A rapid assay for 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D 24-hydroxylase

Journal Article · Mon Oct 01 04:00:00 EDT 1990 · Analytical Biochemistry; (USA)

DOI:https://doi.org/10.1016/0003-2697(90)90141-U· OSTI ID:5924788

Burgos-Trinidad, M; Brown, A J; DeLuca, H F ^[1]

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1. Univ. of Wisconsin-Madison (USA)

A rapid method for the measurement of the 24-hydroxylated metabolites of 25-hydroxy(26,27-3H)vitamin D3 and 1,25-dihydroxy(26,27-3H)vitamin D3 has been developed. This measurement has, in turn, made possible a rapid assay for the 24-hydroxylases of the vitamin D system. The assay involves the use of 26,27-3H-labeled 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 as the substrate and treatment of the enzyme reaction mixture with sodium periodate, which specifically cleaves the 24-hydroxylated products between carbons 24 and 25, releasing tritiated acetone. The acetone is measured after its separation from the labeled substrate by using a reversed-phase cartridge. The results obtained with this assay were validated by comparison with the results obtained with a well-established high-performance liquid chromatography assay. The activity of the enzyme determined by both methods was equal. This assay has been successfully used for the rapid screening of column fractions during purification of the enzyme and in the screening for monoclonal antibodies to the 24-hydroxylase.

• OSTI ID: 5924788
• Journal Information: Analytical Biochemistry; (USA), Journal Name: Analytical Biochemistry; (USA) Vol. 190:1; ISSN ANBCA; ISSN 0003-2697
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETONE
ANIMALS
BIRDS
CHICKENS
CHROMATOGRAPHY
ENZYMES
FOWL
HYDROGEN COMPOUNDS
HYDROXYLASES
ISOTOPE APPLICATIONS
KETONES
LIQUID COLUMN CHROMATOGRAPHY
METABOLISM
METABOLITES
ORGANIC COMPOUNDS
OXIDOREDUCTASES
RADIOASSAY
SEPARATION PROCESSES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES
VITAMIN D
VITAMINS