An improved method for the measurement of 1,25-(OH)2d3 in human plasma

Phillip W. Lambert, David O. Toft, Stephen F. Hodgson, Elizabeth A. Lindmark, Bonnie J. Witrak, Bernard A. Roos

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Here we report a highly sensitive and convenient ligand binding assay for the determination of 1,25(OH)2D3 in small volumes of human plasma. This method involves: (1) extraction of vitamin D3 and its metabolites using methanol-methylene chloride with separation or phases by centrifugation; (2) gel chromatography and high pressure liquid chromatography for the quantitative isolation of 1,25-(OH)2D3: and (3) a sensitive ligand binding assay for 1,25-(OH)2D3 employing cytosol receptor from the intestinal mucosa of rachitic chicks. Using modified rachitogenic chick diets allows early (< 4 wks) harvesting of active receptor for 1,25-(OH)2D3 in high yield. The method includes a rapid and effective procedure for stable and long-term storage of the active cytosol receptor. A convenient dextran-charcoal means is used for the separation of receptor bound from free 1,25-(OH)2D3 resulting in the achievement of a lower (< 5%) background (i.e., nonspecific binding) than reported for other 1,25-(OH)2D3 assays. Analysis of this receptor shows it to be a saturable, single class.of binding sites with a dissociation constant (Kd) of approximately 3.7 × 10-11. The final recovery of 1,25-(OH)2D3. following extraction and chromatography is 80 ± 3% and triplicate determinations can be made on a 3 ml plasma sample. The ligand binding assay routinely detects ± 5pg of 1,25-(OH)2D3, per assay tube and the inter- and intraassay variation, based on repeated determinations of 1,25-(OH)2D3 in pooled normal human plasma, is < 5%. Preliminary studies indicate that our methodology will permit measurement of plasma 1,25-(OH)2D 3 levels in all normal subjects and in pathophysiologic states where 1,25-(OH)2D3levels may be below or above normal values. 1,25-(OH)2D 3 values (pg/ml ± SEMJ in human plasma obtained from both normals and patients with various untreated calcium homeostatic disorders were: normals = 33.5 ± 1.8; end-stage chronic renal failure = 5.1 ± 1.2; primary hypoparathyroidism = 18.3 ± 2.8; primary hyperparathyroidism = 61.4 ± 7.1; and hyperthyroidism with associated hypercalcemia = 42.1 ± 8.4.

Original languageEnglish
Pages (from-to)293-310
Number of pages18
JournalEndocrine Research
Volume5
Issue number4
DOIs
StatePublished - Jan 1 1978
Externally publishedYes

Fingerprint

Ligands
Cytosol
Chronic Kidney Failure
Hypoparathyroidism
Rickets
Primary Hyperparathyroidism
Plasma Volume
Cholecalciferol
Charcoal
Methylene Chloride
Hypercalcemia
Hyperthyroidism
Intestinal Mucosa
Dextrans
Centrifugation
Gel Chromatography
Methanol
Chromatography
Reference Values
Binding Sites

ASJC Scopus subject areas

  • Endocrinology
  • Medicine(all)

Cite this

Lambert, P. W., Toft, D. O., Hodgson, S. F., Lindmark, E. A., Witrak, B. J., & Roos, B. A. (1978). An improved method for the measurement of 1,25-(OH)2d3 in human plasma. Endocrine Research, 5(4), 293-310. https://doi.org/10.1080/07435807809061094

An improved method for the measurement of 1,25-(OH)2d3 in human plasma. / Lambert, Phillip W.; Toft, David O.; Hodgson, Stephen F.; Lindmark, Elizabeth A.; Witrak, Bonnie J.; Roos, Bernard A.

In: Endocrine Research, Vol. 5, No. 4, 01.01.1978, p. 293-310.

Research output: Contribution to journalArticle

Lambert, PW, Toft, DO, Hodgson, SF, Lindmark, EA, Witrak, BJ & Roos, BA 1978, 'An improved method for the measurement of 1,25-(OH)2d3 in human plasma', Endocrine Research, vol. 5, no. 4, pp. 293-310. https://doi.org/10.1080/07435807809061094
Lambert PW, Toft DO, Hodgson SF, Lindmark EA, Witrak BJ, Roos BA. An improved method for the measurement of 1,25-(OH)2d3 in human plasma. Endocrine Research. 1978 Jan 1;5(4):293-310. https://doi.org/10.1080/07435807809061094
Lambert, Phillip W. ; Toft, David O. ; Hodgson, Stephen F. ; Lindmark, Elizabeth A. ; Witrak, Bonnie J. ; Roos, Bernard A. / An improved method for the measurement of 1,25-(OH)2d3 in human plasma. In: Endocrine Research. 1978 ; Vol. 5, No. 4. pp. 293-310.
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