Isolation and identification 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, a new metabolite of 1,25-dihydroxyvitamin D3 produced in rat kidney

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Abstract
A new metabolite of vitamin D3 was produced in vitro by perfusing rat kidneys with 1,25-dihydroxyvitamin D3 (4 .times. 10-6 M). It was isolated and purified from the lipid extract of the kidney perfusate by high-performance liquid chromatography. By means of ultraviolet absorption spectrophotometry, mass spectrometry, chemical derivativation, and chemical synthesis, the new metabolite was identified as 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Along with the new metabolite, three other previously identified metabolites, namely, 1,24,25-trihydroxyvitamin D3, 1,25-dihydroxy-24-oxovitamin D3, and 1,23-25-trihydroxy-24-oxovitamin D3, were also isolated. The new metabolite was also formed when 1,23,25-trihydroxy-24-oxovitamin D3 was used as the substrate. Thus, the new metabolite fits into the following metabolic pathway: 1,25-dihydroxyvitamin D3 .fwdarw. 1,24(R),25-trihydroxyvitamin D3 .fwdarw. 1,25-dihydroxy-24-oxovitamin D13 .fwdarw. 1,23,25-trihydroxy-24-oxovitamin D3 .fwdarw. 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Further, we used 1.alpha.,25-dihydroxy[1.beta.-3H]vitamin D3 in the kidney perfusion system and demonstrated 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 as the major further metabolite of 1,25-dihydroxyvitamin D3, circulating in the final perfusate when kidneys were perfused with 1,25-dihydroxyvitamin D3 (6 .times. 10-10 M) for 4 h. The biological activity of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 (C-23 alcohol) and its metabolic relationship to 1-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D3 (calcitroic acid or C-23 acid), the other previously identified side-chain cleavage metabolite of 1,25-dihydroxyvitamin D3, are unknown and are presently undergoing investigation.