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

Abstract

A method was developed for simultaneous assay of 25-hydroxyvitamin D 1α- 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 1α-hydroxylase and 286 pmol/minute per gram kidney tissue for 24-hydroxylase. The apparent K_m for pig kidney 25-hydroxyvitamin D 1α-hydroxylase (1α-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 1α-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.