Shankar V N, Byford V, Prosser D E, Schroeder N J, Makin H L, Wiesinger H, Neef G, Steinmeyer A, Jones G
Department of Biochemistry, Queen's University, K7L 3N6, Kingston, ON, Canada.
Biochem Pharmacol. 2001 Apr 1;61(7):893-902. doi: 10.1016/s0006-2952(01)00546-9.
We describe here for the first time the effect of introducing a 20-methyl group on the side-chain metabolism of the vitamin D molecule. Using a series of 20-methyl-derivatives of 1alpha,25-(OH)2D3 incubated with two different cultured human cell lines, HPK1A-ras and HepG2, previously shown to metabolize vitamin D compounds, we obtained a series of metabolic products that were identified by comparison to chemically synthesized standards on HPLC and GC-MS. 24-Hydroxylated-, 24-oxo-hydroxylated-, and 24-oxo-23-hydroxylated products of 20-methyl-1alpha,25-(OH)2D3 were observed, but the efficiency of 23-hydroxylation was low as compared with that of the natural hormone and, in contrast to 1alpha,25-(OH)2D3, no truncated 23-alcohol was formed from the 20-methyl analog. These data, taken together with results from other analogs with changes in the vicinity of the C17-C20 positions, lead us to speculate that such changes must alter the accessibility of the C-23 position to the cytochrome P450 involved. Using the HepG2 cell line, we found evidence that the 24S-hydroxylated product of 20-methyl-1alpha,25-(OH)2D3 predominates, implying that the liver cytochrome involved in metabolism is a different isoform. Studies with a more metabolically resistant analog of the series, 20-methyl-Delta(23)-1alpha,25-(OH)2D3, gave the expected block in 23- and 24-hydroxylation, and evidence of an alternative pathway, namely 26-hydroxylation. 20-Methyl-Delta(23)-1alpha,25-(OH)2D3 was also more potent in biological assays, and the metabolic studies reported here help us to suggest explanations for this increased potency. We conclude that the 20-methyl series of vitamin D analogs offers new perspectives into vitamin D analog action, as well as insights into the substrate preferences of the cytochrome(s) P450 involved in vitamin D catabolism.
我们首次在此描述了引入20-甲基基团对维生素D分子侧链代谢的影响。使用一系列1α,25-(OH)₂D₃的20-甲基衍生物与两种不同的人培养细胞系HPK1A-ras和HepG2进行孵育，这两种细胞系先前已被证明能够代谢维生素D化合物，我们获得了一系列代谢产物，并通过与HPLC和GC-MS上的化学合成标准品进行比较来鉴定。观察到了20-甲基-1α,25-(OH)₂D₃的24-羟基化、24-氧代-羟基化和24-氧代-23-羟基化产物，但与天然激素相比，23-羟基化的效率较低，并且与1α,25-(OH)₂D₃不同，20-甲基类似物没有形成截短的23-醇。这些数据与其他在C17-C20位置附近有变化的类似物的结果一起，使我们推测这种变化一定改变了C-23位置对所涉及的细胞色素P450的可及性。使用HepG2细胞系，我们发现有证据表明20-甲基-1α,25-(OH)₂D₃的24S-羟基化产物占主导，这意味着参与代谢的肝脏细胞色素是一种不同的同工型。对该系列中代谢抗性更强的类似物20-甲基-Δ(23)-1α,25-(OH)₂D₃的研究，预期会阻断23-和24-羟基化，并发现了一条替代途径，即26-羟基化。20-甲基-Δ(23)-1α,25-(OH)₂D₃在生物学测定中也更有效，此处报道的代谢研究有助于我们对这种增强的效力提出解释。我们得出结论，维生素D类似物的20-甲基系列为维生素D类似物的作用提供了新的视角，也为参与维生素D分解代谢的细胞色素P450的底物偏好提供了见解。
