Production of 10-oxo-19-nor-25-hydroxyvitamin D3 by solubilized kidney mitochondria from chick and rat.
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.
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000378:metabolism; D000818:Animals; D002084:Butylated Hydroxytoluene / Q000494:pharmacology; D002112:Calcifediol / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000302:isolation & purification; D002117:Calcitriol / Q000302:isolation & purification / Q000378:metabolism; D002645:Chickens; D019826:Cholic Acid; D002793:Cholic Acids; D002851:Chromatography, High Pressure Liquid; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D017137:Nonoxynol; D010655:Phenylenediamines / Q000494:pharmacology; D011092:Polyethylene Glycols; D051381:Rats; D012995:Solubility
D002793:Cholic Acids; D010655:Phenylenediamines; D002084:Butylated Hydroxytoluene; D017137:Nonoxynol; D011092:Polyethylene Glycols; C039030:19-nor-10-keto-25-hydroxyvitamin D3; C004845:N,N'-diphenyl-4-phenylenediamine; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002117:Calcitriol; D019826:Cholic Acid; D002112:Calcifediol