Zerbinati C, Galli F, Regolanti R, Poli G, Iuliano L
Department of Medico-Surgical Sciences and Biotechnologies, Laboratory of Vascular Biology and Mass Spectrometry, Sapienza University of Rome, Latina, Italy.
Department of Internal Medicine, Laboratory of Clinical Biochemistry and Nutrition, University of Perugia, Perugia, Italy.
Clin Chim Acta. 2015 Jun 15;446:156-62. doi: 10.1016/j.cca.2015.04.026. Epub 2015 Apr 25.
Assessing vitamin E status in humans is critical for nutritional evaluation and verification of clinical and biological compliance of supplemented subjects. An accurate analytical method for measuring the two main vitamin E isoforms, i.e. α- and γ-tocopherol (α- and γ-TOH) in small volumes of plasma can facilitate the application of this analysis to clinical trials and in situations where a limited amount of sample is available.
We have developed a micro method, which uses only 5 μL plasma, based on isotope dilution, trimethylsilation and GC-MS. The method was validated according to the guidelines of the International Conference on Harmonization of analytical procedures. The method was also applied to 5 μL of whole blood for the potential use in conditions were the availability of specimens is limited.
Accurate quantitation of α-TOH and γ-TOH was achieved at levels ≥ 0.417 μM and ≥ 0.007 μM, respectively. Within-day coefficient of variation was 1.31% and 4.70% for α-TOH and γ-TOH, respectively. Between-day coefficient of variation was 1.32% and 2.88% for α-TOH and γ-TOH, respectively. Recovery, assessed at three concentration levels, ranged 98-103% and 100-102% for α-TOH and γ-TOH, respectively. The method allowed the detection of α-TOH and γ-TOH in 5 μL whole blood and in membranes of red blood cells washed from 5 μL of blood as well. The analytical performance was assessed in plasma from a cohort of Italian healthy subjects (n = 205). The mean plasma concentrations were 28.01 ± 6.31 and 0.68 ± 0.48 μM (mean ± SD) for α-TOH and γ-TOH, respectively. Alpha-TOH correlated with total cholesterol (r = 0.617, p < 0.0001) and triglycerides (r = 0.420, p < 0.0001) while γ-TOH correlated modestly with total cholesterol (r = 0.213, p < 0.0001) but not with triglycerides. γ-TOH, but not α-TOH, was significantly lower in smokers than in non-smokers (0.72 ± 0.50 vs. 0.56 ± 0.37, μM, mean ± SD, p = 0.017). Given the high sensitivity, the method allowed to be applied to 5 μM whole blood without specific modification.
This micro-method represents an analytical advancement in α- and γ-TOH assay that is available to accurately verify the nutritional status and compliance after supplementation in large-scale settings, and to measure the two vitamers in conditions where sample availability is limited.
评估人体维生素E状态对于营养评估以及验证补充剂使用者的临床和生物学依从性至关重要。一种能够在少量血浆中准确测量两种主要维生素E异构体（即α-生育酚和γ-生育酚，α-TOH和γ-TOH）的分析方法，有助于将该分析应用于临床试验以及样本量有限的情况。
我们开发了一种微量方法，基于同位素稀释、三甲基硅烷化和气相色谱 - 质谱联用技术，仅需5μL血浆。该方法根据国际协调会议分析程序指南进行了验证。该方法还应用于5μL全血，以探讨其在样本获取受限情况下的潜在用途。
分别在α-TOH水平≥0.417μM和γ-TOH水平≥0.007μM时实现了准确定量。α-TOH和γ-TOH的日内变异系数分别为1.31%和4.70%。α-TOH和γ-TOH的日间变异系数分别为1.32%和2.88%。在三个浓度水平评估的回收率，α-TOH和γ-TOH分别为98 - 103%和100 - 102%。该方法能够检测5μL全血以及从5μL血液中洗涤得到的红细胞膜中的α-TOH和γ-TOH。在一组意大利健康受试者（n = 205）的血浆中评估了分析性能。α-TOH和γ-TOH的平均血浆浓度分别为28.01±6.31μM和0.68±0.48μM（均值±标准差）。α-TOH与总胆固醇（r = 0.617，p < 0.0001）和甘油三酯（r = 0.420，p < 0.0001）相关，而γ-TOH与总胆固醇有适度相关性（r = 0.213，p < 0.0001），但与甘油三酯无关。吸烟者的γ-TOH显著低于非吸烟者（0.72±0.50对0.56±0.37，μM，均值±标准差，p = 0.017），而α-TOH则无此差异。鉴于该方法具有高灵敏度，无需进行特定修改即可应用于5μL全血。
这种微量方法代表了α-TOH和γ-TOH测定的分析进展，可用于在大规模环境中准确验证营养状况和补充后的依从性，并在样本获取受限的情况下测量这两种维生素形式。
