title: An improved method for routine determination of vitamin D and its hydroxylated metabolites in serum from children and adults.
abstract: A method for routine determination of vitamin D and its major metabolites 25-hydroxyvitamin D (25(OH)D), 24,25-dihydroxyvitamin D (24,25(OH)2D) and 1,25-dihydroxy-vitamin D (1,25(OH)2D) in serum samples from normal children and adults has been developed. Methodological improvements enable a rapid and accurate analysis of 25(OH)D and also the microscale screening of other metabolites present in large concentrations in serum. Vitamin D and its metabolites are extracted from serum samples using hexane/propan-2-ol, which allows a convenient separation of the water soluble and lipid soluble fractions from each other and also from proteins. Preparative silicic acid chromatography was used to separate vitamin D from its metabolites and also from the major portion of co-eluting lipid contaminants. An automated LC solvent delivery and sample introduction system was used to achieve the rapid separation of metabolites. Vitamin D was further purified using adsorption high-performance liquid chromatography and assayed using reverse phase high-performance liquid chromatography connected with UV detection. The 25(OH)D fraction from the preparative chromatography was measured by a competitive protein-binding assay along with 24,25(OH)2D, which was purified by reverse phase high-performance liquid chromatography along with 1,25(OH)2D. A diluted human serum from a pregnant woman (3rd trimester of pregnancy) was used as source of the binding protein for 25(OH)D and 24,25(OH)2D. 1,25(OH)2D was determined by a competitive protein-binding assay using a diluted cytoplasmic 1,25(OH)2D receptor protein isolated from the intestinal mucosa of rachitic chicks. Vitamin D and its metabolite levels were assayed in serum samples from normal children and adults.
mesh_terms: D015650:24,25-Dihydroxyvitamin D 3; D000293:Adolescent; D000328:Adult; D000368:Aged; D001667:Binding, Competitive; D002112:Calcifediol; D002117:Calcitriol / Q000097:blood; D002352:Carrier Proteins; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004100:Dihydroxycholecalciferols / Q000097:blood; D004872:Ergocalciferols / Q000097:blood; D006801:Humans; D006887:Hydroxycholecalciferols / Q000097:blood; D007223:Infant; D007231:Infant, Newborn; D008875:Middle Aged; D014807:Vitamin D / Q000097:blood*; D014809:Vitamin D-Binding Protein
chemical_list: D002352:Carrier Proteins; D004100:Dihydroxycholecalciferols; D004872:Ergocalciferols; D006887:Hydroxycholecalciferols; D014809:Vitamin D-Binding Protein; D014807:Vitamin D; D015650:24,25-Dihydroxyvitamin D 3; D002117:Calcitriol; D002112:Calcifediol