Original article
Protein oxidation in Type 2 diabetic patients on hemodialysis

https://doi.org/10.1016/j.jdiacomp.2004.11.001Get rights and content

Abstract

Background

Oxidative stress is considered to be a unifying link between diabetes mellitus (DM) and its complications, including nephropathy. There have been many reports on increased production of oxidants and decreased level of antioxidants in diabetic patients. The dialysis procedure contributes to oxidative stress. An increase in oxidative stress may contribute to the development of oxidative protein damage in diabetic patients. Our aim was to reveal the effects of diabetes and hemodialysis (HD) on oxidative modifications of plasma proteins.

Methods

We measured reactive carbonyl derivates (PCO), protein thiol (P-SH), and reduced glutathione (GSH) levels in Type 2 diabetic (DM) and diabetic hemodialysed patients (DHD) and in healthy control participants.

Results

Protein carbonyl (PCO) content increased significantly in all patient groups relative to the controls. The dialysis procedure caused an additional increase in PCO levels in DHD patients before and after dialysis compared with the level in DM patients. There was a significant decrease in P-SH levels in DHD patients compared with the level in healthy participants and DM patients. There was no significant difference in the whole blood GSH levels between the DM patients and control participants. It was significantly higher in DHD patients in comparison to the DM patients.

Conclusions

We conclude that PCO level increases in DM patients, and this increase is more profound in DHD patients, indicating that both diabetes and dialysis contribute to increased protein oxidation. The low P-SH level in DHD patients, but not in DM patients, suggests that dialysis is responsible for this decrease. We propose plasma PCO derivate as a novel specific marker for oxidative protein damage.

Introduction

Type 2 diabetes is an increasing problem worldwide. The prevalence of the disease has been increasing steadily over the last 30 years and is still rising. In the developed world, the prevalence of diagnosed diabetes is approximately 2–3%, and a similar number of people are estimated to have undiagnosed disease (Grimaldi et al., 2000). About 40% of Types 1 and 2 diabetic patients develop diabetic nephropathy and retinopathy as microangiopathy, in addition to cardiovascular complications in the long-term course of their disease (Morbidity in 565 Type 2 Diabetic Patients According to Stage of Nephropathy, Schleiffer, Holken, & Brass, 1998). Oxidative stress has been defined as a loss of balance between free radical production and the antioxidant systems, with negative effects on carbohydrates, lipids, and proteins (Dursun et al., 2002, Klemm et al., 2001, Morena et al., 2002). Oxidative stress has been suggested in diabetes mellitus (DM) and uremic patients on maintenance hemodialysis (HD). The relationship between oxidative stress and diabetic complications has been extensively investigated (Nacitarhan et al., 1995, Ozben et al., 1995). There are a number of hypotheses on the origin of diabetic complications. Oxidative stress is widely thought to play a crucial role in the pathogenesis and progression of late macro- and microangiopathic complications in DM (Morcos et al., 2001, Paolisso & Giugliano, 1998). Although several lines of evidence have suggested that poor glycemic control undoubtedly plays a significant role in diabetic nephropathy, the metabolic events responsible for its development are not well understood (Suzuki & Miyata, 1999).

Increased oxidative stress in diabetic patients may lead to protein oxidation (Telci et al., 2000). The conversion of proteins to protein carbonyl (PCO) derivatives occurs via direct oxidation by reactive oxygen species (ROS), with the eventual formation of oxidized amino acids (Carrard et al., 2002, Stadtman & Levine, 2000). Proteins are also modified indirectly with the reactive carbonyl compounds formed by the autoxidation of carbohydrates and lipids, with the eventual formation of the advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs; Inagi & Miyata, 1999, Miyata et al., 2000, Yegin & Ozben, 1995). In vivo and in vitro studies indicate that AGEs resulting from oxidative and carbonyl stress have a vital role in the pathogenesis of diabetic nephropathy and the progression of renal failure.

Oxidative protein damage cannot be repaired, except for the oxidation of methionine and cysteine (Stadtman, 2002). These oxidations cause irreversible modifications in proteins. The structure and activity of oxidized proteins change profoundly in comparison with their native forms. Oxidative modification of proteins in vivo may affect a variety of cellular functions. The best marker for intracellular oxidative stress-dependent cellular damage is the PCO content. The unique advantage of the carbonyl measurement as a good marker of oxidative stress is the fact that it covers a much wider range of oxidative damage than other markers do (Cakatay et al., 2003, Carrard et al., 2002, Evans et al., 1999, Reznick & Packer, 1994). The other markers, such as nitrotyrosine, hydroxylation of aromatic, and hydrophobic amino acids, are at a very low level in comparison with the carbonyl content.

Protein thiol (P-SH) groups and tripeptide glutathione (GSH) are particularly important for antioxidative defense in the cells. Reduced glutathione (GSH) is the most widespread cellular thiol compound and an important intracellular antioxidant (Stadtman & Levine, 2000). Free radicals may cause the oxidation of P-SH groups. P-SH groups in proteins may serve an antioxidant function by several mechanisms. P-SH may scavenge oxidants, thus sparing antioxidants and/or cellular constituents from attack (Cakatay et al., 2003, Dubey et al., 1996, Takenaka et al., 1991). Therefore, the measurement of sulphydryl groups in proteins, as well as PCO content, may be useful.

