Isolation and identification of 1 alpha- and 23-hydroxylated metabolites of 25-hydroxy-24-oxovitamin D3 from in vitro incubates of chick kidney homogenates.

Five major metabolites (peaks I-V) of 25-hydroxy-24-oxovitamin D3 (25(OH)24-oxo-D3) have been isolated in pure form from in vitro incubates containing kidney homogenates of vitamin D-deficient chicks and chicks given 65 nmol of vitamin D3; peaks II, III, and V are from vitamin D-deficient chicks and peaks I, II, and IV are from vitamin D-supplemented birds. The structures of the metabolites were unequivocally identified as 23,25-dihydroxy-24-oxo-vitamin D3 (peak I), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) (peak II), 1 alpha, 25-dihydroxy-24-oxovitamin D3 (peak III), 23,24,25-trihydroxyvitamin D3 (peak IV), and 1 alpha,24,25-trihydroxyvitamin D3 (peak V) by means of ultraviolet absorption spectrometry, mass spectrometry, and specific chemical reactions. It is concluded that 25(OH)24-oxo-D3 is further hydroxylated at the 1 alpha-position in the kidney of vitamin D-deficient chicks and at the 23-position in that of vitamin D-supplemented animals. Formation of 24,25(OH)2D3 from 25(OH)24-oxo-D3 in both vitamin D-deficient and -supplemented animals provides evidence for the presence of an enzyme to reduce the 24-oxo group irrespective of the vitamin D status.