Immediate stabilization of human blood for delayed quantification of endogenous thiols and disulfides.
Immediate stabilization of human blood for delayed quantification of endogenous thiols and disulfides.
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DOI：
10.1016/j.jchromb.2016.02.009
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发表时间：
2016-04-15
期刊：
影响因子：
--
通讯作者：
Rossi R
中科院分区：
文献类型：
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作者：
Giustarini D;Galvagni F;Orlandini M;Fanti P;Rossi R
当暴露于氧化剂时，内源性硫醇经历快速和可逆的氧化成二硫化物，因此是氧化应激的合适生物标志物。然而，血液中硫醇的准确分析经常受到样品操作过程中人为氧化的影响，这会虚假地提高二硫化物水平。在这里，我们描述了一种经过验证的分析前方法，该方法可以防止样品操作过程中硫醇的人为氧化，以及在−80°C下储存的生物样品中硫醇的氧化衰减数月。将N-乙基马来酰亚胺添加至来自健康供体的血液样品中用于稳定全血、红细胞、血小板和血浆二硫化物，而添加柠檬酸盐缓冲液，然后用H2O稀释血浆用于稳定血浆硫醇。当通过UV/维斯HPLC定期分析时，所有生物样品中硫醇和二硫化物的浓度均稳定至少6个月。仅需3 ml血液即可分析不同血液组分中的硫醇和二硫化物。该预分析方法可用于动物和人体前瞻性研究。其易于实施使得该方法适用于多中心研究，其中血液样本由不同的地点和人员收集，并运送到特定的专业实验室。
Endogenous thiols undergo rapid and reversible oxidation to disulfides when exposed to oxidants and are, therefore, suitable biomarkers of oxidative stress. However, accurate analysis of thiols in blood is frequently compromised by their artifactual oxidation during sample manipulation, which spuriously elevates the disulfide levels. Here, we describe a validated pre-analytical procedure that prevents both artifactual oxidation of thiols during sample manipulation and their oxidative decay for months in biosamples that are stored at −80°C. Addition of N-ethylmaleimide to blood samples from healthy donors was used to stabilize whole blood, red blood cells, platelets and plasma disulfides, whereas addition of citrate buffer followed by dilution of plasma with H2O was used to stabilize plasma thiols. The concentrations of thiols and disulfides were stable in all biosamples for at least 6 months when analyzed by UV/Vis HPLC at regular intervals. Only 3 ml of blood were needed to perform the analyses of thiols and disulfides in the different blood fractions. This pre-analytical procedure is reliable for use in both animal and human prospective studies. Its ease of implementation makes the method suitable for application to multicenter studies where blood samples are collected by different sites and personnel and are shipped to specific specialized laboratories.