An Improved Method for the Measurement of 1,25-(OH)2D3in Human Plasma

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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.