Department of Chemistry, College of Science, United Arab Emirates University (UAEU), Al Ain 15551, United Arab Emirates.
Department of Biology, College of Sciences, United Arab Emirates University (UAEU), Al Ain 15551, United Arab Emirates.
Molecules. 2021 Aug 26;26(17):5182. doi: 10.3390/molecules26175182.
The most common forms of vitamin D in human and mouse serum are vitamin D3 and vitamin D2 and their metabolites. The aim of this study is to determine whether diet and sunlight directly affect the circulating concentrations of vitamin D metabolites in a mouse model. We investigated the serum concentrations of eight vitamin D metabolites-vitamin D (vitamin D3 + vitamin D2), 25OHD (25OHD3 + 25OHD2), 1α25(OH)D (1α25(OH)D2, and 1α25(OH)D3)-including their epimer, 3-epi-25OHD (3-epi-25OHD3 and 3-epi-25OHD2), and a bile acid precursor 7alpha-hydroxy-4-cholesten-3-one (7αC4), which is known to cause interference in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
The LC-MS/MS method was validated according to FDA-US guidelines. The validated method was used for the analysis of mouse serum samples. Forty blood samples from mice were collected and divided into three groups. The first group, the DDD mice, were fed a vitamin D-deficient diet (25 IU VD3/kg of diet) and kept in the dark; the second group, the SDD mice, were maintained on a standard-vitamin D diet (1000 IU VD3) and kept in the dark; and the third group, SDL, were fed a standard-vitamin D diet (1000 IU VD3) but kept on a normal light/dark cycle. LC-MS/MS was used for the efficient separation and quantitation of all the analytes.
The validated method showed good linearity and specificity. The intraday and interday precision were both <16%, and the accuracy across the assay range was within 100 ± 15%. The recoveries ranged between 75 and 95%. The stability results showed that vitamin D metabolites are not very stable when exposed to continuous freeze-thaw cycles; the variations in concentrations of vitamin D metabolites ranged between 15 and 60%. The overlapping peaks of vitamin D, its epimers, and its isobar (7αC4) were resolved using chromatographic separation. There were significant differences in the concentrations of all metabolites of vitamin D between the DDD and SDL mice. Between the groups SDD (control) and SDL, a significant difference in the concentrations of 3-epi-25OHD was noted, where C3 epimer was about 30% higher in SDL group while no significant differences were noted in the concentrations of vitamin D, 25OHD, 1α25(OH)D, and 7αC4 between SDD and SDL group.
A validated method, combined with a simple extraction technique, for the sensitive LC-MS/MS determination of vitamin D metabolites is described here. The method can eliminate the interferences in LC-MS/MS analysis caused by the overlapping epimer and isobar due to them having the same molecular weights as 25OHD. The validated method was applied to mouse serum samples. It was concluded that a standard-vitamin D diet causes an increase in the proportion of all the vitamin D metabolites and C3 epimers and isobar, while UV light has no pronounced effect on the concentrations of the majority of the vitamin D metabolites except 3-epi-25OHD. Further studies are required to confirm this observation in humans and to investigate the biochemical pathways related to vitamin D's metabolites and their epimers.
在人和鼠血清中，最常见的维生素 D 形式是维生素 D3 和维生素 D2 及其代谢物。本研究的目的是确定饮食和阳光是否直接影响小鼠模型中维生素 D 代谢物的循环浓度。我们研究了 8 种维生素 D 代谢物的血清浓度-维生素 D（维生素 D3 + 维生素 D2）、25OHD（25OHD3 + 25OHD2）、1α25（OH）D（1α25（OH）D2 和 1α25（OH）D3）-包括它们的表异构体 3-epi-25OHD（3-epi-25OHD3 和 3-epi-25OHD2）和一种胆汁酸前体 7α-羟基-4-胆甾烯-3-酮（7αC4），已知它会在液相色谱-串联质谱（LC-MS/MS）分析中引起干扰。
根据 FDA-US 指南验证了 LC-MS/MS 方法。该验证方法用于分析小鼠血清样本。从 40 只小鼠中采集血液样本并将其分为三组。第一组，DDD 小鼠，喂食缺乏维生素 D 的饮食（25 IU VD3/kg 饮食）并保持在黑暗中；第二组，SDD 小鼠，维持标准维生素 D 饮食（1000 IU VD3）并保持在黑暗中；第三组，SDL，喂食标准维生素 D 饮食（1000 IU VD3）但保持正常的光/暗循环。LC-MS/MS 用于所有分析物的有效分离和定量。
验证后的方法显示出良好的线性和特异性。日内和日间精密度均<16%，整个检测范围内的准确度在 100±15%范围内。回收率在 75%至 95%之间。稳定性结果表明，维生素 D 代谢物在连续冻融循环中不太稳定；维生素 D 代谢物浓度的变化范围在 15%至 60%之间。使用色谱分离解决了维生素 D、其表异构体和等重物（7αC4）的重叠峰。DDD 和 SDL 小鼠之间所有维生素 D 代谢物的浓度存在显著差异。在 SDD（对照）和 SDL 组之间，3-epi-25OHD 的浓度存在显著差异，其中 C3 表异构体在 SDL 组中约高 30%，而 25OHD、1α25（OH）D 和 7αC4 的浓度在 SDD 和 SDL 组之间没有显著差异。
本文描述了一种经过验证的方法，结合了一种简单的提取技术，用于敏感的 LC-MS/MS 测定维生素 D 代谢物。该方法可以消除由于表异构体和等重物（25OHD）具有相同的分子量而在 LC-MS/MS 分析中引起的干扰。该验证方法应用于小鼠血清样本。结果表明，标准维生素 D 饮食会增加所有维生素 D 代谢物和 C3 表异构体和等重物的比例，而紫外线对大多数维生素 D 代谢物的浓度没有明显影响，除了 3-epi-25OHD。需要进一步的研究来证实这一观察结果在人类中，并研究与维生素 D 代谢物及其表异构体相关的生化途径。
