Yeung B, Vouros P, Siu-Caldera M L, Reddy G S
Department of Chemistry, Northeastern University, Boston, MA 02115, USA.
Biochem Pharmacol. 1995 Apr 18;49(8):1099-710. doi: 10.1016/0006-2952(95)98507-6.
1,25-Dihydroxy-16-ene vitamin D3 is a synthetic analog of 1,25-dihydroxyvitamin D3, the most physiologically active metabolite of vitamin D3. The renal metabolism of 1,25-dihydroxy-16-ene vitamin D3 had been studied previously using a perfused rat kidney system [Reddy et al., Bioorg Med Chem Lett 3: 1879-1884, 1993], and its C-24 oxidative metabolic pathway had been found to be different from that of 1,25-dihydroxyvitamin D3 by HPLC. To further delineate the differences between the C-24 oxidative metabolic pathways of 1,25-dihydroxyvitamin D3 and 1,25-dihydroxy-16-ene vitamin D3 in this present study we investigated the C-24 oxidation pathway of 1,25-dihydroxy-16-ene vitamin D3 using a novel detection approach based on on-line capillary liquid chromatography coupled to electrospray tandem mass spectrometry. Two types of tandem mass spectrometric detection were employed to characterize the metabolites in the kidney perfusate: (a) the preliminary screening of metabolites by parent scan, which led to the tentative discovery of the production of 1,23,25-trihydroxy-24-oxo-16-ene vitamin D3, a new metabolite of 1,25-dihydroxy-16-ene vitamin D3, and (b) the pharmacokinetic studies of the substrate, 1,25-dihydroxy-16-ene vitamin D3 and its metabolites by multiple reaction monitoring. In the latter, the mass spectrometric sensitivity for quantification was found to be about 20-fold better than UV detection. The current work concluded that the C-24 oxidative metabolic pathway of 1,25-dihydroxy-16-ene vitamin D3 closely mimicked that of its natural counterpart. Furthermore, the use of mass spectrometry permitted the clearance rate of the starting substrate to be studied at a more physiological level (ng/mL or submicromolar level), which had not been possible previously by HPLC-UV detection.
1,25-二羟基-16-烯维生素D3是维生素D3最具生理活性的代谢产物1,25-二羟基维生素D3的合成类似物。先前已使用灌注大鼠肾脏系统研究了1,25-二羟基-16-烯维生素D3的肾脏代谢[雷迪等人，《生物有机与药物化学快报》3: 1879 - 1884，1993]，并且通过高效液相色谱法发现其C - 24氧化代谢途径与1,25-二羟基维生素D3不同。在本研究中，为了进一步阐明1,25-二羟基维生素D3和1,25-二羟基-16-烯维生素D3在C - 24氧化代谢途径上的差异，我们使用基于在线毛细管液相色谱与电喷雾串联质谱联用的新型检测方法研究了1,25-二羟基-16-烯维生素D3的C - 24氧化途径。采用了两种类型的串联质谱检测来表征肾脏灌注液中的代谢产物：(a) 通过母离子扫描对代谢产物进行初步筛选，这导致初步发现了1,23,25-三羟基-24-氧代-16-烯维生素D3的产生，它是1,25-二羟基-16-烯维生素D3的一种新代谢产物，以及(b) 通过多反应监测对底物1,25-二羟基-16-烯维生素D3及其代谢产物进行药代动力学研究。在后者中，发现用于定量的质谱灵敏度比紫外检测大约高20倍。当前的研究得出结论，1,25-二羟基-16-烯维生素D3的C - 24氧化代谢途径与其天然对应物的代谢途径非常相似。此外，质谱的使用使得能够在更生理的水平（纳克/毫升或亚微摩尔水平）研究起始底物的清除率，这在以前通过高效液相色谱 - 紫外检测是不可能的。
