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A Microassay for 1,25-Dihydroxyvitamin D Not requiring High Performance Liquid Chromatography: Application to Clinical Studies^*

TIMOTHY A. REINHARDT, RONALD L. HORST, JOHN W. ORF and BRUCE W. HOLLIS

National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture Ames, Iowa 50010;
Immuno-Nuclear Corp. Stillwater, Minnesota 550
Case Western Reserve University Cleveland, Ohio 44106

Address all correspondence and requests for reprints to: Dr. T. A. Reinhardt, National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 70, Ames, IA 50010.

This report describes a microassay for 1,25––dihydroxyvitamin D [1,25–(OH)₂D] in plasma which does not require high performance liquid chromatography. The assay involves rapid extraction and preliminary purification on a C-18 Sep-Pak cartridge, followed by final purification on a silica Sep-Pak using hexane-isopropanol. Quantitation of 1,25-(OH)₂D is achieved using a nonequilibrium assay employing 1,25-(OH)₂D receptor from calf thymus. The method is sensitive to 1.5 pg/tube, with B₅₀ occurring at 9 pg/tube and a useful assay range of 1.5–40 pg/tube. The intra- and interassay coefficients of variation are 6.5% and 11.5%, respectively, and the method is linear over a wide range of sample dilutions. In addition, this assay measures both 1,25-(OH)₂D₂ and 1,25-(OH)₂D₃ with equal affinity. The importance of using an assay with equal affinity for 1,25-(OH)₂D₂ and 1,25-(OH)₂D₃ is demonstrated by the findings that 25-hydroxyvitamin D₂ (250HD2) constituted 38.9% of the total 25-OHD found in clinical samples (12.6 ± 0.7 ng/ml 25-OHD2 vs. 20.1 ± 0.5 ng/ml 25-OHD₃; n = 807). Results of this new assay have been compared to those of the assay of Horst et al. (21), which employs Sephadex LH-20 and high performance liquid chromatography sample purification. The correlation coefficient was r² = 0.96, and the slope was 1.05. Using this new assay, plasma 1,25-(OH)₂D concentrations were as follows: normal adults, 37.4 ± 2.2 pg/ml (n = 22); chronic renal failure, 10.6 ± 1.5 pg/ml (n = 7); anephrics, undetectable (n = 10); infant cord blood, 22.9 ± 4.4 pg/ml (n = 7); and hyperparathyroidism, 68.9 ± 5.0 pg/ml (n = 13). This assay should be particularly useful in pediatric or other studies in which sample size is limited.

^* Mention of a trade name, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. which will be discussed. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

Recipient of a Research Career Development Award from the USPHS.

Received May 27, 1983.

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