calcitroic-acid and 1-25-dihydroxyergocalciferol

1alpha-Hydroxy-23 carboxy-24,25,26,27-tetranorvitamin D(3) (calcitroic acid) is known to be the major water-soluble metabolite produced during the deactivation of 1,25-(OH)(2)D(3). This deactivation process is carried out exclusively by the multicatalytic enzyme CYP24 and involves a series of oxidation reactions at C(24) and C(23) leading to side-chain cleavage and, ultimately, formation of the calcitroic acid. Like 1,25-(OH)(2)D(3), 1alpha,25-1,25-(OH)(2)D(2) is also known to undergo side-chain oxidation and side-chain cleavage to form calcitroic acid (Zimmerman et al. [2001]. 1,25-(OH)(2)D(2) differs from 1,25-(OH)(2)D(3) by the presence of a double bond at C(22) and a methyl group at C(24). To date, there have been no studies detailing the participation of CYP24 in the production of calcitroic acid from 1,25-(OH)(2)D(2). We, therefore, studied the metabolism of 1,25-(OH)(2)D(3) and 1,25-(OH)(2)D(2) using a purified rat CYP24 system. Lipid and aqueous-soluble metabolites were prepared for characterization. Aqueous-soluble metabolites were subjected to reverse-phase high-pressure liquid chromatography (HPLC) analysis. As expected, 1,23(OH)(2)-24,25,26,27-tetranor D and calcitroic acid were the major lipid and aqueous-soluble metabolites, respectively, when 1,25-(OH)(2)D(3) was used as substrate. However, when 1,25-(OH)(2)D(2) was used as substrate, 1,24(R),25-(OH)(3)D(2) was the major lipid-soluble metabolite with no evidence for the production of either 1,23(OH)(2)-24,25,26,27-tetranor D or calcitroic acid. Apparently, the CYP24 was able to 24-hydroxylate 1,25-(OH)(2)D(2), but was unable to effect further changes, which would result in side-chain cleavage. These data suggest that the presence of either the double bond at C(22) or the C(24) methyl group impedes the metabolism of 1,25-(OH)(2)D(2) to calcitroic acid by CYP24 and that enzymes other than CYP24 are required to effect this process.

Topics: Animals; Calcitriol; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Ergocalciferols; Rats; Steroid Hydroxylases; Vitamin D3 24-Hydroxylase

Calcitroic acid (1 alpha-hydroxy-23 carboxy-24,25,26,27-tetranorvitamin D(3)) is known to be the major water-soluble metabolite produced during the deactivation of 1 alpha,25-dihydroxyvitamin D(3). This deactivation process involves a series of oxidation reactions at C(24) and C(23) leading to side-chain cleavage and, ultimately, formation of the calcitroic acid. Like 1 alpha,25-dihydroxyvitamin D(3), 1 alpha,25-dihydroxyvitamin D(2) is also known to undergo side-chain oxidation; however, to date there has been no evidence suggesting that 1 alpha,25-dihydroxyvitamin D(2) undergoes side-chain cleavage. To investigate this possibility, we studied 1 alpha,25-dihydroxyvitamin D(2) metabolism in HPK1A-ras cells as well as the well characterized perfused rat kidney system. Lipid and aqueous-soluble metabolites were prepared for characterization. Aqueous-soluble metabolites were subjected to reverse-phase HPLC analysis. The major aqueous-soluble metabolite from both the kidney and cell incubations comigrated with authentic calcitroic acid on two reverse-phase HPLC columns of different chemistry. The putative calcitroic acid from the cell and kidney incubations was methylated and found to comigrate with methylated authentic standard on straight-phase and reverse-phase HPLC columns. The identity of the methylated metabolite from cell incubations was also confirmed by mass spectral analysis. These data show, for the first time, that calcitroic acid is a major terminal product for the deactivation of 1 alpha,25-dihydroxyvitamin D(2). Intermediates leading to the formation of the calcitroic acid in the 1 alpha,25-dihydroxyvitamin D(2) metabolism pathway are currently being studied.

Topics: Animals; Calcitriol; Cell Line; Chromatography, High Pressure Liquid; Ergocalciferols; Humans; In Vitro Techniques; Kidney; Mass Spectrometry; Oxidation-Reduction; Perfusion; Rats; Solubility