Metabolism of vitamin D2 in pig liver homogenates: evidence for a free radical reaction.
In vitro hydroxylation of vitamin D2 at carbon-24 (C-24) was demonstrated with pig liver homogenate. The putative 24-hydroxyvitamin D2 (24-OHD2) comigrated with standard 24-OHD2 on a Zorbax Sil column developed in hexane/isopropanol (98/2). Rechromatography in methylene chloride/methanol (99.8/0.2) resolved the putative 24-OHD2 into two components. The identity of these compounds was determined to be 24(R)-OHD2 and 24(S)-OHD2 (epimers) by low resolution mass spectroscopy and proton NMR spectroscopy. The fact that epimers of 24-OHD2 were produced from vitamin D2 in the absence of pig liver homogenate in vitro was strong evidence for the participation of free radicals in the reaction. Further support for free radical involvement was provided by the following observations: (a) hydroxyl free radical scavengers such as alpha-tocopherol, catalase, and ethanol reduced the amount of 24-OHD2 produced by 18-64%; (b) use of autoclaved homogenate in the incubation mixture had little or no effect on the amount of 24-OHD2 produced; and (c) the failure of the enzyme-substrate saturation curve to level off as expected with high levels of vitamin D2 (100-2000 micrograms = 50-1009 microM). Maximum production of 24-OHD2 was obtained at pH 4.75 and represented a sevenfold increase relative to the amount produced at pH 7.4. The omission of citrate or the addition of electron transport inhibitors, cyanide or antimycin, had little or no effect on the reaction. These data suggested that C-24 hydroxylation of vitamin D2 in vitro was a free radical-mediated process not involving the electron transport system. In vitro hydroxylation at C-24 appeared to be driven by free radicals, and the dominance of this reaction made it difficult to determine whether there was an enzyme involved in the reaction.
Engstrom
G W
GW
National Animal Disease Center, U.S. Department of Agriculture, Ames, Iowa 50010.
Koszewski
N J
NJ
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004872:Ergocalciferols / Q000031:analogs & derivatives / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D005609:Free Radicals; D006900:Hydroxylation; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D013250:Steroid Hydroxylases / Q000032:analysis; D013552:Swine
D004872:Ergocalciferols; D005609:Free Radicals; C028139:24-hydroxyvitamin D2; D013250:Steroid Hydroxylases