Vol. 99, No. 1,198l March 16, 1981 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 302-307 A NEW IN VIVO METABOLITE OF VITAMIN D3 : 1,25,26-TRIHYDROXYVITAMIN D3 T. A. Reinhardt*, J. L. Napoli, D. C. Beitz, E. T. Littledike, and R. L. Horst National Animal Disease Center, Agricultural Research, Science and Education Administration, U. S. Department of Agriculture, P.O. Box 70, Ames, IA 50010; Department of Biochemistry, The University of Texas Health Science Center, Dallas, TX 75235; and Department of Animal Science, Iowa State University, Ames, IA 50011 Received January 26, 1981 SUMMARY A new metabolite of vitamin D3 was detected with a modified receptor- binding assay in plasma of cows given large parenteral doses of vitamin D3. This metabolite has been identified as 1,25,26’-trihydroxyvitamin D3 by comigration of the in vivo-isolated metabolite with synthetic 1,25(S) ,26- -__ trihydroxyvitamin D3 on two different high-pressure liquid chromatography systems and by mass spectroscopy. INTRODUCTION Metabolism of vitamin D3 to more polar and active metabolites has been well documented (1). Several new metabolites of vitamin D3 have recently been described in detail (l-4); however, it has been pointed out that * Address all correspondence to Dr. Timothy A. Reinhardt, National Animal Disease Center, AR-SEA, USDA, P.O. Box 70, Ames, IA 50010. This investigation was supported in part by a NIH National Research Service Award AM06382 (TAR) from the NIAMD, by a NIH Grant AM26535 (JLN), by the Robert A. Welch Foundation Grant I-769 (JLN), and by a shared instrumentation grant GM27506 (UTHSCD) . Journal paper No. J-10158 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA. Project No. 2393. Disclaimer Statement Mention of a trade name, proprietary product, or vendor does not constitute a guarantee or warranty by the U. S. Department of Agriculture or cooperating agencies and does not imply its approval to the exclusion of other products or vendors that may be suitable. Abbreviations used: 1,25- (OH) 2D3, 1,25-dihydroxyvitamin D 3; 1,24(R) ,25-(OH)3D3, 1,24(R) ,25-trihydroxyvitamin D3; 1,25,26-(OH)gD3, 1,25,26-trihydroxyvitamin D 3; HPLC , high-pressure liquid chromatography. 0006-291 X/81/050302-06$01.00/0 Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. 302