An improved radioreceptor assay for 1,25-dihydroxyvitamin D in human plasma

Shigeharu Dokoh, J. Wesley Pike, John S. Chandler, Jennifer M. Mancini, Mark R. Haussler

Research output: Contribution to journalArticle

75 Scopus citations

Abstract

We describe a modified assay technique for quantitating 1,25-dihydroxyvitamin D in plasma. The method involves a rapid extraction of the hormone using minicolumn (made of granular diatomaceous earth) chromatography followed by single-step purification on high-performance liquid chromatography. Quantitation of plasma 1,25-dihydroxyvitamin D is achieved by a radioligand receptor assay employing lyophilized cytosolic receptor protein from chick intestine and high-specific-activity 1,25-dihydroxy[3H]vitamin D3 (166 Ci/mmol). A new incubation medium including an ethanol extract of vitamin D-deficient chick serum yields high specific binding and improves the precision of the radioassay. Bound and free hormone are separated with dextran-coated charcoal of equivalent particle size. The method is sensitive to 0.5 pg/tube with a practical detection range of 1-20 pg/tube, permitting duplicate assay of endogenous 1,25-dihydroxyvitamin D in plasma volumes as small as 0.5 ml. The intra- and interassay coefficient of variation are 5 and 9%, respectively, and the method is valid over a wide-range sample dilution. This assay technique was applied to the measurement of plasma 1,25-dihydroxyvitamin D hormone concentration in normal young adults (55.2 ± 13.6 pg/ml; n = 20) and in patients with chronic renal failure (13.5 ± 5.2 pg/ml; n = 9) and primary hyperparathyroidism (83.3 ± 18 pg/ml; n = 10).

Original languageEnglish (US)
Pages (from-to)211-222
Number of pages12
JournalAnalytical Biochemistry
Volume116
Issue number1
DOIs
StatePublished - Sep 1 1981

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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