School of Molecular Sciences, The University of Western Australia, Perth, WA 6009, Australia.
School of Molecular Sciences, The University of Western Australia, Perth, WA 6009, Australia.
J Steroid Biochem Mol Biol. 2023 Oct;233:106368. doi: 10.1016/j.jsbmb.2023.106368. Epub 2023 Jul 24.
Vitamin D is found in two forms in humans, D3 produced in the skin and D2 solely from the diet. Both 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH)D) are oxidised and inactivated by CYP24A1, a tightly regulated mitochondrial enzyme that controls serum levels of these secosteroids. The pathways of oxidation of 25(OH)D2 and 1,25(OH)D2, particularly 25(OH)D2, by human CYP24A1 are not well characterized. The aim of this study was to further elucidate these pathways, and to compare the kinetics of metabolism of 25(OH)D2 and 1,25(OH)D2 with their vitamin D3 counterparts. We used expressed and partially purified human CYP24A1 with substrates dissolved in the membrane of phospholipid vesicles, to mimic the inner mitochondrial membrane. We found that the major pathways for side chain oxidation of 25(OH)D2 and 1,25(OH)D2 were identical and that predominant intermediates of 25(OH)D2 metabolism could be converted to the corresponding intermediates in the pathway of 1,25(OH)D2 oxidation by 1α-hydroxylation by CYP27B1. The initial steps in the CYP24A1-mediated oxidation involved hydroxylation at the C24R position, and another unknown position where the alcohol was oxidised to an aldehyde. The 24R-hydroxylation was followed by hydroxylation at C26 or C28, or cleavage between C24 and C25 to produce the 24-oxo-25,26,27-trinor derivative. All of these products were further oxidised, with 24-oxo-25,26,27-trinor-1(OH)D2 giving a product tentatively identified as 24-oxo-25,26,27-trinor-1,28(OH)D2. The catalytic efficiency (k/K) of CYP24A1 for initial 25(OH)D2 hydroxylation was similar to that for 25(OH)D3, indicating that they have similar rates of inactivation at low substrate concentrations, supporting that vitamins D2 and D3 are equally effective in maintaining serum 25(OH)D concentrations. In contrast, the k/K value for 1,25(OH)D3 was almost double that for 1,25(OH)D2 indicating a lower rate of inactivation of 1,25(OH)D2 at a low substrate concentration, suggesting that it has increased metabolic stability in vivo.
维生素 D 在人体内有两种形式，D3 由皮肤产生，D2 仅来自饮食。25-羟维生素 D（25(OH)D）和 1,25-二羟维生素 D（1,25(OH)D）都被 CYP24A1 氧化失活，CYP24A1 是一种受严格调控的线粒体酶，控制这些甾体激素的血清水平。人 CYP24A1 对 25(OH)D2 和 1,25(OH)D2 的氧化途径，特别是 25(OH)D2 的氧化途径，尚未得到很好的描述。本研究的目的是进一步阐明这些途径，并比较 25(OH)D2 和 1,25(OH)D2 与它们的维生素 D3 对应物的代谢动力学。我们使用表达和部分纯化的人 CYP24A1 与溶解在磷脂囊泡膜中的底物一起使用，以模拟线粒体内膜。我们发现 25(OH)D2 和 1,25(OH)D2 侧链氧化的主要途径是相同的，并且 25(OH)D2 代谢的主要中间产物可以通过 CYP27B1 的 1α-羟化转化为 1,25(OH)D2 氧化途径中的相应中间产物。CYP24A1 介导的氧化的初始步骤涉及 C24R 位置的羟化，以及另一个未知位置，其中醇被氧化为醛。24R-羟化后，C26 或 C28 羟化，或 C24 和 C25 之间裂解，生成 24-氧代-25,26,27-三降衍生物。所有这些产物都进一步氧化，24-氧代-25,26,27-三降-1(OH)D2 生成的产物暂定鉴定为 24-氧代-25,26,27-三降-1,28(OH)D2。CYP24A1 对初始 25(OH)D2 羟化的催化效率（k/K）与 25(OH)D3 相似，表明它们在低底物浓度下失活的速度相似，这支持维生素 D2 和 D3 在维持血清 25(OH)D 浓度方面同样有效。相比之下，1,25(OH)D3 的 k/K 值几乎是 1,25(OH)D2 的两倍，表明 1,25(OH)D2 在低底物浓度下失活的速度较低，这表明它在体内具有增加的代谢稳定性。
