A colorimetric method to measure oxidized, reduced and total glutathione levels in erythrocytes

Abstract Background The most important antioxidant for erythrocytes is glutathione. In this study, a non-enzymatic recycling spectrophotometric method was developed to measure oxidized and reduced glutathione (GSH) levels in erythrocytes. The newly developed method consists of two steps. In the first step, the levels of GSH were determined in the sample. Subsequently, total glutathione levels were measured by reducing the oxidized glutathione (GSSG) with sodium borohydride (NaBH4). Half of the difference between total glutathione and GSH gives the GSSG. Results The new method was linear between 0 and 3000 μmol/L (r2 = 0.999). The percentage recovery measured by the developed method was 100.2 ± 2.4%. The total precision of the total glutathione, GSH and GSSG was 1.26%, 1.02% and 6.65%, respectively. GSH levels were significantly lower in patients with type 2 diabetes mellitus (DM), while GSSG levels were significantly higher (p < 0.05). Hemoglobin A1c (HbA1c) levels were negatively correlated with GSH levels, whereas positively correlated with GSSG (p < 0.05). Conclusions In conclusion, this method is easy to apply in routine practice with high accuracy, precision and reproducibility.

[1]  Shiwen Xu,et al.  Oxidative stress, inflammation, and glycometabolism disorder‐induced erythrocyte hemolysis in selenium‐deficient exudative diathesis broilers , 2019, Journal of cellular physiology.

[2]  E. A. de Almeida,et al.  Prolonged erythrocyte auto-incubation as an alternative model for oxidant generation system. , 2019, Toxicology in vitro : an international journal published in association with BIBRA.

[3]  A. Holmgren,et al.  Enzymatic glutaredoxin-dependent method to determine glutathione and protein S-glutathionylation using fluorescent eosin-glutathione. , 2019, Analytical biochemistry.

[4]  H. Stenlund,et al.  A standardized protocol for comparable analysis of GSH/GSSG by UHPLC-ESI-MSMS for human plasma. , 2019, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[5]  Min Liu,et al.  Superwettable Microwell Arrays Constructed by Photocatalysis of Silver-Doped-ZnO Nanorods for Ultrasensitive and High-Throughput Electroanalysis of Glutathione in Hela Cells. , 2018, ACS applied materials & interfaces.

[6]  F. Laurindo,et al.  Implications of plasma thiol redox in disease. , 2018, Clinical science.

[7]  F. Bennett,et al.  Glutathione metabolism in type 2 diabetes and its relationship with microvascular complications and glycemia , 2018, PloS one.

[8]  I. Clarot,et al.  Glutathione: Antioxidant Properties Dedicated to Nanotechnologies , 2018, Antioxidants.

[9]  S. Hashemy,et al.  Evaluation of serum levels of oxidized and reduced glutathione and total antioxidant capacity in patients with head and neck squamous cell carcinoma , 2018, Journal of cancer research and therapeutics.

[10]  T. Lőrincz,et al.  The Problem of Glutathione Determination: a Comparative Study on the Measurement of Glutathione from Plant Cells , 2018 .

[11]  Pablo Cannata,et al.  Role of glutathione biosynthesis in endothelial dysfunction and fibrosis , 2017, Redox biology.

[12]  Zhongming Huang,et al.  A facile label-free colorimetric method for highly sensitive glutathione detection by using manganese dioxide nanosheets , 2017 .

[13]  R. Compton,et al.  Rapid Method for the Quantification of Reduced and Oxidized Glutathione in Human Plasma and Saliva. , 2017, Analytical chemistry.

[14]  M. Comini.  Measurement and meaning of cellular thiol:disufhide redox status , 2016, Free radical research.

[15]  O. Erel,et al.  A novel and automated assay for thiol/disulphide homeostasis. , 2014, Clinical biochemistry.

[16]  J. Winther,et al.  Quantification of thiols and disulfides. , 2014, Biochimica et biophysica acta.

[17]  G. Wittmann,et al.  Determination of glutathione and glutathione disulfide in biological samples: an in-depth review. , 2009, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[18]  X. Guan,et al.  Determination of thiols and disulfides via HPLC quantification of 5-thio-2-nitrobenzoic acid. , 2008, Journal of pharmaceutical and biomedical analysis.

[19]  J. Winther,et al.  Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4'-dithiodipyridine. , 2007, Analytical biochemistry.

[20]  M. Rema,et al.  Increased glutathionylated hemoglobin (HbSSG) in type 2 diabetes subjects with microangiopathy. , 2005, Clinical biochemistry.

[21]  G. Pes,et al.  Plasma thiols redox status by laser‐induced fluorescence capillary electrophoresis , 2004, Electrophoresis.

[22]  E. Camera,et al.  Analytical methods to investigate glutathione and related compounds in biological and pathological processes. , 2002, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[23]  D. Giustarini,et al.  Blood glutathione disulfide: in vivo factor or in vitro artifact? , 2002, Clinical chemistry.

[24]  G. Koren,et al.  Glutathione Stability in Whole Blood: Effects of Various Deproteinizing Acids , 2001, Therapeutic drug monitoring.

[25]  A. Proietti,et al.  Reduction of oxidative stress by oral N-acetyl-L-cysteine treatment decreases plasma soluble vascular cell adhesion molecule-1 concentrations in non-obese, non-dyslipidaemic, normotensive, patients with non-insulin-dependent diabetes , 1998, Diabetologia.

[26]  C. Spickett,et al.  Erythrocyte glutathione balance and membrane stability during preeclampsia. , 1998, Free radical biology & medicine.

[27]  E. Flagg,et al.  Glutathione in human plasma: decline in association with aging, age-related macular degeneration, and diabetes. , 1998, Free radical biology & medicine.

[28]  J. Viña,et al.  Blood glutathione as an index of radiation-induced oxidative stress in mice and humans. , 1997, Free radical biology & medicine.

[29]  C. Sergeant,et al.  The enzymatic antioxidant system in blood and glutathione status in human immunodeficiency virus (HIV)-infected patients: effects of supplementation with selenium or beta-carotene. , 1996, The American journal of clinical nutrition.

[30]  M. Hu,et al.  Measurement of protein thiol groups and glutathione in plasma. , 1994, Methods in enzymology.

[31]  H. Sies,et al.  Identification and quantitation of glutathione in hepatic protein mixed disulfides and its relationship to glutathione disulfide. , 1983, Biochemical pharmacology.

[32]  F. Tietze.  Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. , 1969, Analytical biochemistry.

[33]  H Güntherberg,et al.  The true oxidized glutathione content of red blood cells obtained by new enzymic and paper chromatographic methods. , 1966, Analytical biochemistry.

[34]  R. Belcher,et al.  A COLORIMETRIC MICRO-METHOD FOR THE DETERMINATION OF GLUTATHIONE. , 1965, The Biochemical journal.

[35]  G. Ellman,et al.  Tissue sulfhydryl groups. , 1959, Archives of biochemistry and biophysics.