Elsevier

Clinical Biochemistry

Volume 38, Issue 10, October 2005, Pages 892-899
Clinical Biochemistry

Increased glutathionylated hemoglobin (HbSSG) in type 2 diabetes subjects with microangiopathy

https://doi.org/10.1016/j.clinbiochem.2005.06.009Get rights and content

Abstract

Objective:

Protein glutathionylation is considered an important post-translational modification in the pathogenesis of complex diseases. The aim of this study was to examine whether hemoglobin (Hb) is modified by reduced glutathione (GSH) via oxidation of the thiol groups present in diabetes and its associated microangiopathy and to determine whether oxidative imbalance has any correlation with glutathionylated Hb (HbSSG) levels.

Methods:

The study group consisted of a total of 130 subjects which included non-diabetic healthy control subjects (n = 30) and type 2 diabetic patients with (n = 53) and without (n = 47) microangiopathy. All subjects were assessed for glycemic and lipidemic status, while diabetic subjects were also assessed for the diagnosis of retinopathy and nephropathy. RBC lysates from all the subjects were analyzed by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) for HbSSG β-globin chains. Levels of GSH and thiobarbituric acid substances (TBARS) levels were measured by spectrophotometric and fluorimetric methods, respectively.

Results:

The positivity for HbSSG in diabetic subjects with microangiopathy was significantly higher (69%) compared to diabetics without microangiopathy (22%) and control subjects (14%). In univariate regression analysis, HbSSG levels were significantly associated with the duration of diabetes, HbA1c, and TBARS levels. GSH levels were negatively correlated (r = −0.57, P < 0.001) with HbSSG in diabetic subjects. A significant inverse correlation (r = −0.42, P < 0.001) between the GSH levels and HbA1c levels was also seen in diabetic subjects.

Conclusions:

This is perhaps the largest LC-MS-based study to demonstrate that HbSSG levels are markedly increased in diabetic subjects with microangiopathy. Since diabetic subjects also exhibited increased lipid peroxidation and decreased GSH levels, it appears that enhanced oxidative stress may account for the increased HbSSG concentrations and altered reduction–oxidation (redox) signaling.

Introduction

Several experimental, epidemiological, and clinical studies support the view that oxidative stress plays a significant role in the development of vascular complications in diabetics [1], [2]. The importance of reactive oxygen species (ROS) in the pathogenesis of diabetes and its complications are well recognized and a considerable body of evidence implicates ROS in defective cellular signaling [3], [4]. However, the molecular mechanisms by which ROS alter cellular signaling are not well understood. Reactive thiols on cysteine (Cys) residues of selected proteins are among the most sensitive sites to be modified by ROS. Oxidants react with these redox-sensitive thiols to form thiyl radicals, which subsequently react with other thiols to form mixed-disulfide bonds. In mammalian cells, glutathione is the most abundant low molecular thiol, and hence the most likely to bind to protein thiols to form mixed-disulfides, a process termed S-glutathionylation [5], [6].

Since a number of functionally critical proteins within the cell possess accessible Cys residues, glutathionylation may be considered an important post-translational modification in the pathogenesis of complex diseases such as diabetes. Therefore, we hypothesize that, under conditions of increased oxidative stress and changes in glutathione levels, many critical proteins are liable to undergo glutathionylation in patients with diabetes and its associated complications. In recent years, glutathionylated proteins including HbSSG have been investigated as possible biomarkers of oxidative stress using the HPLC or LC-MS detection systems [6], [7], [8], [9], [10]. Since the blood concentrations of glutathionylated proteins may reflect alterations in redox signaling and oxidation status [10], [11], in the present study, we have used erythrocytes as a cellular model to test HbSSG as a possible biomarker of increased oxidative stress.

Section snippets

Methods

The study group was comprised of a total of 130 subjects, which included non-diabetic healthy control subjects (n = 30) and type 2 diabetic patients with (n = 53) and without (n = 47) microangiopathy. The diabetic subjects were selected from out-patients attending the Dr. Mohans' M.V Diabetes Specialties Centre (MVDSC), a tertiary referral center for diabetes care at Chennai (formerly Madras) in Southern India. Control subjects were recruited from the on-going Chennai Urban Rural

Results

Table 1 shows the clinical characteristics of the study groups. Diabetic subjects without microangiopathy had higher systolic blood pressure compared to control subjects. Diabetic subjects with microangiopathy had significantly higher systolic and diastolic blood pressures, serum triglyceride, and LDL-C levels compared to control subjects.

Fig. 1a shows an ESI mass spectrum of a non-diabetic (control) subject's hemoglobin. Deconvolution of the charge states (transformed mass scale) distinctively

Discussion

Currently, there is a growing awareness of the importance of oxidants as signaling molecules that directly impact the function of tissues in a regulatory fashion by altering the structure of proteinaceous cysteinyl thiols. Multiple modes of protein–cysteine oxidation, such as S-thiolation, S-nitrosylation, sulfenic acid, sulfinic acid, sulfenyl-amide formation, reactive oxidized lipid derivates, and intra- and intermolecular protein disulfides are already known to be important in redox

Acknowledgments

This work was supported by a research grant from the Department of Science and Technology (DST), New Delhi, India. The mass spectrometry facilities at the Indian Institute of Science are funded by a program support grant from the Department of Biotechnology, India. S.S. acknowledges a postdoctoral fellowship from the Department of Biotechnology, India. We thank the Chennai Willington Corporation Foundation for their support for the CURES field studies. This is paper No. 15 from the CURES study.

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