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Sensitive enzymatic cycling assay for glutathione and glutathione disulfide content
Xinyue Lin1, Yajun Chu1, Qianhe Xu1
1School of Pharmacy, and State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, 730000, China.
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.
