Radioligand receptor assay 25 hydroxyvitamin D₂/D₃ and 1α,25 dihydroxyvitamin D₂/D₃. Application to hypervitaminosis D

A competitive protein binding assay for measurement of the plasma concentration of 1α,25 dihydroxyvitamin D3 [1α,25 (OH)2D3] has been extended to include the immediate precursor of this hormone, 25 hydroxyvitamin D3 (25 OHD3). In addition, the assay system is capable of measuring the two metabolic products of ergocalciferol, namely, 25 hydroxyvitamin D2 (25 OHD2) and 1α,25 dihydroxyvitamin D2 [1α,25 (OH)2D2]. The target tissue assay system consists of a high affinity cytosol receptor protein that binds the vitamin D metabolites and a limited number of acceptor sites on the nuclear chromatin. By utilizing a series of chromatographic purification steps, a single plasma sample can be assayed for any of the four vitamin D metabolites either individually or combined. Therefore, the assay procedure allows for both the quantitative and qualitative assessment of the total active vitamin D level in a given plasma sample. To show that the binding assay was capable of measuring 1α,25 (OH)2D2 as well as 1α,25 (OH)2D3, two groups of rats were raised. One group, supplemented with vitamin D3, produced assayable material that represented 1α,25 (OH)2D3. The other group, fed only vitamin D2 in the diet, yielded plasma containing only 1α,25 (OH)2D2 as the hormonal form of the vitamin. The circulating concentrations of the two active sterols were nearly identical (15 ng/100 ml) in both groups, indicating that the competitive binding assay can be used to measure both hormonal forms in plasma. In a separate experiment, 1α,25 (OH)2D2 was generated in an in vitro kidney homogenate system using 25 OHD2 as substrate. Comparison of this sterol with 1α,25 (OH)2D3 in the assay system showed very similar binding curves; the D2 form was slightly less efficient (77%). Comparison of the respective 25 hydroxy forms (25 OHD2 vs. 25 OHD3) at concentrations 500 fold that of 1α,25 (OH)2D3, again suggested that the binding of the D2 metabolite was slightly less efficient (71%). Finally, the assay was employed to measure the total active vitamin D metabolite pools in the plasma of normal subjects and patients with varying degrees of hypervitaminosis D. The normal plasma levels of 25 OHD and 1α,25 (OH)2D measured in Tucson adults were 25-40 ng/ml and 2.1-4.5 ng/100 ml, respectively. Both sterols were predominantly (>90%) in the form of vitamin D3 metabolites in this environment. Typical cases of hypervitaminosis D exhibited approximately a 15 fold increase in the plasma 25 OHD concentration, and a dramatic changeover to virtually all metabolites existing in the form of D2 vitamins. In contrast, the circulating concentration of 1α,25 (OH)2D was not substantially enhanced in vitamin D intoxicated patients. The authors therefore conclude that hypervitaminosis D is not a result of abnormal plasma levels of 1α,25 (OH)2D but may be caused by an excessive circulating concentration of 25 OHD.