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

Abstract

A new metabolite of vitamin D3 was produced in vitro by perfusing rat kidneys with 1,25-dihydroxyvitamin D3 (4 X 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 derivatization, 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----1,24(R),25-trihydroxyvitamin D3----1,25-dihydroxy-24-oxovitamin D3----1,23,25-trihydroxy-24-oxovitamin D3----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 X 10(-10) M) for 4 h. The biological activity of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 (C-3 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.

Target cell metabolism of 1,25-dihydroxyvitamin D3 to calcitroic acid. Evidence for a pathway in kidney and bone involving 24-oxidation

“…One pathway begins with 24-hydroxylation and leads to 24-oxo-1,23,25(OH)3D3 Mayer et al, 1983b;Jones et al, 1987), while the other begins with 23-hydroxylation and leads to 1,25(OH)2D3-26,23-lactone Yamada et al, 1984). The demonstrations that the 24-hydroxylation pathway continues further with the formation of a side-chain cleaved molecule, a C23 alcohol Reddy et al, 1987;) provided a strong indicator that the 24-hydroxylation pathway would turn out to be the source of the intermediates leading to calcitroic acid. In this paper we report the formation of calcitroic acid from 1,25(OH)2D3 in two target cell systems in vitro and we provide evidence for the role of the 24-hydroxylation pathway in this metabolic process. A preliminary account of aspects of this work was presented to the 7th Workshop on Vitamin D, Rancho Mirage, California, U.S.A.; 24-29 April, 1988. EXPERIMENTAL Materials UMR-106 cells (Partridge et al, 1980) were obtained from J.N.M.…”

Further Metabolism of 1α, 25-Dihydroxyvitamin D3in Target Cells

“…Esvelt et al [3] showed that calcitroic acid is found in a variety of tissues in the body. Subsequent work using a series of in vitro cell systems including intestinal and kidney homogenates and the perfused rat kidney revealed the existence of a number of side chain-modified metabolites [4,5,6] derived from 1,24,254011)3D,, which might be precursors of calcitroic acid. Work in our laboratory [7] demonstrating the existence of a C23-alcohol derived from 25-OH-D3 strengthened the theory that calcitroic acid was derived from the C-24 oxidation pathway.…”

Metabolism of 2-Methyl Analogs of 1α,25-Dihydroxyvitamin D₃ in Rat Osteosarcoma Cells (UMR 106)

“…The C-24 oxidation pathway, initiated by hydroxylation at C-24 is the major side chain modification pathway, which leads to the formation of the final product, calcitroic acid. 1,[10][11][12] The newly discovered A-ring modification pathway is the C-3 epimerization pathway through which 1a,25(OH) 2 D 3 is converted into 1a-25-dihydroxy-3-epi-vitamin D 3 [1a,25(OH) 2 -3-epi-D 3 ]. [13][14][15] In the present study, we compared the metabolic fate of the four 1a,2-methyl diastereomers with that of 1a,25(OH) 2 D 3 in rat osteosarcoma cells (UMR 106) which have been shown to express both the C-24 oxidation and the C-3 epimerization pathways.…”