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Measurement of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in mammalian kidney.

Publication ,  Journal Article
Lobaugh, B; Drezner, MK
Published in: Anal Biochem
March 1983

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.

Duke Scholars

Published In

Anal Biochem

DOI

ISSN

0003-2697

Publication Date

March 1983

Volume

129

Issue

2

Start / End Page

416 / 424

Location

United States

Related Subject Headings

  • Vitamin D Deficiency
  • Substrate Specificity
  • Steroid Hydroxylases
  • Parathyroid Glands
  • Mice, Inbred C57BL
  • Mice
  • Kidney
  • In Vitro Techniques
  • Biochemistry & Molecular Biology
  • Animals
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Lobaugh, B., & Drezner, M. K. (1983). Measurement of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in mammalian kidney. Anal Biochem, 129(2), 416–424. https://doi.org/10.1016/0003-2697(83)90571-7
Lobaugh, B., and M. K. Drezner. “Measurement of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in mammalian kidney.Anal Biochem 129, no. 2 (March 1983): 416–24. https://doi.org/10.1016/0003-2697(83)90571-7.
Lobaugh B, Drezner MK. Measurement of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in mammalian kidney. Anal Biochem. 1983 Mar;129(2):416–24.
Lobaugh, B., and M. K. Drezner. “Measurement of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in mammalian kidney.Anal Biochem, vol. 129, no. 2, Mar. 1983, pp. 416–24. Pubmed, doi:10.1016/0003-2697(83)90571-7.
Lobaugh B, Drezner MK. Measurement of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in mammalian kidney. Anal Biochem. 1983 Mar;129(2):416–424.
Journal cover image

Published In

Anal Biochem

DOI

ISSN

0003-2697

Publication Date

March 1983

Volume

129

Issue

2

Start / End Page

416 / 424

Location

United States

Related Subject Headings

  • Vitamin D Deficiency
  • Substrate Specificity
  • Steroid Hydroxylases
  • Parathyroid Glands
  • Mice, Inbred C57BL
  • Mice
  • Kidney
  • In Vitro Techniques
  • Biochemistry & Molecular Biology
  • Animals