An improved competitive protein-binding assay for 25-hydroxy vitamin D

Abstract

A competitive protein binding assay for 25-hydroxy vitamin D in serum is described using serum from a pregnant woman as source of binding protein. Introduction of a polyvinyl alcohol to the reaction buffer enhanced the solubility of 25-hydroxy vitamin D in aqueous solutions and nearly eliminated loss of material to the walls of reaction tubes. Addition of a protein fraction obtained by gel filtration of human serum, shown to bind only vitamin D, enhanced the specificity and reproducibility of the assay without interfering with the binding properties of 25-hydroxy vitamin D. Serum samples were extracted by chloroform-methanol. Comparison of column chromatography on silicic acid and Sephadex LH-20 showed that both systems gave good separation of vitamin D and 25-hydroxy vitamin D and with a similar recovery of about 80%. The silicic acid columns were less time consuming, either to handle and cheaper in use, and was therefore preferred as the routine separation method. The assay has a detection limit of 0.4 nmol/l in serum, and an intra- and inter-assay coefficient of variation of 6.8 and 8.1%, respectively.

Cross-sectional analysis of universal vitamin D supplementation in former East Germany during the first year of life

“…One of these was the commonly used Nichols-RIA, this method did not include previous chromatography [41,42]. Studies revealed that the Aksnes method is comparable with most of the leading methods at the time [13]. In comparison to the method of Bouillon et al [43] it revealed higher values for 13 year olds but good agreement with values from patients treated with pharmacological doses of vitamin D [13].…”

“…Studies revealed that the Aksnes method is comparable with most of the leading methods at the time [13]. In comparison to the method of Bouillon et al [43] it revealed higher values for 13 year olds but good agreement with values from patients treated with pharmacological doses of vitamin D [13]. We are not aware of studies comparing the Aksnes method to liquid chromatography-tandem mass spectrometry (LC-MS/MS).…”

“…This is outweighed by the large number of participants and the standardised, population-based approach for every visit as outlined in official notes [11,12]. We used a competitive protein binding assay with [ 3 H]-25(OH)D developed by Aksnes [13,40]. Preparation of the samples included lipid extraction with chloroform/methanol, chromatography similar to high-performance liquid chromatography (HPLC) followed by gel-filtration, separation of beta-lipoproteins of serum proteins and binding studies.…”

“…Biochemical parameters of vitamin D and calcium metabolism 25(OH)D, calcium, phosphate and ALP were included if available. The vitamin D analysis was based on a 25(OH)D radioimmunoassay using the micromethod by Aksnes [13]. Addition of a protein fraction obtained by gel filtration of human serum, shown to bind only vitamin D, enhanced the specificity and reproducibility of the assay without interfering with the binding properties of 25(OH)D. Serum samples were extracted by chloroform-methanol.…”

Vitamin D Metabolism in Hypophosphatasia

“…Serum 25-hydroxyvitamin D (250HD) was determined employing a protein binding assay after purification on silicic acid columns (7). The dihydroxyvitamin D compounds 1 ,25(OH)2D, 24,25(OH)2D and 25.26(OH)2D were isolated and purified by high pressure liquid chromatography employing silicic acid columns, and quantitated by a modification of the chick duodenal The serum-levels for calcium, phosphorus, the various vitamin D metabolites and cyclic AMP during different stages and dietary regimens of the 9-month study period are charted on Fig.…”