (17,18).Cholate/Emulgen and was blocked by the antioxidants, diphenyl-p-phen9-11-solubilizedand partially purified cytochrome P-450 fracylenediamine,andbutylatedhydroxytoluene,impli- tion from rat kidney mitochondria did not require a source of cating a free radical or peroxidative synthmeteicha- reducing equivalents to produce 1,25-(OH)z-D3.In addition, nism
Rat kidney mitochondria solubilized with choilnahteibition studies indicateda peroxidative mechanism fothr is or with cholate and Emulg9en11 produced lO-oxo-19nor-25-OH-D3 but no detectable la,25-(OH)z-D3
Summary
Vitamin D-deficient animals, the kidney 25-0H-D~ la-hydroxylase activity is maximal (7). Washed rat mitochondria were solubilized with cholate and Emulgen 911 and partially purified by chromatography on a column of dichlorophenylphenoxyethylamine-Sepharose6B as described by Warner (17). Adrenodoxin reductase was purified from beef adrenal mitochondria by the affinity chromatography method of Sugiyama and Yamano (26). Assay of 25-oH-D3Metabolism-The standard assay mixture contained 150 pg of protein from cholate-solubilized chick or rat preparation or 33 pgof cholate/Emulgen 911-solubilized and partially purified rat preparation in 25 pl, 165 pl of 15 mM Tris acetate, pH. Aliquots of 4 ml wereplaced tion solvent(191) (A)or on Zorbax-ODS reverse phase column using in 125-ml Erlenmeyer flasks and incubated for 60 min a t 37"C. methanokwater (41) ( E ) .Arrows indicate elutionposition of 10-oxo- Samples were extracted by the Bligh and Dyer method (27) and. The purified material was analyzed by mass 'spectrometry using a Kratos
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