Bioassay of 1,25-dihydroxyvitamin D in human plasma purified by partition, alkaline extraction, and high-pressure chromatography

Paula H Stern*, Thomas E. Phillips, Thalia Mavreas

*Corresponding author for this work

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

A method is described which permits the assay of 1,25-dihydroxyvitamin D in 1 ml of normal human plasma. Samples are extracted with benzene or isopropyl ether. The organic phase is then back-extracted with 0.1 m Na2HPO4, pH 10.5. The final purification step involves high-pressure liquid chromatography on μPorosil with a solvent system of 13% isopropanol in hexane. Recovery through the entire purification procedure averages 76.8%, the purified extract is assayed for 1,25-dihydroxyvitamin D by determining its effect on the resorption of fetal rat bones in vitro. 1,25-Dihydroxyvitamin Da standards in the 4- to 25 pg/ml range gave responses which fitted a logarithmic dose-response curve with an average r2 of 0.986. Repeat assays with normal human plasma gave a coefficient of variation ( SD x) between experiments of 16.1 ± 0.3%. Normal human serum contains 33.4 ± 6.4 pg/ml (x ± SD) 1,25-dihydroxyvitamin D by this assay. Values averaging less than normal are found in plasmas of patients with renal failure and in patients with pseudohypoparathyroidism. Average values greater than normal were found in patients with hyperparathyroidism. 1,25-Dihydroxyvitamin D was not detectable in plasma of anephric patients.

Original languageEnglish (US)
Pages (from-to)22-30
Number of pages9
JournalAnalytical Biochemistry
Volume102
Issue number1
DOIs
StatePublished - Jan 1 1980

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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