The isolation and identification of 5,6-trans-25-hydroxyvitamin D3 from the plasma of rats dosed with vitamin D3. Evidence for a novel mechanism in the metabolism of vitamin D3.

Abstract

We isolated 5,6-trans-25-hydroxyvitamin D3 from the plasma of vitamin D-deficient rats which had received 5,6-cis-vitamin D3 orally.The relative amounts of 5,6-tmmd5-hydroxyvitamin D3 and 5,6-cis-25-hydroxyvitamin D3 in plasma were 1:s.Control experiments in which 5,6-cis-25-hydroxyvitamin D3 was added to plasma or water showed that less than 0.5% of the added 25-hydroxyvitamin DS was converted to 5,6trans-25-hydroxyvitamin D3 during the isolation procedure ( p < 0.001, control versu8 experimental).We synthesized 5,6-tr~m~-25-hydroxyvitamin D3 from 25hydroxyvitamin D3 and demonstrated that authentic 5,6-tnuzs-25-hydroxyvitamin D3 co-eluted with the isolated putative 5,6-trans-25-hydroxyvitamin D3 on a high performance liquid chromatography system.In addition, the isolated metabolite demonstrated mass spectral, ultraviolet absorption, and protein-binding properties similar to those of synthetic 5,6-tmns-25hydroxyvitamin D3.As 5,6-tmm-25-hydroxyvitamin D3 binds more efficiently to intestinal cytosol-binding protein than 25-hydroxyvitamin D3, this observation could explain, in part, how vitamin D3 and 25-hydroxyvitamin D3 are effective in large doses when administered to anephric patients or patients with advanced renal failure or hypoparathyroidism.This observation may also hold true for other analogs of vitamin D. Further, our observation provides strong evidence for the existence of 5,6-cis-tnuts isomerization in vivo.Whether this process is enzymatic or nonenzymatic is unknown, and the site of formation of the 5,6-tnurs metabolite has not been determined.Vitamin D3 plays an important role in normal calcium and phosphorus physiology (1).D : is metabolized in the liver to