Measurement of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in mammalian kidney.
Until recently measurement of 25-OH-D3-1 alpha-hydroxylase activity in mammalian kidney has not been possible due to the presence of a protein which inhibits the enzyme by reducing available substrate. However, utilization of sufficient unlabeled 25-OH-D3 (80 nmol/ml renal homogenate) to overcome the effect of the inhibitor while maintaining optimal concentration for 1-hydroxylation has made quantitation of enzyme activity possible. We have modified this existing technique in order to increase the sensitivity and to permit detailed study of 1 alpha-hydroxylate regulation in mouse kidney. The modifications that we have incorporated include (i) simplifying the purification scheme for obtaining measurable 1,25-(OH)2D3 by reducing to one the necessary number of high-performance liquid chromatography steps and (ii) quantifying 1,25-(OH)2D3 by radioligand assay. The sensitivity of the assay is 10 pg, which, corrected for fractionation and recovery (50-60%), allows the measurement of 0.5 fmol 1,25-(OH)2D3 produced per milligram kidney per minute. Moreover, reliability and precision of the assay have been confirmed by demonstrating that samples from carefully matched, identically treated mice have reproducible enzyme activity (interassay coefficient of variation = 9.1%, n = 5) and show appropriate dilution characteristics. We have also demonstrated appropriate modulation of enzyme activity by known effectors of 1-hydroxylation. Kidneys from D-deficient mice exhibit significantly higher enzyme activity (15.28 +/- 1.17, n = 21) than do normal mouse kidneys (5.14 +/- 0.26, n = 33). In contrast, enzyme activity is suppressed significantly in kidneys obtained from calcium-loaded (1.20 +/- 0.04, n = 5) and parathyroidectomized animals (2.94 +/- 0.29, n = 5). Our assay now permits the indepth study of 1 alpha-hydroxylase regulation in mammalian (mouse) kidneys.
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D000818:Animals; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D010280:Parathyroid Glands / Q000502:physiology; D013250:Steroid Hydroxylases / Q000302:isolation & purification*; D013379:Substrate Specificity; D014808:Vitamin D Deficiency / Q000201:enzymology
D013250:Steroid Hydroxylases; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase