Radioligand receptor assay for 25-hydroxyvitamin D2/D3 and 1 alpha, 25-dihydroxyvitamin D2/D3.

Abstract

A B S T R A C T A competitive protein binding assay for measurement of the plasma concentration of la,25-dihydroxyvitamin D3 [la,aD3] has been extended to include the immediate precursor of this hormone, 25-hydroxyvitamin Ds (25-OHD3). In addition, the assay system is capable of measuring the two metabolic products of ergocalciferol, namely, 25-hydroxyvitamin D2 (25-OHD2) and la,25-dihydroxyvitamin D2 [la,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 25-OHD2 as substrate. Comparison of this sterol with la,25-(OH) 2D3 in the assay system showed very similar binding curves; the Da form was slightly less efficient (77%). Comparison of the respective 25-hydroxy forms (25-OHD2 vs. 25-OHD3) at concentrations 500-fold that of la,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 la,25-(OH) 2D measured in Tucson adults were 25-40 ng/ml and 2.1-4.5 ng/100 ml, respectively. Both sterols were predominately (> 90%) in the form of vitamin D3 metabolites in this environment.

Evidence for abnormal regulation of circulating 1 alpha,25-dihydroxyvitamin D in patients with sarcoidosis and normal calcium metabolism.

“…The findings are consistent with those obtained in patients with vitamin D intoxication, who show increases in serum 25-OHD and not la,25(OH)2D (12). In the rat, vitamin D intoxication with hypercalcemia is characterized by increases in serum 25-OHD and decreases in serum la,25(OH)2D (13).…”

“…We demonstrated previously in two patients with sarcoid and a history of hypercalcemia that vitamin D, 250 ug/d for 12 d, increased both serum la,25(OH)2D and urinary calcium. Serum calcium remained within the normal range.…”

“…Mean plasma la,25(OH)2D is increased and serum immunoreactive parathyroid hormone is suppressed in patients with sarcoidosis and hypercalcemia who do not have primary hyperparathyroidism (1,2). Further vitamin D, 0.25 mg (10,000 U)/d for 12 d, increases serum la,25(OH)2D abnormally in patients with normocalcemia and a history of hypercalcemia but does not alter serum la,25(OH)2D abnormally in subjects or patients with sarcoid who have normal calcium metabolism (1). Thus, the increased sensitivity to vitamin D in sarcoid results from increases in serum la,25(OH)2D.…”

Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein

“…In the face of vitamin D toxicity, conversion of vitamin D to the active 1,25(OH) 2 D form is normally downregulated, and 25(OH)D or one of its metabolic products has been proposed to be responsible for the direct toxic effect (45,46). An alternative hypothesis proposes that the toxicity may be secondary to competition from the high levels of 25(OH)D for DBP binding, with resultant elevations of free 1,25(OH) 2 D (47).…”

Progressively Increased Serum 1,25-Dihydroxyvitamin D2 Concentration in a Hypoparathyroid Patient with Protracted Hypercalcemia due to Vitamin D2 Intoxication.

“…In contrast, other investigators have reported that serum 1,25-(OH)2D levels are slightly or greatly increased [6][7][8][9], suggesting that 1,25-(OH)2D is the agent causing the toxicity [10]. Since previous assays for vitamin D metabolites were tedious and time-consuming [1], and required a large amount of serum, studies involving serial measurement of serum 1,25-(OH)2D levels have rarely been reported [8].…”