Studies on Status of Oxidative Stress related Molecules and Enzymes in Obese with and without Diabetes in the Northern region of India

 

Sukhpal Singh1, 2, Amita Mahajan2, 3, Jaspreet Kaur1*

1Department of Biotechnology, University Institute of Engineering and Technology, Panjab University, Chandigarh-160014, India.

2Department of Life Sciences,  I.K. Gujral Punjab Technical University, Jalandhar, Punjab-144603, India.

3University School of Sciences, Rayat Bahra University, Mohali, Punjab-140104, India.

*Corresponding Author E-mail: jaspreet_uiet@pu.ac.in

 

ABSTRACT:

This study was done to assess the interrelationship of “oxidative stress-related molecules & enzymes” with obesity and diabetes. Xanthine oxidase (XO), malondialdehyde  (MDA) representing lipid peroxidation and protein oxidative stress parameters such as advanced oxidation of protein products (AOPP), protein carbonyls (PC) and antioxidant enzyme activities in obese with or without type 2 diabetes mellitus (T2DM) were studied. This study enrolled 41 healthy controls, 41 obese non-diabetics, and 41obese T2DM patients. Biochemical parameters related to diabetes, oxidative stress and antioxidant enzymes were measured in all groups. The levels of XO, MDA, AOPP, and PC were significantly increased in obese with and without T2DM as compared to healthy controls (P < 0.001). However levels of antioxidants such as reduced glutathione (GSH) and  activity of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx) were significantly decreased in obese with and without T2DM as compared to healthy control (P < 0.001 or P < 0.05). Positive correlations were found among XO with MDA (P = 0.006), PC (P = 0.012) in obese non-diabetic subjects and with MDA (P < 0.001), AOPP (P = 0.026), and PC (P = 0.003) in obese T2DM patients. Positive correlation were seen in MDA with AOPP (P = 0.017), PC (P = 0.046) in obese non-diabetic subjects and MDA with AOPP (P < 0.001), PC (P < 0.001) in obese T2DM patients. Also observed a significant correlation between AOPP and PC (P < 0.001) in both groups of obese. The present study results suggest that increased activity of xanthine oxidase, higher lipid peroxide generation, and higher levels of protein oxidative stress parameters (AOPP and PC) may be a most considerable mechanism in reducing the activity of antioxidant enzymes in obese with and without diabetic groups as compared to control group. However, extent of derangements is lower in obese subjects as compared to diabetic patients, thereby suggesting that obese subjects have a higher risk of diabetes as well as early onset of diabetes. 

 

KEYWORDS: Xanthine oxidase, Malondialdehyde, Protein carbonyl, Oxidative stress, Antioxidants.

 

 


INTRODUCTION:

Diabetes and obesity concurrence called diabesity has emerged as an epidemic disease of the 21st century. Its increasing pervasiveness represents a huge economic burden on health services affecting both colonization and non-colonization countries. The International Diabetes Federation (IDF), diabetes Atlas 2017, reported 425 million diabetic adults worldwide, another 212 million (1 in 2) people were estimated to be undiagnosed diabetic. The increase in type 2 diabetes has been reported to be mainly due to an upturn in obesity1.

The statistical data of population attributable factor (PAF) indicated that 90% of T2DM is due to overweight and obesity2. According to World Health Organization (WHO) global facts on overweight and obesity in 2016, more than 1.9 billion adults were overweight and of these over 650 million were obese, which was about 13 % of the world’s adult population3. Many studies have also reported that obesity is linked to chronic low grade inflammation in hypertrophied adipose tissue that promotes the initiation and progression of insulin resistance and metabolic disorders2, 4. Therefore obesity may promote insulin resistance and is the major factor responsible for T2DM5.  Inflammation of adipose tissue in obese has been further linked to the increased levels of reactive oxygen species (ROS) such as hydroxyl radicals, superoxide anions, hydrogen peroxide that are involved7.

 

The studies have found that ROS plays a major role in the development of xanthine oxidase generated superoxide anion radicals in purines, carbonyl and chloraminated oxidants (hypochlorous acid and chloramines) in protein and lipid peroxides in lipids. Xanthine oxidoreductase (XOD) is a purine catabolism enzyme, converting hypoxanthine into xanthine and uric acid by oxidation8. In-vivo it acts as a pro-oxidant in a cell and as an antioxidant in plasma9. The defense mechanism of the body prevents the cell membrane and organelles from harmful effects of ROS by enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and non-enzymatic antioxidants such as reduced glutathione10-14. However, in disease condition such as diabetes, there is an accumulation of ROS which results in increased oxidants and a decrease in antioxidant capacity.

