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 250HD(3) and its active metabolite 1 alpha,25(OH)(2)D-3 progressed in parallel. In spite of many advances in this area of vitamin D research, the unequivocal identification of the end products of 250HD(3) metabolism through C-24 oxidation pathway has not been achieved. It is now well established that both 250HD(3) and 1 alpha,25(OH)(2)D-3 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 1 alpha,25(OH)(2)D-3 metabolism through C-24 oxidation pathway is 1 alpha-OH-23- COOH-24,2 5,26,27-tetranor D-3 or calcitroic acid; the metabolism of 250HD(3) into 23-COOH-24,25,26,27-tetranor D-3 has been assumed. Furthermore, a previous study indicated 24-COOH25,26,27-trinor D-3 as a water soluble metabolite of 24R,25(OH)(2)D-3 produced in rat kidney homogenates. Therefore, 24-COOH-25,26,27-trinor D3 was also assumed as another end product of 250HD3 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 250HD(3) at low substrate concentration (3 x 10(-10) M) using [1,2-H-3]250HD(3) as the substrate in the perfused rat kidneys isolated from both normal and vitamin D-3 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 250HD(3) (5 x 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 D-3 and 24-COOH-25,26,27-trinor D-3 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)(2)D-3 was used as the starting substrate. Previously, the trivial name of cholacalcioic acid was assigned to 24-COOH-25,26,27-trinorvitamin D-3. Using the same guidelines, we now assign the trivial name of calcioic acid to 23-COOH-24,25,26,27-tetranor D-3. In summary, for the first time our study provides unequivocal evidence to indicate that both calcioic and cholacalcioic acids as the end products of 250HD(3) metabolism in rat kidney through C-24 oxidation pathway. (c) 2006 Elsevier Inc. All rights reserved.