谷胱甘肽和谷胱甘肽二硫化物含量的灵敏酶循环测定

IF 6 2区化学 Q1 CHEMISTRY, ANALYTICAL

Analytica Chimica Acta Pub Date : 2025-06-20 DOI:10.1016/j.aca.2025.344348

Xinyue Lin , Yajun Chu , Qianhe Xu , Qiuying Nie , Cui Xu , Jianguo Fang , Junmin Zhang

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引用次数: 0

摘要

开发可靠的技术来准确检测和测量生物样品中谷胱甘肽（GSH）和谷胱甘肽二硫（GSSG）的含量是至关重要的。然而，目前对于制备和分析样品以获得准确定量结果的最佳方法尚未完全达成共识。结果筛选了n -乙基马来酰亚胺（NEM）作为掩蔽剂，5-磺基水杨酸（SSA）作为蛋白质去除剂，建立了测定GSH和GSSG的标准、稳定的方法。随后，该方法在细胞、小鼠血液和组织中得到验证。采用标准浓度曲线测定GSH抑制剂丁硫氨酸亚砜（BSO）治疗前后HeLa和LO2细胞中GSH和GSSG的水平。结果表明，与BSO孵育后，总GSH和GSH/GSSG比值显著低于对照组。同样，BSO验证也已成功应用于小鼠组织和血液的定量。与BSO孵育3 h后，肝脏和肾脏中GSH含量显著降低，小鼠血浆中GSH浓度约为50 μM。本工作展示了一种灵敏、准确的酶循环检测细胞、组织和血液中GSH和GSSG含量的方法，有助于建立不同样品类型中GSH与GSSG比例的共识。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

Sensitive enzymatic cycling assay for glutathione and glutathione disulfide content

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Sensitive enzymatic cycling assay for glutathione and glutathione disulfide content

Background

It is crucial to develop reliable techniques for accurately detecting and measuring glutathione (GSH) and glutathione disulfide (GSSG) content in biological samples. However, there is currently not yet a full consensus on the best approach for preparing and analyzing samples to obtain accurate quantitative results.

Results

This study screened N-ethylmaleimide (NEM) as a masking agent and 5-sulfosalicylic acid (SSA) as a protein removal agent to establish a standard and stable method for GSH and GSSG determination. Subsequently, the method was validated in cells, mouse blood and tissues. Standard concentration curves were used to determine the levels of GSH and GSSG in HeLa and LO2 cells before and after treatment with the GSH inhibitor, buthionine sulfoximine (BSO). The results showed that after incubation with BSO, the total GSH and GSH/GSSG ratios were significantly lower than those in the control group. Likewise, BSO verification has also been successfully applied to the quantification of mouse tissue and blood. After incubation with BSO for 3 h, the GSH content in the liver and kidneys significantly decreased, and the GSH concentration in mouse plasma was about 50 μM.

Significance

This work demonstrates a sensitive and accurate enzymatic cycling method for the detection of GSH and GSSG content in cells, tissues and blood, helping to build consensus on GSH to GSSG ratios in different sample types.

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来源期刊

Analytica Chimica Acta 化学-分析化学

CiteScore

10.40

自引率

6.50%

发文量

1081

审稿时长

38 days

期刊介绍： Analytica Chimica Acta has an open access mirror journal Analytica Chimica Acta: X, sharing the same aims and scope, editorial team, submission system and rigorous peer review. Analytica Chimica Acta provides a forum for the rapid publication of original research, and critical, comprehensive reviews dealing with all aspects of fundamental and applied modern analytical chemistry. The journal welcomes the submission of research papers which report studies concerning the development of new and significant analytical methodologies. In determining the suitability of submitted articles for publication, particular scrutiny will be placed on the degree of novelty and impact of the research and the extent to which it adds to the existing body of knowledge in analytical chemistry.