Hashizume Takanori, Xu Yang, Mohutsky Michael A, Alberts Jeffrey, Hadden Chad, Kalhorn Thomas F, Isoherranen Nina, Shuhart Margaret C, Thummel Kenneth E
Department of Pharmaceutics, University of Washington, Seattle, WA, United States.
Biochem Pharmacol. 2008 Mar 1;75(5):1240-50. doi: 10.1016/j.bcp.2007.11.008. Epub 2007 Nov 22.
The biological effects of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) are terminated primarily by P450-dependent hydroxylation reactions. However, the hormone is also conjugated in the liver and a metabolite, presumably a glucuronide, undergoes enterohepatic cycling. In this study, the identity of human enzymes capable of catalyzing the 1,25(OH)2D3 glucuronidation reaction was investigated in order to better understand environmental and endogenous factors affecting the disposition and biological effects of vitamin D3. Among 12 different UGT isozymes tested, only UGT1A4 >> 2B4 and 2B7 supported the reaction. Two different 1,25(OH)2D3 monoglucuronide metabolites were generated by recombinant UGT1A4 and human liver microsomes. The most abundant product was identified by mass spectral and NMR analyses as the 25-O-glucuronide isomer. The formation of 25-O-glucuronide by UGT1A4 Supersomes and human liver microsomes followed simple hyperbolic kinetics, yielding respective Km and Vmax values of 7.3 and 11.2 microM and 33.7 +/- 1.4 and 32.9 +/- 1.9 pmol/min/mg protein. The calculated intrinsic 25-O-glucuronide M1 formation clearance for UGT1A4 was 14-fold higher than the next best isozyme, UGT2B7. There was only limited (four-fold) inter-liver variability in the 25-O-glucuronidation rate, but it was highly correlated with the relative rate of formation of the second, minor metabolite. In addition, formation of both metabolites was inhibited >80% by the selective UGT1A4 inhibitor, hecogenin. If enterohepatic recycling of 1,25(OH)2D3 represents a significant component of intestinal and systemic 1,25(OH)2D3 disposition, formation of monoglucuronides by hepatic UGT1A4 constitutes an important initial step.
1α,25 - 二羟基维生素D3（1,25(OH)2D3）的生物学效应主要通过依赖细胞色素P450的羟基化反应而终止。然而，该激素在肝脏中也会发生共轭反应，并且一种代谢产物，推测为葡糖醛酸苷，会进行肠肝循环。在本研究中，对能够催化1,25(OH)2D3葡糖醛酸化反应的人类酶的特性进行了研究，以便更好地了解影响维生素D3代谢和生物学效应的环境及内源性因素。在测试的12种不同的尿苷二磷酸葡萄糖醛酸基转移酶（UGT）同工酶中，只有UGT1A4 >> 2B4和2B7支持该反应。重组UGT1A4和人肝微粒体产生了两种不同的1,25(OH)2D3单葡糖醛酸苷代谢产物。通过质谱和核磁共振分析确定，最丰富的产物为25 - O - 葡糖醛酸苷异构体。UGT1A4重组微粒体和人肝微粒体形成25 - O - 葡糖醛酸苷的过程遵循简单的双曲线动力学，其Km值和Vmax值分别为7.3和11.2 microM，以及33.7±1.4和32.9±1.9 pmol/min/mg蛋白。计算得出UGT1A4形成25 - O - 葡糖醛酸苷M1的内在清除率比第二有效的同工酶UGT2B7高14倍。25 - O - 葡糖醛酸化速率在肝脏之间仅有有限的（四倍）变异性，但它与第二种次要代谢产物的相对形成速率高度相关。此外，选择性UGT1A4抑制剂海柯皂苷元对两种代谢产物形成的抑制率均>80%。如果1,25(OH)2D3的肠肝循环是肠道和全身1,25(OH)2D3代谢的重要组成部分，那么肝脏UGT1A4形成单葡糖醛酸苷就是一个重要的起始步骤。
