25-Hydroxyvitamin D 1 alpha- and 24-hydroxylase activities in pig kidney homogenates: effect of vitamin D deficiency.
A method was developed for simultaneous assay of 25-hydroxyvitamin D 1 alpha- 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 microM substrate (25-hydroxycholecalciferol) concentration and an incubation time of 15 minutes at 37 degrees 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 alpha-hydroxylase and 286 pmol/minute per gram kidney tissue for 24-hydroxylase. The apparent Km for pig kidney 25-hydroxyvitamin D 1 alpha-hydroxylase (1 alpha-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 alpha-hydroxylase activity, severe hypocalcemia, low plasma, 1,25-dihydroxycholecalciferol, very low plasma (undetectable) 24,25-dihydroxycholecalciferol concentration, and no detectable 24-hydroxylase activity compared to those fed the vitamin D-replete diet.
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D013250:Steroid Hydroxylases / Q000032:analysis / Q000378:metabolism*; D013552:Swine / Q000378:metabolism*; D014018:Tissue Distribution; D014807:Vitamin D / Q000008:administration & dosage / Q000378:metabolism; D014808:Vitamin D Deficiency / Q000201:enzymology*; D014809:Vitamin D-Binding Protein; D065668:Vitamin D3 24-Hydroxylase
D002352:Carrier Proteins; D014809:Vitamin D-Binding Protein; D014807:Vitamin D; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002118:Calcium