Glutathione Detection Method Based on Electron Paramagnetic Resonance Spectroscopy
10.16476/j.pibb.2024.0097
- VernacularTitle:基于电子顺磁共振技术的谷胱甘肽检测方法
- Author:
Zhi-Wen WANG
1
;
Jian KUANG
1
;
Ao-Kun LIU
2
;
Ruo-Tong WEI
2
;
Lu YU
2
;
Chang-Lin TIAN
1
Author Information
1. Department of Chemical Physics, University of Science and Technology of China, Hefei230026, China
2. High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei230031, China
- Publication Type:Journal Article
- Keywords:
glutathione assay;
electron paramagnetic resonance;
ABTS
- From:
Progress in Biochemistry and Biophysics
2024;51(11):3034-3045
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveGlutathione (γ-glutamyl-L-cysteinylglycine, GSH) is the most abundant non-protein compound containing sulfhydryl (―SH) groups in cells. It serves as a source of reducing equivalents, effectively neutralizing harmful reactive substances, and playing a crucial role in maintaining cellular redox balance. Therefore, sensitive detection and accurate measurement of GSH levels in tissues are of great importance. In this work, we presents a novel method for GSH detection utilizing electron paramagnetic resonance (EPR) spectroscopy. MethodsInitially, ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate acid)) solution was mixed with K2S2O8 solution and reacted in the dark for 12 to 16 h to prepare ABTS·+ solution, which was then quantified using UV-Vis spectroscopy. Subsequently, the concentration of glutathione (GSH) was determined based on the changes in the EPR signal of ABTS·+. On this basis, the optimal reaction time and temperature were explored to establish a standard equation correlating the EPR signal intensity of ABTS·+ with GSH concentration. Finally, the derived standard curve was employed to quantitatively analyze the GSH concentration in whole blood from C57BL/6J mice, and the results were compared with those reported in the literature to verify the accuracy of the method. ResultsThe experimental results demonstrate that this method has a linear detection range from50 nmol/L to 15 μmol/L for GSH, spanning two orders of magnitude, with a limit of detection (LOD) at0.50 nmol/L. The measured GSH content in mouse whole blood is (10 660±706) nmol/g Hb, which agrees with the value of (11 200±237) nmol/g Hb as previously reported. Furthermore, a similar method was developed for detection of glutathione disulfide (GSSG) at higher reaction temperature. ConclusionThis article presents a novel assay for the rapid detection of GSH using the intensity of EPR signal from ABTS·+ as indicator. This method demonstrates enhanced detection sensitivity and a broader linear range compared to conventional colorimetric methods. Furthermore, we have extended the application of this method to detect GSH content in blood samples efficiently and accurately, offering valuable information for assessing tissue redox balance, thus holding significant potentials.
