Micro-method for the determination of glutathione in human blood.
Micro-method for the determination of glutathione in human blood.
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DOI：
10.1016/j.jchromb.2014.02.018
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发表时间：
2014-08-01
影响因子：
3
通讯作者：
Rossi, Ranieri
中科院分区：
文献类型：
--
作者：
Giustarini, Daniela;Fanti, Paolo;Matteucci, Elena;Rossi, Ranieri
本文建立了一种用指签法测定微量血（低至0.5 μL）中谷胱甘肽（GSH）的可见范围分光光度法。样品在稳定溶液中稀释1至300 (v/v)，然后测定血红蛋白浓度并进行酸脱蛋白。然后使用DTNB，即5,5 ' -二硫代-二（2-硝基苯甲酸），在pH为7.8的水溶液中，用比色法在透明上清中测量谷胱甘肽。制备DTNB试剂并将其保持在pH 6.2直到加入之前，从而避免了试剂的自发分解。该方法快速，易于适应大规模研究，并且避免了GSH的人为氧化，这是一种常见的方法缺点。日间相对标准偏差（RSD）为1.7 ~ 3.4%，日间相对标准偏差（RSD）为2.2 ~ 4.2%，日间相对误差（RE）为- 1.4% ~ 2.3%，日间相对误差为- 2.8% ~ 1.6%，GSH回收率为97.5% ~ 100%。新的比色显微法通过可靠的高效液相色谱法进行了验证。该方法适用于外周血氧化应激的微创检查。
A new procedure is described for the visible-range spectrophotometric analysis of glutathione (GSH) in microvolumes of blood (as low as 0.5 μL) collected by fingerstick. Samples are diluted 1 to 300 (v/v) in a stabilizing solution, followed by determination of hemoglobin concentration and by acid deproteination. GSH is then measured in the clear supernatant by colorimetry using DTNB, i.e., 5,5’-dithio-bis(2-nitrobenzoic acid), in aqueous solution at pH 7.8. The DTNB reagent is prepared and kept at pH 6.2 until just prior its addition, thus avoiding spontaneous decomposition of the reagent. The assay is rapid, easy to adapt to large-scale studies and it avoids artifactual oxidation of GSH, a common methodological shortcoming. The method is precise with 1.7 to 3.4% intra-day relative standard deviation (RSD) and 2.2 to 4.2% inter-day RSD, and accurate with −1.4% to 2.3% intra-day relative error (RE) and −2.8% to 1.6% inter-day RE. GSH is recovered by 97.5 to 100% at all tested concentrations. The new colorimetric micro-method was validated by a reliable previously reported HPLC method. The procedure is suitable for minimally invasive investigation of oxidative stress in peripheral blood.
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影响因子：
4.2
作者：
Wu, GY;Fang, YZ;Turner, ND
通讯作者：
Turner, ND
影响因子：
14.8
作者：
Giustarini, Daniela;Dalle-Donne, Isabella;Rossi, Ranieri
通讯作者：
Rossi, Ranieri
影响因子：
3.9
作者：
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通讯作者：
ELLMAN, GL
影响因子：
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作者：
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通讯作者：
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影响因子：
168.9
作者：
BLAND, JM;ALTMAN, DG
通讯作者：
ALTMAN, DG