0021-972XI841580 1-0091$02.00 10 Journal of ~1~1 Endocrinology and Metabolism Copyrigh~,,84 by The Endocrine Society ~~ Vol.58,No.l Printed in U.S.A. ings that 25-hydroxyvitamin D2 (250HD2) constituted 38.9% of the total 25-0HD found in clinical samples (12.6 :t 0.7 ng/ml 25-0HD2 V8. 20.1 :t 0.5 ng/ml 25-0HD3; 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 r = 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 :t 2.2 pg/ml (n = 22); chronic renal failure, 10.6 :t 1.5 pg/ml (n = 7); anephrics, undetectable (n = 10); infant cord blood, 22.9 :t 4.4 pg/ml (n = 7); and hyperparathyroidism, 68.9 :t 5.0 pg/ml (n = 13). This assay should be particularly useful in pediatric or other studies in which sample size is limited. (J Clin Endocrinol Metab 58: 91, 1984) ABSTRACT. This report describes a microassay for 1,25-di- hydroxyvitamin 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 Boo 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 assaymeasuresboth 1,25-(OH)2D2 and 1,25-(OH)2D3with equal affinity. The importance of using an assay with equal affinity for 1,25-(OH)2D2and 1,25-(OH)2D3is demonstrated by the find- V IT AMIN D is metabolized to several compounds, and ultimately, a small portion is metabolized to a major biologically active metabolite, 1,25-dihydroxyvi- tamin D [1,25-(OH)2D] (1-3). This metabolite is a steroid hormone produced by the kidney whose production is affected by factors that control mineral and skeletal metabolism (3-8). Additionally, the production of 1,25- (OH)2D is altered by a number of diseases, such as parathyroid gland disorders (1, 5,9-11), renal failure (1, 5, 6), certain bone diseases (1, 12), sarcoidosis (13, 14), and parturient hypocalcemia (15, 16). Because changes in circulating 1,25-(OH)2D are of pivotal physiopatho- logical importance in these diseases of calcium metabo- lism, considerable effort has been directed toward devel- oping specific assays for 1,25-(OH)2D in plasma (17-23). Vitamin D is unique among the steroid prohormones, in that it exists in two distinct chemical forms: vitamin D2 and vitamin D3. Because of technical limitations, the contribution of vitamin D2 to the D status of the patient has been largely neglected by clinical researchers. These limitations include: 1) unavailability of standard prepa- rations of vitamin D2 metabolites, 2) different chromato- graphic motilities between the vitamin D2 and vitamin D3 analogs (21), and 3) different apparent affinities for the specific binding proteins and receptors used for mea- surement of vitamin D metabolites (19, 20, 22-24). This underestimation of vitamin D2 metabolites was not an- ticipated to be a problem, since vitamin D3 metabolites constitute 90-95% of vitamin D metabolites, except un- der conditions of extremely high vitamin D2 intake (20). This concept needs to be reevaluated in light of a 5-yr retrospective analysis of data collected in our laboratory, which will be discussed. The purpose of this study was 3-fold: 1) to develop an assay for 1,25-(OH)2D which would measure the vitamin D2 and vitamin D3 forms simultaneously and equally, 2) ReceivedMay 27, !983. 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 .0. Box 70, Ames, IA 500!0. * Mention of a trade name, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Depart- ment of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. t Recipient of a Research Career Development Award from the USPHS. ~ Application to Clinical Studies* TIMOTHY A~NHARDT, RONALD L 1 HORST, JOHN w I ORF, AND BRUCE W.\r'r6LLISt ~ ~ National Anim}jr[j-~~er, Agricultural Research Service, U.S. Department of Agriculture (T.A.R., R.L.H.), Ames, Iowa 50010; Immuno-Nuclear Corp. (J. W.O.), StiUwater, Minnesota 55082; and Case Western Reserve University (B. W.H.), Cleveland, Ohio 44106