Garcia Solange Cristina, Schott Karen, Charão Mariele, Moro Angela, Bulcão Rachel, Grotto Denise, Valentini Juliana, Bohrer Denise, Cardoso Simone, Pomblum Valdeci
Laboratory of Toxicology (LATOX), Department of Clinical and Toxicology Analysis, Center of Healthy Sciences, Federal University of Santa Maria, 97105-900, Santa Maria, RS, Brazil.
Biomed Chromatogr. 2008 May;22(5):460-8. doi: 10.1002/bmc.954.
Reduced glutathione (GSH) is a well-known multifunctional antioxidant. Its depletion is linked to a number of pathologies, such as renal insufficiency. Feasible methodologies in clinical chemistry are vital. Therefore a methodology for GSH quantification was optimized and validated by HPLC-UV. Important aspects such as acid deproteinization and GSH stability were established. The erythrocytes were hemolyzed, deproteinized, derivatized with 5,5-dithio-bis (2-nitrobenzoic) acid and analyzed using HPLC, on an RP18 gradient elution, lambda=330 nm. The method was applied to hemodialysis patients (n=75) compared with healthy subjects (n=40). The assay was linear from 0.5 to 3.0 mm (r2>0.99). The intra- and inter-run reproducibilities were obtained with CV%<10%. The accuracy (bias %) ranged from 1.32 to -6.38%, and the recovery was >94%. The derivatized sample was stable for 60 days at -20 degrees C. The GSH levels in hemodialysis patients showed a significant increase compared with healthy subjects (p<0.05) and an inverse correlation with age (r=-0.286; p=0.013) was found. This method used UV detection, reduction of the phosphate concentration in the mobile phase and effective protein removal with trichloroacetic acid. The method proved to be reproducible, precise, accurate and stable. Thus, it can be suggested for routine laboratory tests for the verification of physiopathological conditions.
还原型谷胱甘肽（GSH）是一种著名的多功能抗氧化剂。其消耗与许多病理状况有关，如肾功能不全。临床化学中可行的方法至关重要。因此，通过高效液相色谱 - 紫外检测法（HPLC - UV）对GSH定量方法进行了优化和验证。确定了诸如酸脱蛋白和GSH稳定性等重要方面。将红细胞进行溶血、脱蛋白，用5,5 - 二硫代双（2 - 硝基苯甲酸）进行衍生化，然后使用HPLC在RP18梯度洗脱、波长λ = 330 nm条件下进行分析。该方法应用于75例血液透析患者，并与40例健康受试者进行比较。该检测方法在0.5至3.0 mmol范围内呈线性（r2>0.99）。批内和批间重复性的变异系数（CV%）<10%。准确度（偏差%）范围为1.32%至 - 6.38%，回收率>94%。衍生化后的样品在 - 20℃下可稳定保存60天。血液透析患者的GSH水平与健康受试者相比显著升高（p<0.05），并且发现与年龄呈负相关（r = - 0.286；p = 0.013）。该方法采用紫外检测，降低流动相中的磷酸盐浓度，并使用三氯乙酸有效去除蛋白质。该方法被证明具有可重复性、精确性、准确性和稳定性。因此，可推荐用于常规实验室检测以验证生理病理状况。
