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

Murat Alisik,Salim Neselioglu,Ozcan Erel

doi:10.1515/labmed-2019-0098

Murat Alisik, Salim Neselioglu + Show 1 more

Open Access

https://doi.org/10.1515/labmed-2019-0098

Copy DOI

Abstract

AbstractBackgroundThe 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.ResultsThe 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).ConclusionsIn conclusion, this method is easy to apply in routine practice with high accuracy, precision and reproducibility.

Highlights

Glutathione, apart from being an important antioxidant, is the main low-molecular-weight thiol-containing peptide with its cysteine residue present in most living cells
This study aimed to develop a spectrophotometric method based on sodium borohydride (NaBH4) as a reductant
20% trichloroacetic acid (TCA) was used and this provides the acidity of the supernatant

Summary

Introduction

Glutathione, apart from being an important antioxidant, is the main low-molecular-weight thiol-containing peptide with its cysteine residue present in most living cells. Its. Erythrocytes are exposed to oxidative stress from a wide range of sources compared to other somatic cells. Erythrocytes are exposed to oxidative stress from a wide range of sources compared to other somatic cells As they are O2-transferring cells that contain hemoglobin, they produce O2− and H2O2 with continuous autoxidation [3]. Measuring GSH and GSSG levels of erythrocytes has been a topic of interest all the time. The most important antioxidant for erythrocytes is glutathione. A non-enzymatic recycling spectrophotometric method was developed to measure oxidized and reduced glutathione (GSH) levels in erythrocytes. Total glutathione levels were measured by reducing the oxidized glutathione (GSSG) with sodium borohydride (NaBH4). The total precision of the total glutathione, GSH and GSSG was 1.26%, 1.02% and 6.65%, respectively. Conclusions: In conclusion, this method is easy to apply in routine practice with high accuracy, precision and reproducibility

Objectives

Methods

Results

Discussion

Conclusion