25-Hydroxyvitamin D 1 alpha- and 24-hydroxylase activities in pig kidney homogenates: effect of vitamin D deficiency

J Nutr. 1984 Jan;114(1):119-26. doi: 10.1093/jn/114.1.119.


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.

MeSH terms

  • 25-Hydroxyvitamin D3 1-alpha-Hydroxylase
  • Animals
  • Calcium / pharmacology
  • Carrier Proteins / pharmacology
  • Cytochrome P-450 Enzyme System*
  • Kidney / enzymology*
  • Kinetics
  • Steroid Hydroxylases / analysis
  • Steroid Hydroxylases / metabolism*
  • Swine / metabolism*
  • Tissue Distribution
  • Vitamin D / administration & dosage
  • Vitamin D / metabolism
  • Vitamin D Deficiency / enzymology*
  • Vitamin D-Binding Protein
  • Vitamin D3 24-Hydroxylase


  • Carrier Proteins
  • Vitamin D-Binding Protein
  • Vitamin D
  • Cytochrome P-450 Enzyme System
  • Steroid Hydroxylases
  • Vitamin D3 24-Hydroxylase
  • 25-Hydroxyvitamin D3 1-alpha-Hydroxylase
  • Calcium