血浆中游离还原型及氧化型谷胱甘肽的荧光测定法

文章建立了快速、可同时检测血浆中游离的还原型和氧化型谷胱甘肽(即GSH和GSSG)的方法, 并评价了血浆中GSH/GSSG的氧化还原状态。利用二硫苏糖醇将GSSG还原成两分子的GSH，使GSH在pH 8.0时与邻苯二甲醛(OPA)结合生成荧光产物GSH-OPA，用荧光分光光度仪测定其荧光值，从而对GSH和GSSG进行定量分析。并运用此方法测量青年健康志愿者静脉血中的游离GSH和GSSG。该法最低可检测出测定管中11.4 pmol·L-1GSH以及5.7 pmol·L-1GSSG。测量GSH批内和批间变异系数分别4.6%和3.9%，GSSG批内和批间变异系数分别为3.5%和4.1%，GSH，GSSG加标准品后的回收率分别为(99.77±5.70)%和(99.28±4.73)%。测定健康青年志愿者血浆中游离GSH，GSSG的含量分别为(16.5±2.4) nmol·mL-1和(1.7±0.35) nmol·mL-1。

A Novel Fluorophotometric Method for Assaying Free Glutathione of Both Reduced and Oxidized in Plasma

To investigate the measurement of free glutathione of both reduced or oxidized (i.e. GSH and GSSG) in plasma, and to evaluate the redox state of GGSH/GSSG in human plasma, both GSH and GSSG in plasma were measured by fluorophotometry based on the facts that one molar GSSG can be reduced to two molar GSH by dithiothreitol(DTT) under the condition of the pH being about 6.0, and GSH can provide both primary amine and thiol groups to react with the two carbonyl groups of O-phthaldehyde (OPA) to form a fluorescent ternary isoindole complex at pH 8.0. This method can at least measure 16 picomole GSH and 8 picomole GSSG respectively in the tube. The variation coefficient (CV) for intra-ssay and intera-ssay is about 4. 6% and 3.9% for GSH and 3.5% and 4.1% for GSSG respectively. The recovery of GSH and GSSG added to the plasma is (99.77% +/- 5.70)% and (99.28% +/- 4.73)% respectively. The concentration of GSH and GSSG in the plasma of young healthy volunteer is (16.5 +/- 2.4) nmol x mL(-1) and (1.7 +/- 0.35)nmol x mL(-1) respectively, without significant difference between male and female. This measurement method is simple with great sensitivity and selectivity for rapid measuring GSH and GSSG in human plasma simultaneously.