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Table of Content - Volume 11 Issue 3 - September 2019

 

A study on role of protein oxidation and lipid peroxidation as markers in NIDDM patients

 

M B C R Naidu1, K Sudheer2*, Pradeep Kumar Vegi3

 

1Associate Professor, 3Assistant Professor, Department of Biochemistry, GEMS, Srikakulam, Andhra Pradesh, INDIA.

2Associate Professor, Department of General Medicine, GEMS, Srikakulam, Andhra Pradesh, INDIA.

Email: rd@gems.edu.in

 

Abstract               Aim and Objective: The link between hyperglycemia, enhanced free radical activity, and the complications of diabetes is unknown. The purpose of this study is to evaluate the levels of Protein Carbonyl, malondialdehyde (MDA) measured as thio-barbituric acid-reactive malondialdehyde (MDA) measured as thio-barbituric acid-reactive substances (TBARS), an index of protein oxidation, lipid peroxidation and microalbumin in patients of type 2 diabetes without complications compare with normal subjects of the same population. Methodology: We recruited 60 type 2 diabetic subjects without complications and with poor metabolic control and 60 age-matched controls with good metabolic control. Levels of Protein Carbonyl, glucose, total cholesterol, HbA1C, and MDA as TBARS, micro-albumins were determined. Results: Diabetic patients had higher levels of blood glucose (P<0.001), HbA1C (P<0.001), Protein Carbonyl( P<0.001), microalbumin ( P<0.05)and MDA (P<0.001) than control subjects. The total cholesterol of the control subjects and diabetic patients did not differ. There was no correlation between the family history in diabetics and elevation in either HbA1C or MDA levels. Conclusion: To clarify the levels of protein oxidation markers such as protein carbonyl (PCO), increased levels of Protein Carbonyl, MDA may be a useful marker of oxidative stress. The enhanced lipid peroxidation and protein oxidation leads to an increase in free-radical activity in type 2 diabetics. This increase in free-radical activity in type 2 diabetes mellitus along with insulin resistance can lead to activation of stress-sensitive pathways, which may play an important role in the complications of diabetes.

Key Word: protein oxidation, lipid peroxidation.

 

INTRODUCTION

Diabetes mellitus is a chronic metabolic syndrome, lifelong progressive disease, caused by an absolute or relative insulin deficiency, and is characterized by high circulating glucose. The International Diabetes Federation estimates that 366 million people had diabetes in 2011, and that by 2030, this figure will have risen to a staggering 552 million worldwide1, 2. One of the more debilitating aspects of diabetes is the numerous complications that can arise from the disease. The chronic hyperglycemia of diabetes is associated with significant long-term sequel, particularly damage, dysfunction, and failure of various organs especially the kidneys, eyes, nerves, heart3,4 and blood vessels. There is considerable evidence that hyperglycemia results in the generation of reactive oxygen species (ROS), ultimately leading to increased oxidative stress in a variety of tissues, and playing an important role in diabetic complications. Oxidative stress leads to protein, lipid, and DNA modifications that cause cellular dysfunction and this could have teratogenic or carcinogenic consequences 5- 8. Accumulation of protein carbonyls have been observed in many pathological conditions [9]. Protein carbonyl content is actually the most general indicator and by far the most commonly used marker for protein oxidation. The appearance of carbonyl groups such as aldehyde or ketone groups in proteins as the result of several oxidative modification reactions, Carbonylated proteins tend to be more hydrophobic and resistant to proteolysis10. Protein carbonyl is generated by oxidative modifications of proteins either by α-amidation pathway or by oxidation of glutamyl side chains10,11, reactions with aldehydes like malondialdehyde produced during lipid peroxidation, oxidation of reducing sugars and reaction of oxidized product with lysine residues of proteins12,13. The enhanced oxidative stress in hyperglycemia may modify endothelial function by variety of mechanisms leading to dysfunction14. This could contribute to the pathogenesis of microalbuminuria either directly by causing increase in glomerular pressure and the synthesis of a leaky glomerular basement membrane or indirectly by influencing glomerular mesangial and epithelial cell function in a paracrine fashion. Importantly the molecular pathways by which endothelial dysfunction causes microalbuminuria has yet to be worked out. So, high amount of carbonyl production resulting from oxidative stress may cause complications in diabetes16.

 

MATERIALS AND METHODS

The present study was carried out on the subjects of Out Patient Department of GEMS, Srikakulam. Age matched subjects were included for study. Present study comprised of two groups.

Study Design: A Case-control study consisting of 60 controls and 60 Type II Diabetes Mellitus patients is undertaken to investigate the effect of Type II DM on Clinical parameters namely, HbA1c, Cholesterol, TG, Serum Protein, Serum Albumin, TBARS and Protein Carbonyl. Diabetic Patients with atherosclerosis, chronic infections, and acute renal failure were excluded from the study. The healthy subject did not show any inflammatory conditions, abnormalities in lipid and carbohydrate metabolism or kidney disorders in routine medical checkups. 5 ml Fasting blood samples of patients and control were collected in plain bulb and Sodium fluoride bulb. Precaution was taken to avoid any traces of hemolysis. After an hour clear serum was separated by centrifugation at 3000 rpm for 5 min and serum samples were analyzed for HbA1c by ion exchange resin method, Total protein conc. by Biuret method and Protein carbonyl assay by Spectrophotometric DNPH meth17. Plasma samples were analyzed for blood glucose by routine GOD-POD method and Glycosylated Hemoglobin in hemolysate was determined by particle enhanced immunoturbidimetric method using commercial kits.

