New tritium-release assay for 25-hydroxyvitamin D-1 alpha-hydroxylase

Journal Article · Sat Aug 01 04:00:00 EDT 1987 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(87)90514-8· OSTI ID:5856828

Brown, A J; Perlman, K; Schnoes, H K; DeLuca, H F

A new, rapid assay for 1 alpha-hydroxylase has been developed using 25-hydroxy-(1 alpha-/sup 3/H)vitamin D3 as the substrate. Using the solubilized and reconstituted chick 1 alpha-hydroxylase, conversion of this substrate to 1,25-dihydroxyvitamin D3 causes the release of tritium into the aqueous medium. This /sup 3/H/sub 2/O can be easily separated from the labeled substrate by passing the reaction mixture through a reverse-phase silica cartridge. The release of tritium is stereospecific as evidenced by the lack of /sup 3/H/sub 2/O formed when 25-hydroxy-(1 beta-/sup 3/H)vitamin D3 is used as the substrate. In parallel reactions containing the 25-hydroxy-(26,27-/sup 3/H)vitamin D3 substrate, production of labeled 1,25-dihydroxyvitamin D3 was assessed by extraction and high-performance liquid chromatography and found to agree very closely with the amount of /sup 3/H/sub 2/O produced from 25-hydroxy-(1 alpha-/sup 3/H)vitamin D3, validating the accuracy of the new assay. Finally, a major advantage of the tritium-release assay for 1 alpha-hydroxylase is that the results are not affected by further metabolism of the 1,25-dihydroxyvitamin D formed in the incubations.

• Research Organization: Univ. of Wisconsin, Madison
• OSTI ID: 5856828
• Journal Information: Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 164:2; ISSN ANBCA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BIRDS
CELL CONSTITUENTS
CHICKENS
CHROMATOGRAPHY
ENZYMES
FOWL
HYDROGEN COMPOUNDS
HYDROXYLASES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
METABOLISM
MITOCHONDRIA
ORGANOIDS
OXIDOREDUCTASES
OXYGEN COMPOUNDS
RADIOENZYMATIC ASSAY
SEPARATION PROCESSES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES
VITAMIN D
VITAMINS
WATER