23-Carboxy-24,25,26,27-tetranorvitamin D 3 (calcioic acid) and 24-carboxy-25,26,27-trinorvitamin D 3 (cholacalcioic acid): End products of 25-hydroxyvitamin D 3 metabolism in rat kidney through C-24 oxidation pathway

Abstract

During the past two and half decades the elucidation of the metabolic pathways of 25OHD 3 and its active metabolite 1α,25(OH) 2D 3 progressed in parallel. In spite of many advances in this area of vitamin D research, the unequivocal identification of the end products of 25OHD 3 metabolism through C-24 oxidation pathway has not been achieved. It is now well established that both 25OHD 3 and 1α,25(OH) 2D 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 1α,25(OH) 2D 3 metabolism through C-24 oxidation pathway is 1α-OH-23- COOH-24,25,26,27-tetranor D 3 or calcitroic acid; the metabolism of 25OHD 3 into 23-COOH-24,25,26,27-tetranor D 3 has been assumed. Furthermore, a previous study indicated 24-COOH-25,26,27-trinor D 3 as a water soluble metabolite of 24 R,25(OH) 2D 3 produced in rat kidney homogenates. Therefore, 24-COOH-25,26,27-trinor D 3 was also assumed as another end product of 25OHD 3 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 25OHD 3 at low substrate concentration (3 × 10 −10 M) using [1,2- 3H]25OHD 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 25OHD 3 (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 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 24 R,25(OH) 2D 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 25OHD 3 metabolism in rat kidney through C-24 oxidation pathway.