Assay of 25-hydroxy vitamin D3-1 α-hydroxylase in pig kidney mitochondria using isotope dilution-mass spectrometry

Abstract An assay of 1α-hydroxylation of 25-hydroxy vitamin D 3 in pig kidney mitochondria, based on selected ion monitoring, has been developed. Trideuterium-labeled 1,25-dihydroxy vitamin D 3 was synthesized and used as internal standard. This standard was added immediately after incubation of 25-hydroxy vitamin D 3 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 D 3 was linear with the amount of mitochondrial protein and time of incubation. Substrate saturation was obtained at about 20 μ m of 25-hydroxy vitamin D 3 . The maximal rate of conversion obtained under the conditions employed was about 0.1 pmol/mg protein · minute.