毛细管电泳-激光诱导荧光法测定细胞中谷胱甘肽

谷胱甘肽(GSH)在抵抗氧化应激和重金属解毒过程中发挥着重要作用，建立灵敏、准确的GSH定量分析方法对于研究细胞重金属毒性机制具有深远意义。该研究以肝癌细胞(HepG2)为研究对象，以活性基团为芳香邻二醛的2,3-萘二甲醛(NDA)为标记试剂，建立了一种高灵敏度的测定细胞中GSH含量的毛细管电泳-激光诱导荧光检测方法(CE-LIF)。实验考察了缓冲溶液的种类、pH、添加剂等对GSH与NDA的反应速率和NDA-GSH检测灵敏度的影响。比较了pH为7.4和9.2的三羟甲基氨基甲烷(Tris)缓冲溶液、pH为9.2的硼砂和Tris缓冲溶液中NDA-GSH的灵敏度和反应速率，结果显示在pH为9.2的硼砂缓冲溶液中NDA-GSH的灵敏度最高且反应速率最快。进一步比较了4种添加剂对NDA-GSH灵敏度的影响，结果显示以β-环糊精(β-CD)作为添加剂效果最好。在最优的实验条件下，GSH与NDA可以在5 min内达到反应平衡，3 min内检测到NDA-GSH电泳信号。采用外标法对细胞中的GSH进行定量分析，方法线性范围为0.01～20.00 mmol/L, GSH的检出限和定量限分别为0.006μm...

Determination of glutathione in cells by capillary electrophoresis-laser induced fluorescence

Glutathione (GSH) is vital for oxidative stress resistance and heavy metals detoxification. It is significant to develop a sensitive and accurate quantitative GSH approach for the toxicity mechanism for studying heavy metals in cells. A high-sensitive capillary electrophoresis-laser induced fluorescence (CE-LIF) detection approach was proposed in this study to detect GSH content in cells. The approach employed HepG2 cells as an object and 2,3-naphthalenedicarboxaldehyde (NDA) with the active group of aromatic o-dialdehyde as a labeling reagent. The effects of buffer solution types, pH, additives on the GSH reaction rate with NDA, and the sensitivity of NDA-GSH were systematically investigated. The sensitivity of NDA-GSH and the reaction rate of GSH with NDA were compared in tris(hydroxymethyl)aminomethane (Tris) buffer solution at pH 7.4 or 9.2 and borate-Tris buffer solution at pH 9.2. The results revealed that the NDA-GSH sensitivity was the highest and the reaction rate of GSH and NDA was the fastest in borate buffer solution at pH 9.2. The effects of the four additives on the sensitivity of NDA-GSH were further compared. The best additive was revealed to be β-cyclodextrin (β-CD). GSH reacted with NDA to reach equilibrium within 5 min under the optimal experimental conditions, and the electrophoretic signal of NDA-GSH could be seen in 3 min. Quantitative analysis of GSH in HepG2 cells was performed using an external standard approach by determining a series of GSH standard solutions. The results revealed that the approach had a good linear relationship with the peak area vs. concentration (0.01-20.00 mmol/L) of GSH. The limit of detection (LOD) and limit of quantification (LOQ) of GSH were determined using signal-to-noise ratios of 3 (S/N=3) and 10 (S/N=10), which were 0.006 μmol/L and 0.020 μmol/L, respectively. The approach’s spiked recoveries were 95.7%-112.6%, with relative standard deviations of the approach being 3.8%-5.0% (n=3). This approach offers high sensitivity, good stability, accuracy, and reliability. To study the relationship between the toxicity of arsenic and chromium on HepG2 cells and the content of GSH in HepG2 cells, the effects of arsenic and chromium with different valences on cell viability were analyzed. The results illustrated that the cytotoxicity of potassium dichromate (Cr(Ⅵ)) was the strongest. The variations of GSH content in HepG2 cells stimulated with arsenite (As(Ⅲ)), arsenate (As(Ⅴ)), chromium chloride (Cr(Ⅲ)), and Cr(Ⅵ) were analyzed by the proposed approach and analysis of intracellular GSH imaging. The results revealed that the stimulation group i. e. analyzed doses (low-dose 2 mg/L, high-dose 5 mg/L) of As(Ⅲ), As(Ⅴ), and Cr(Ⅲ) had no obvious effect on GSH content in HepG2 cells compared with the control group, whereas high-dose Cr(Ⅵ) can significantly reduce GSH content in HepG2 cells. Considering the analysis of cytotoxicity of As(Ⅲ), As(Ⅴ), Cr(Ⅲ), and Cr(Ⅵ), it shows that the content of GSH in HepG2 cells is related to cytotoxicity, and the content of GSH will decrease with the increase in cytotoxicity.