The Cytoreceptor Assay for 1,25-Dihydroxyvitamin D and Its Application to Clinical Studies*

Abstract

We have developed a method for the measurement of 1,25-dihydroxyvitamin D [1,25-(OH)₂D] which is based on the selective internalization and subsequent binding of 1,25-(OH)₂D to its specific receptor inside cultured target cells. This technique provides the means of assaying 1,25-(OH)₂D directly in blood extracts without prior biochemical isolation of this hormone from other metabolites of vitamin D. The displacement of 1,25-(OH)₂-[₃H]D₃ bound to the intracellular receptor by the extracted 1,25-(OH)₂D provides a measurement of its concentration in the blood samples. The method is sensitive to 2 pg/tube and can be performed with 1- to 2-ml samples. The intra- and interassay coefficients of variation are 10.2% and 15.3%, respectively. Other major metabolites of vitamin D, at their circulating levels, do not interfere with the 1,25-(OH)₂D measurement. Moreover, 1,25-(OH)₂D levels determined by this direct method correlate closely (r = 0.93) with the values determined by the traditional high pressure liquid chromatographic assays on the same samples. Using the cytoreceptor assay we found that normal adults (n = 79) had a mean (±SD) 1,25-(OH)₂D level of 33.8 10.64 pg/ml; normal children (n = 40) had higher 1,25-(OH)₂D levels than the adults (P < 0.0005), with a mean level of 43.3 ± 13.9 pg/ml. In normals subjects, 1,25-(OH)₂D levels correlated negatively with age (r = −0.40; P < 0.001). Pregnant women (n = 6) in the third trimester had elevated levels, with a mean of 96.5 ± 17.1, anephric patients (n = 3) had undetectable levels, and patients on hemodialysis (n = 11) had a mean of 11.5 ± 6.7 pg/ml, significantly (P < 0.0005) lower than normal. Adult patients with primary hyperparathyroidism (n = 25) had a mean 1,25-(OH)₂D level of 50.2 ± 15.3 pg/ml, significantly (P < 0.0005) higher than normal; in this group, hormone levels correlated positively with calcium absorption (r = 0.81; P < 0.001) and serum calcium (r = 0.53; P < 0.02 and negatively with serum phosphate (r = −0.49; P < 0.05). (J Clin Endocrinol Metab56: 751,1983)