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  • Title: Assay of 25-hydroxy vitamin D3-1 alpha-hydroxylase in pig kidney mitochondria using isotope dilution-mass spectrometry.
    Author: Holmberg I, Saarem K, Pedersen JI, Björkhem I.
    Journal: Anal Biochem; 1986 Dec; 159(2):317-22. PubMed ID: 3548480.
    Abstract:
    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.
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