QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Engstrom, G. W. Articles by Littledike, E. T. Search for Related Content PubMed PubMed Citation Articles by Engstrom, G. W. Articles by Littledike, E. T.

25-Hydroxyvitamin D 1- and 24-Hydroxylase Activities in Pig Kidney Homogenates: Effect of Vitamin D Deficiency

National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 70, Ames, IA 50010

A method was developed for simultaneous assay of 25-hydroxyvitamin D 1- and 24-hydroxylase in pig kidney homogenates. The products of these enzymes, 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol, were extracted from the in vitro incubation mixtures, isolated and purified by gel filtration and high-performance liquid chromatography, identified by ultraviolet absorption spectrometry, and high performance liquid chromatography with a second solvent system and quantified relative to authentic standards with a 254-nm detector system. Assay conditions included 12.5 µM substrate (25-hydroxycholecalciferol) concentration and an incubation time of 15 minutes at 37°C, which was on the linear portion of time curves for both enzymes. Maximum enzyme velocity was 1548 pmol/minute per gram kidney tissue for 1-hydroxylase and 286 pmol/minute per gram kidney tissue for 24-hydroxylase. The apparent K_m for pig kidney 25-hydroxyvitamin D 1-hydroxylase (1-hydroxylase) was 445 nM and for 25-hydroxyvitamin D 24-hydroxylase (24-hydroxylase) was 833 nM. We also demonstrated the use of this assay in pigs fed a semisynthetic diet with or without vitamin D. Pigs fed vitamin D-deficient diet had a 5- to 10-fold increase in 1-hydroxylase activity, severe hypocalcemia, low plasma 1,25-dihydroxycholecalciferol, very low plasma (undetectable) 24,25-dihydroxy-cholecalciferol concentration, and no detectable 24-hydroxylase activity compared to those fed the vitamin D-replete diet.

KEY WORDS: • assay • kidney hydroxylases • pig

Manuscript received 17 May 1983.