Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10756534 | Biochemical and Biophysical Research Communications | 2014 | 6 Pages |
Abstract
The total serum concentration of 25-hydroxyvitamins D (25-hydroxyvitamin D3 and 25-hydroxyvitamin D2) is currently used as an indicator of vitamins D status. Vitamins D insufficiency is claimed to be associated with multiple diseases, thus accurate and precise reference methods for the quantification of 25-hydroxyvitamins D are needed. Here we present a novel enzyme-assisted derivatisation method for the analysis of vitamins D metabolites in adult serum utilising 25-[26,26,26,27,27,27-2H6]hydroxyvitamin D3 as the internal standard. Extraction of 25-hydroxyvitamins D from serum is performed with acetonitrile, which is shown to be more efficient than ethanol. Cholesterol oxidase is used to oxidize the 3β-hydroxy group in the vitamins D metabolites followed by derivatisation of the newly formed 3-oxo group with Girard P reagent. 17β-Hydroxysteroid dehydrogenase type 10 is shown to oxidize selectively the 3α-hydroxy group in the 3α-hydroxy epimer of 25-hydroxyvitamin D3. Quantification is achieved by isotope-dilution liquid chromatography-tandem mass spectrometry. Recovery experiments for 25-hydroxyvitamin D3 performed on adult human serum give recovery of 102-106%. Furthermore in addition to 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3 and other uncharacterised dihydroxy metabolites, were detected in adult human serum.
Keywords
RIC24,25-(OH)2D324,25-dihydroxyvitamin D3β-NAD+Girard P reagent25-Hydroxyvitamin D225-OHD3vitamin D External Quality Assessment SchemeDEQASNISTMSNSPECyPSRM1α,25-(OH)2D31α,25-dihydroxyvitamin D325-hydroxyvitamin D3LC–MSnβ-nicotinamide adenine dinucleotideSolid phase extractionUltra violetSerumCytochrome P450Coefficient of VariationMass spectrometrystandard reference materialNational Institute of Standards and TechnologyPyridineliquid chromatographyReconstructed ion chromatogramcholesterol oxidase
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Authors
Jonas Abdel-Khalik, Peter J. Crick, Graham D. Carter, Hugh L. Makin, Yuqin Wang, William J. Griffiths,