Statistical Methods: Student t test (unpaired) has been used to test the homogeneity of samples between controls and cases. Chi-square test has been used to find the homogeneity of samples based on sex. Two tailed Student t test has been used to find the significance of Anthropometry and Clinical parameters between Control and cases. The Effect Size has been used to find the effect of Type II DM on Clinical parameters when compared to Control. The Statistical software namely SPSS 11.0 and Systat 8.0 were used for the analysis of the data. The assay of carbonyl groups in proteins provides a convenient technique for detecting and quantifying oxidative modification of proteins. 2, 4- dinitrophenylhyrdazine (DNPH) reacts with protein carbonyls to produce hydrazones. Hydrazones can be detected spectrophotometrically at an absorbance of 370 nm or by fluorescence17.

 

RESULTS AND DISCUSSION

In normal individuals (control group) glucose mean level was 92.77 10.34mg/dl. In Type 2 diabetic patients the glucose was significantly raised being 178.75 38.30 mg/dl. The mean HbA1c in normal is 5.590.24mg/dl, while the mean value of HbA1c in Type 2 diabetic patients is 7.540.87 mg/dl. HbA1c slightly increases in Type 2 diabetic patients are given table I.

 

Table 1: Clinical characteristics of type 2 diabetic patients with health control of mean SD values

Clinical Parameters

Control (n=60)

Type 2 DM (n=60)

P- value

 

(Mean SD)

(Mean SD)

 

 

FBS (mg/dl)

92.7710.34

178.7538.30

0.000**

HbA1c

5.590.24

7.540.87

0.000**

Cholesterol (mg/dl)

181.8732.34

188.0720.90

0.215

Triglycerides (mg/dl)

119.9727.17

171.8519.21

0.000**

Serum HDL (mg/dl )

41.96 ± 2.58

40.08 ± 3.29

0.045

Serum LDL (mg/dl )

109.16 ± 25.54

128.08 ±29.09

0.001

Serum Protein (gm/dl)

6.610.46

6.570.50

0.608

Serum Albumin (gm/dl)

3.790.42

3.670.53

0.178

TBARS (nmole/dl)

55.6413.37

114.7011.69

0.000**

Protein carbonyl (nmole/ml)

12.172.72

21.763.35

0.000**

Micro albumin (mg/L)

15.91 ± 10.90

40.19 ± 29.18

0.05*

 


