Original articleProtein oxidation in Type 2 diabetic patients on hemodialysis
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.
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