23-Carboxy-24,25,26,27-tetranorvitamin D3 (calcioic acid) and 24-carboxy-25,26,27-trinorvitamin D3 (cholacalcioic acid): End products of 25-hydroxyvitamin D3 metabolism in rat kidney through C-24 oxidation pathway

During the past two and half decades the elucidation of the metabolic pathways of 25OHD3 and its active metabolite 1α,25(OH)2D3 progressed in parallel. In spite of many advances in this area of vitamin D research, the unequivocal identification of the end products of 25OHD3 metabolism through C-24 oxidation pathway has not been achieved. It is now well established that both 25OHD3 and 1α,25(OH)2D3 are metabolized through the same C-24 oxidation pathway initiated by the enzyme 24-hydroxylase (CYP24A1). Based on the information that the end product of 1α,25(OH)2D3 metabolism through C-24 oxidation pathway is 1α-OH-23- COOH-24,25,26,27-tetranor D3 or calcitroic acid; the metabolism of 25OHD3 into 23-COOH-24,25,26,27-tetranor D3 has been assumed. Furthermore, a previous study indicated 24-COOH-25,26,27-trinor D3 as a water soluble metabolite of 24R,25(OH)2D3 produced in rat kidney homogenates. Therefore, 24-COOH-25,26,27-trinor D3 was also assumed as another end product of 25OHD3 metabolism through C-24 oxidation pathway. We embarked on our present study to provide unequivocal proof for these assumptions. We first studied the metabolism of 25OHD3 at low substrate concentration (3 × 10−10 M) using [1,2-3H]25OHD3 as the substrate in the perfused rat kidneys isolated from both normal and vitamin D3 intoxicated rats. A highly polar water soluble metabolite, labeled as metabolite X was isolated from the kidney perfusate. The amount of metabolite X produced in the kidney of a vitamin D intoxicated rat was about seven times higher than that produced in the kidney of a normal rat. We then produced metabolite X in a quantity sufficient for its structure identification by perfusing kidneys isolated from vitamin D intoxicated rats with high substrate concentration of 25OHD3 (5 × 10−6 M). Using the techniques of electron impact and thermospray mass spectrometry, we established that the metabolite X contained both 23-COOH-24,25,26,27-tetranor D3 and 24-COOH-25,26,27-trinor D3 in a ratio of 4:1. The same metabolite X containing both acids in the same ratio of 4:1 was also produced when 24R,25(OH)2D3 was used as the starting substrate. Previously, the trivial name of cholacalcioic acid was assigned to 24-COOH-25,26,27-trinorvitamin D3. Using the same guidelines, we now assign the trivial name of calcioic acid to 23-COOH-24,25,26,27-tetranor D3. In summary, for the first time our study provides unequivocal evidence to indicate that both calcioic and cholacalcioic acids as the end products of 25OHD3 metabolism in rat kidney through C-24 oxidation pathway.