The elevated glucose levels can induce oxidative stress by various mechanisms including non-enzymatic, enzymatic and mitochondrial pathways. Non-enzymatically, hyperglycemia can directly cause increased ROS generation under autoxidation and generate OH∙ radicals18. This reacts with proteins to form AGEs which can generate ROS at various stages and enhance the metabolism of glucose through polyol pathway to produce O219. The enzymatic sources of enhanced generation of free radicals in diabetes include NOS, NADPH oxidase and xanthine oxidase. The mitochondrial electron transport chain at complex-II is another source of free radicals20. The increased oxidative stress acts on signal transduction pathways via NF-kB affects gene expression of antioxidant enzymes Hyperglycemia can also glycate the proteins and inactivate it. Thus hyperglycemia reduces the antioxidant potential. HbA1c has been thought to represent the average glycemia over the past six to eight weeks22,23. Glycation of hemoglobin occurs over the entire 120 days life span of RBCs24 but within these 120 days, recent glycemia has the largest influence on the HbA1c value 25. The HbA1c value >6.5 in study group patient show the poor glycaemic control, in spite of insulin treatment. The glycation of proteins like HbA1c and increased free radical generation could promote the development of further complications. Griesmacher et al25 showed that type 2 DM patients with HbA1c levels >6.5 % had a positive correlation between HbA1c and TBARS levels. However, such a correlation was not observed in the study group. Serum total Cholesterol, triglycerides, LDL-Cholesterol was 181.8732.34 mg /dl, 119.97 27.17 mg/dl and 109.16 ± 25.54 mg/dl. In Type 2 diabetic patients the mean levels of serum total Cholesterol was significantly raised, the value being 188.07 20.90 mg / dl, 171.8519.21 mg/dl and 128.08 ± 29.09 mg/dl. The HDL Cholesterol mean level in normal individuals was 41.96 ± 2.58. In Type 2 diabetic patients the HDL- Cholesterol was 40.08 ± 3.29 significantly decreased are given table I. An increase in the fasting plasma levels of cholesterol, free fatty acids and triglycerides are observed commonly in type II diabetes which in turn are known to generate ROS26. ROS can stimulate oxidation of LDL. This oxidized LDL is not recognized by the LDL receptor and hence taken by scavenger receptors leading to form cell formation and atherosclerotic plaques27-29. The Total proteins and serum albumin mean level in normal individuals was 6.610.46 gm%and 3.790.42 gm%. In Type 2 diabetic patients the Total proteins and serum albumin was 6.570.50 and3.670.53 significantly decreased. The maintenance of protein redox status is of great importance for cell function and structural changes to proteins may be responsible for diabetic complications. Reactive oxygen species have shown to react with several amino acid residues, generating denatured and non functioning proteins30. Serum proteins like ceruloplasmin and transferrin are considered as markers of free radical scavenger, and their role as antioxidants is related on their metal binding ability. In type 2 diabetes decreased transferrin levels and no alteration in ceruloplasmin levels were observed31. Albumin is a plasma protein and is a powerful extracellular antioxidant. Albumin modified by amadori glucose has however been shown to modulate signal transduction that contributes to the development of diabetic complications32. It rapidly scavenges hypochlorous and peroxynitrous acids and slowly reacts with hydrogen peroxide. Albumin inhibits oxidative damage by binding to heme and copper ions33. In normal individuals (control group) TBARS mean level was 55.6413.37 nmole/dl. In Type 2 diabetic patients the TBARS was significantly raised being 114.7011.69 nmole/dl. Increased free radicals found in diabetes and impaired glucose tolerance is related to chronically elevated glucose levels34. The measurement of the free radicals is difficult due to their high reactivity, very short half-life and low conc. Therefore indirect markers are commonly used to evaluate secondary products of lipid peroxidation such as TBARS. TBARS assay is easy to perform and commonly used but it lacks specificity35. Most studies reported elevated plasma TBARS levels in patients with impaired glucose tolerance, early hyperglycemia and also in type 2 diabetes patients compared to a normoglycemic population 36-38. Protein carbonyl content in serum of diabetic patients 21.763.35 nmole/ml shows highly significance increase as compared with control group 12.172.72 nmole/ml, when measured in terms of total protein in DM 6.570.50 gm/dl shows significance decreases as compared with control group 6.610.46 gm/dl (table I). The Protein carbonyl levels were significantly increased in diabetes without complication when compared with controls. These results suggest that impaired glycemic control is related to protein oxidation. The glycation cascade further releases free radicals, which may be responsible for more oxidative attack, which leads to complications of diabetes39,40. Significant increases in urine microalbumin levels were found in diabetic patients (40.19 ± 29.18 mg/L) when compared with healthy subjects (15.91 ± 10.90mg/L). Microalbuminuria in type 2 diabetic patients might be promoted by an insufficient counter-regulation of the antioxidant system in the event of increased glyco-oxidation / glycation of proteins which is induced due to carbonyl stress41,11 Thus, monitoring of changes in protein oxidation has shown practical application in a type 2 diabetes mellitus and its complications. Increased levels of TBARS and protein carbonyl may be due to hyperglycemia derived from reactive oxygen species activity and increased superoxide anions, other oxidants together with decreased levels of antioxidants42, 43. Abnormally high levels of free radicals which are formed in diabetes by glucose oxidation, non enzymatic glycation of proteins and subsequent oxidative degradation of glyceated proteins can lead to damage of cellular organelles, increased lipid peroxidation and development of insulin resistance. These consequences of oxidative stress can promote the development of complications of diabetes. Further studies on oxidative stress and antioxidant markers on patients of type 2 diabetes would help to elucidate mechanisms leading to these complexities and also expand treatment options.

CONCLUSION

Diabetes mellitus has been known to be a state of excess generation of free radicals contributed by several mechanisms, including hyperglycemia and antioxidant status, causing oxidative stress. This oxidative stress exacerbates the development and progress of diabetes and its complications. Oxidative stress plays an important role in the development of complications in type 2 diabetes. In this study, an analysis of the biochemical parameters in type-2 diabetes mellitus on insulin treatment and without any complications and age matched controls was carried out and compared. Type 2 diabetes were obese and had increased levels of fasting blood glucose, HbA1c, serum triglycerides, TBARS and protein carbonyl when compared with the controls. The elevated free radical activity as shown by TBARS (index of lipid peroxidation) and protein carbonyl levels (index of protein oxidation) suggests the role of hyperglycemia in the production of oxidative stress. This state of heightened oxidative stress can decrease antioxidant mechanisms and lead to the development of insulin resistance and cellular damage. In type 2 diabetic patients might be promoted by an insufficient counter-regulation of the antioxidant system in the event of increased glycooxidation/ glycation of proteins which is induced due to carbonyl stress [44]. Thus, monitoring of changes in protein oxidation has shown practical application in a type 2 diabetes mellitus and its complications45. Thus free radical activity in type 2 diabetes mellitus along with insulin resistance can lead to activation of stress sensitive pathways, which may play an important role in the complications of diabetes. Further studies have to be done to diagnose early changes in type 2 diabetics in order to identify high risk subjects and to prevent further complications.

 

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