Assay of 25-hydroxy vitamin D3-1 alpha-hydroxylase in pig kidney mitochondria using isotope dilution-mass spectrometry.
An assay of 1 alpha-hydroxylation of 25-hydroxy vitamin D3 in pig kidney mitochondria, based on selected ion monitoring, has been developed. Trideuterium-labeled 1,25-dihydroxy vitamin D3 was synthesized and used as internal standard. This standard was added immediately after incubation of 25-hydroxy vitamin D3 with the mitochondrial fraction. The incubation extracts were purified by high-performance liquid chromatography. After formation of the trimethylsilyl derivative, the product was quantitated by mass fragmentography using the ion at m/z 452 and m/z 455. With the use of this assay it was found that formation of 1,25-dihydroxy vitamin D3 was linear with the amount of mitochondrial protein and time of incubation. Substrate saturation was obtained at about 20 microM of 25-hydroxy vitamin D3. The maximal rate of conversion obtained under the conditions employed was about 0.1 pmol/mg protein X minute.
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000032:analysis*; D000818:Animals; D008401:Gas Chromatography-Mass Spectrometry; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D011865:Radioisotope Dilution Technique; D013250:Steroid Hydroxylases / Q000032:analysis*; D013552:Swine
D013250:Steroid Hydroxylases; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase