A rapid and sensitive in vitro assay of 25-hydroxyvitamin D3-1 alpha-hydroxylase and 24-hydroxylase using rat kidney homogenates.
A sensitive and rapid in vitro assay of 25-hydroxyvitamin D3 [25-(OH)D3]-1 alpha- and 24-hydroxylase activities was developed using rat kidney homogenates. A potent inhibitor of the enzymes in rat plasma was removed by thoroughly perfusing rats with saline. Kidney homogenates prepared from vitamin D-deficient rats preferentially produced tritiated 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] from 25(OH) [3H]D3. Addition of 10 microliter or more of rat plasma to 3 ml of 10% kidney homogenates suppressed 1 alpha-hydroxylase activity dose-dependently. Thyroparathyroidectomy (TPTX) of vitamin D-deficient rats greatly abolished 1 alpha-hydroxylase activity. Administration of parathyroid hormone to the TPTX rats increased 1 alpha-hydroxylase activity and that of 1 alpha,25(OH)2D3 enhanced 24-hydroxylase markedly. Since this assay is technically simple, rapid and sensitive, it will be useful in studying the regulatory mechanism in the renal metabolism of 25(OH)D3 in mammals.
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D000818:Animals; D002117:Calcitriol / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D008297:Male; D010281:Parathyroid Hormone / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013250:Steroid Hydroxylases / Q000032:analysis*; D065668:Vitamin D3 24-Hydroxylase
D010281:Parathyroid Hormone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002117:Calcitriol