Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection
Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection
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DOI：
10.1039/c1an00004g
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发表时间：
2011-01-01
期刊：
影响因子：
4.2
通讯作者：
Conlan, Xavier A.
中科院分区：
文献类型：
--
作者：
McDermott, Geoffrey P.;Francis, Paul S.;Conlan, Xavier A.
谷胱甘肽(GSH)和谷胱甘肽二硫键(GSSG)的测定是评估细胞氧化还原状态的重要工具。在这里，我们报道了一种基于快速色谱分离和酸性高锰酸钾化学发光检测的直接测定细胞内GSH的方法，该方法通过引入硫醇封闭和二硫键还原而扩展到GSSG。重要的是，这个简单的程序避免了GSH的衍生化(从而最大限度地减少了自动氧化)，并克服了从“总GSH”得出GSSG浓度时遇到的问题。线性范围为7.5×10~(-7)~1×10~(-5)M，检出限为5×10~(-7)M。用葡萄糖氧化酶(0、15、30、100、250和500 mU·L~(-1))处理培养的肌肉细胞24 h后，测定GSH和GSSG的含量。与未经处理的对照组相比，100或250 mU mL(-1)葡萄糖氧化酶处理的肌管中这两种分析物浓度更高，而500 mU mL(-1)处理的肌管中这两种分析物浓度显著降低(p<；0.05)，考虑到过氧化氢和细胞存活率的测量，这是合理的。然而，100、250和500 mU/L葡萄糖氧化酶处理的肌管GSH/GSSG比值呈剂量依赖性下降，反映了细胞内ROS的增加。
Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from 'total GSH'. The linear range and limit of detection for both analytes were 7.5 x 10(-7) to 1 x 10(-5) M, and 5 x 10(-7) M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL(-1)), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL(-1) glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL(-1) (p < 0.05), which was rationalised by considering measurements of H2O2 and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL(-1) glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.