The aim of our study was to reveal oxidative modifications of plasma proteins by measuring DNPH-reactive carbonyl derivates (PCO) and P-SH levels in DM and diabetic hemodialysed (DHD) patients. Reduced glutathione was also measured to show the effect of diabetes and dialysis. The effect of dialysis procedure on these parameters was also investigated in specimens taken before and after a single dialysis treatment.

Section snippets

Participants

The assays were performed in four groups as follows:

  • Group 1: age- and sex-matched healthy individuals (n=20). Only those who proved to be in a good state of health and free from any signs of chronic disease, by a careful clinical examination and biochemical and hematological laboratory tests, were included in the study. They were nonsmokers and did not consume alcohol regularly. The intake of analgesics or antiinflammatory drugs was cut a few weeks before blood sampling, and they were not

Results

PCO content increased significantly in DM patients relative to the controls (P<.05). There was an additional increase in DHD patients before and after HD compared with the DM patients and control participants (P<.05). Dialysis procedure contributed to the increase in PCO levels, and the highest PCO level was observed in the DHD patients after dialysis (Fig. 1).

There was no significant difference in the plasma P-SH levels between DM patients and control participants. In contrast, a significant

Discussion

Our data confirm that plasma carbonyl level is a relevant marker of protein oxidation in both DM and diabetic HD patients. Our data are in agreement with other investigators and confirm the presence of increased oxidative protein damage in DM patients with and without nephropathic complications (Aso et al., 2000, Bhatia et al., 2003, Danielski et al., 2003, Miyata & Kurokawa, 2003, Telci et al., 2000). Increased oxidative stress in these patients is the most potential cause of protein oxidation

Acknowledgments

This work was supported by the Research Fund of Akdeniz University.

References (32)

  • S. Nacitarhan et al.

    Serum and urine malondialdehyde levels in NIDDM patients with and without hyperlipidemia

    Free Radical Biology & Medicine

    (1995)
  • A.Z. Reznick et al.

    Oxidative damage to proteins: Spectrophotometric method for carbonyl assay

    Methods in Enzymology

    (1994)
  • T. Schleiffer et al.

    Morbidity in 565 type 2 diabetic patients according to stage of nephropathy

    Journal of Diabetes and its Complications

    (1998)
  • E.R. Stadtman

    Importance of individuality in oxidative stress and aging

    Free Radical Biology & Medicine

    (2002)
  • Y. Takenaka et al.

    The effect of alpha-tocopherol as an antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites

    Archives of Biochemistry and Biophysics

    (1991)
  • Y. Aso et al.

    Serum concentrations of advanced glycation endproducts are associated with the development of atherosclerosis as well as diabetic microangiopathy in patients with type 2 diabetes

    Acta Diabetologica

    (2000)
  • Cited by (39)

    • A rhodol-derived probe for intracellular biothiols imaging and rapid labelling of sulfhydryl-containing proteins

      2022, Sensors and Actuators B: Chemical
      Citation Excerpt :

      Biological thiols, including low molecular weight thiols and thiol-containing proteins, are crucial to biological and physiological processes, such as protein folding, detoxification, signal transduction, and metabolism in cellular functions [1–3]. Furthermore, thiol oxidation has been reported as the potential cause of many diseases, including diabetes mellitus, Parkinson’s disease, and Alzheimer’s disease [4–6]. Therefore, accurate, sensitive and quantitative approaches for detecting biothiols, especially thiol-containing proteins, are strongly required and of great significance for diseases diagnosis and treatment.

    • In vitro upregulation of erythrocytes glucose uptake by Rhaphnus sativa extract in diabetic patients

      2012, Biochimie
      Citation Excerpt :

      These consequences of oxidative stress can promote the development of complications of DM [6,28]. Many studies showed that, the biomarkers of oxidative stress increase in diabetes and its complications [29–31]. MDA and conjugated dienes are the two markers used widely.

    • Oxidative stress biomarkers and chromogranin A in uremic patients: Effects of dialytic treatment

      2010, Clinical Biochemistry
      Citation Excerpt :

      Dialysis treatment does not significantly modify plasma levels of oxidative stress biomarkers as compared with uremic patients, indicating that dialysis treatment is not the major determinant of oxidative stress in uremic patients. In fact, despite dialysis treatment is generally considered ‘per se’ a cause of increased glycation end products, oxidative stress and generalized multi-organ stress [22,23], we found no differences in plasma oxidative stress biomarker levels between uremic patients treated or not treated with dialysis. In our study we have found an increase in protein and DNA oxidation, indicated by –SH group reduction and by increased 8-OHdG serum levels, respectively, while no differences were observed in LDL oxidation.

    • Oxidative stress parameters as possible urine markers in patients with diabetic nephropathy

      2009, Journal of Diabetes and its Complications
      Citation Excerpt :

      A significant decrease in plasma thiol levels was found in diabetic patients on hemodialysis compared to control participants and patients with DM who have no macro- and microvascular complications. Increased plasma thiol levels have been observed after a single dialysis session in these patients, but the level was still lower than in healthy participants and patients with DM (Dursun, Dursun, Suleymanlar, & Ozben, 2005; Dursun, Timur, Dursun, Suleymanlar, & Ozben, 2005). A similar result after dialysis was reported by Jackson, Loughrey, Lightbody, McNamee, and Young (1995).

    View all citing articles on Scopus
    View full text