Michaelsen Jan Thomas, Dehnert Sabine, Giustarini Daniela, Beckmann Bibiana, Tsikas Dimitrios
Institute of Clinical Pharmacology, Hannover Medical School, Hannover, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Oct 15;877(28):3405-17. doi: 10.1016/j.jchromb.2009.06.043. Epub 2009 Jul 4.
Glutathione exists in biological samples in the reduced form (GSH), as its disulfide (GSSG) and as a mixed disulfide (GSSR) with thiols (RSH). GSH is the most abundant low-molecular-mass thiol and plays important roles as a cofactor and as a main constituent of the intracellular redox status. Due to its own sulfhydryl (SH) group, GSH reacts readily with o-phthaldialdehyde (OPA) to form a highly stable and fluorescent isoindole derivative (GSH-OPA), which allows for sensitive and specific quantitative determination of GSH in biological systems by HPLC with fluorescence (FL) detection. In the present article we report on the utility of the novel, strongly disulfide bond-reducing thiol N-acetyl-cysteine ethyl ester (NACET) for the specific quantitative analysis of GSH and GSSG in the cytosol of red blood cells (RBC) as GSH-OPA derivative with FL (excitation/emission 338/458nm) or UV absorbance (338nm) detection. Unlike in aqueous solution, the derivatization of GSH in RBC cytosol yielded two closely related derivatives in the absence of NACET and only the GSH-OPA derivative in the presence of NACET. The HPLC method was optimized and validated for human RBC and applied to measure GSH and GSSG in RBC of healthy subjects. Basal GSH and GSSG concentrations were determined to be 2340+/-350microM and 11.4+/-3.2microM, respectively, in RBC of 12 healthy young volunteers (aged 23-38 years). The method was also applied to study the effects of nitrite on the glutathione status in intact and lysed human RBC. Nitrite at mM-concentrations caused instantaneous and considerable GSSG formation in lysed but much less pronounced in intact RBC. GSH externally added to lysed RBC inhibited nitrite-induced methemoglobin formation. Our findings suggest that nitric oxide/nitrite-related consumption rate of GSH, and presumably that of NADH and NADPH, could be of the order of 600micromol/day in RBC of healthy subjects.
谷胱甘肽在生物样品中以还原形式（GSH）、其二硫化物形式（GSSG）以及与硫醇（RSH）形成的混合二硫化物形式（GSSR）存在。GSH是最丰富的低分子量硫醇，作为辅因子和细胞内氧化还原状态的主要成分发挥着重要作用。由于其自身的巯基（SH）基团，GSH很容易与邻苯二甲醛（OPA）反应形成高度稳定的荧光异吲哚衍生物（GSH - OPA），这使得通过带有荧光（FL）检测的高效液相色谱法（HPLC）能够灵敏且特异定量测定生物系统中的GSH。在本文中，我们报道了新型的、具有强二硫键还原能力的硫醇N - 乙酰 - 半胱氨酸乙酯（NACET）用于红细胞（RBC）胞质溶胶中GSH和GSSG作为GSH - OPA衍生物的特异性定量分析，采用FL（激发/发射338/458nm）或紫外吸光度（338nm）检测。与在水溶液中不同，在无NACET时，RBC胞质溶胶中GSH的衍生化产生了两种密切相关的衍生物，而在有NACET时仅产生GSH - OPA衍生物。该HPLC方法针对人RBC进行了优化和验证，并应用于测量健康受试者RBC中的GSH和GSSG。在12名健康年轻志愿者（年龄23 - 38岁）的RBC中，基础GSH和GSSG浓度分别测定为2340±350μM和11.4±3.2μM。该方法还应用于研究亚硝酸盐对完整和裂解的人RBC中谷胱甘肽状态的影响。毫摩尔浓度的亚硝酸盐在裂解的RBC中导致瞬时且大量的GSSG形成，但在完整的RBC中则不太明显。向裂解的RBC中外部添加的GSH抑制了亚硝酸盐诱导的高铁血红蛋白形成。我们的研究结果表明，在健康受试者的RBC中，一氧化氮/亚硝酸盐相关的GSH消耗速率，可能还有NADH和NADPH的消耗速率，可能约为600μmol/天。
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