 

It has been seen that obese human has increased the level of oxidative stress in the adipose tissue4, 15-16. The study has also reported that increased production of ROS in cultured 3T3-L1 adipocytes and excess release of free fatty acids from excess accumulated fat leads to insulin resistance4, 5. This overproduction of ROS can cause oxidative damage to nucleic acid, lipids, proteins and sometimes cell death by necrosis or apoptosis17.

 

After extensive literature survey no combined study of oxidative stress molecules such as xanthine oxidase, lipid peroxidation, and oxidative protein damage and its relation to the antioxidant enzymes have not been completely elucidated in obese non-diabetic subjects and obese T2DM patients from Chandigarh region and the surrounding region of north India. Therefore the present study was to investigate the relationship among oxidative stress parameters such as XO, MDA, AOPP, and PCO, and antioxidant molecules such as reduced glutathione and enzymatic activity of SOD, CAT, GST, and GPx in various groups of subjects. Also related parameters like glucose level, glycated hemoglobin, and lipid profile were measured in all the subjects.

 

Materials and methods:

Study design:

The present study enrolled a total of 123 subjects, consisting of 41 healthy controls with a BMI ≤ 25 kg/m2, 41 obese non-diabetic subjects with BMI ≥ 30 kg/m2 and 41 obese T2DM patients with BMI ≥ 30 kg/m2. The patients involved in this study were outpatients of special diabetic clinics. The study was approved by the Govt. Multispecialty hospital, Chandigarh (MS-II-2015/4631) India. All the participants were informed about the objectives of the study and written informed consent was obtained from all subjects involved in this study.

 

Inclusion and exclusion criteria:

Anthropometric parameters age (years), weight (kg) and heights (m2) were recorded. BMI was calculated by using parameters weight and height. The subjects in the age of 30-50 years were involved in the study. The selection of healthy control subjects who had normal BMI was from the health staff and caretakers of patients enrolled in the study. Obese non-diabetic subjects and obese T2DM patients were recruited from outpatients of special diabetology clinic. The diabetic patients were selected according to the World Health Organization (WHO) with fasting glucose ≥ 126mg/dl and level of glycated hemoglobin (HbA1C) ≥ 6.5%. The onset of diabetes was at least for five years. Diabetic patients under the treatment of oral hypoglycemic drugs were involved. Exclusion criteria for patients in this study were subjects having exogenous insulin, Type 1 diabetic patients, antioxidant supplements, anti-inflammatory drugs, anti-obesity therapies, inflammatory or malignant disease, and history of hospitalization in preceding six months, pregnant and lactating women.          

 

Blood sample collection and hemolysate preparation:

After 10-12 hrs of overnight fasting, peripheral venous blood was withdrawn aseptically from the antecubital vein of the subjects. Blood samples were collected into fluoride-oxalate, EDTA and plain sterile vacutainers. Fluoride oxalate vacutainers were used for plasma glucose estimation. EDTA vacutainers were used for plasma AOPP, PCO, and glycated hemoglobin. After collection of plasma remaining blood samples were used for the preparation of erythrocyte hemolysate by the method of Lohr and Waller18. Plasma and serum aliquots were stored at -80 C till further used for investigation.

 

Biochemical estimation:

Determination of diabetic profile such as fasting blood glucose (FBG), postprandial blood glucose (PPBG), glycated hemoglobin and lipid profile such as total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDLC) levels were estimated by Erba Transasia Mannheim diagnostic kits. Low density lipoprotein cholesterol (LDL-C) and Very low density lipoprotein cholesterol (VLDL-C) was calculated indirectly by Friedwald’s formula19. Protein concentration was measured in the erythrocyte hemolysate by the method Lowry et al.20.

 

Assay of xanthine oxidase activity:

The amplite xanthine oxidase assay kit (AAT Bioquest, Inc., USA) was used for the measurement of serum XO activity. In this method, XO catalyzes the oxidation of xanthine or hypoxanthine to superoxide and uric acid. The superoxide spontaneously degrades to hydrogen peroxide which can be measured with the amplite red substrate in dual mode with OD ratio at the wavelength of 570nm to 610nm. The content of XO was evaluated by using a standard curve and expressed as mU/ml.

 

Estimation of  malondialdehyde:

MDA level in the erythrocyte hemolysate was measured by Beuge and Aust method21. The amount of MDA produced in the reaction mixture was expressed as nmoles of MDA/mg protein and calculated by using the molar extinction coefficient of MDA-TBA chromophore (1.56 x 105 M-1 cm-1).

 

Determination of AOPP:

Determination of AOPP in plasma was measured by spectrophotometric assay of Witko-Sarsat22. A standard curve was prepared using chloramines –T (0-100 µmol/L) absorbance at 340nm. AOPP concentration was expressed as µmol/L of chloramines-T equivalents.

 

Determination of Protein Carbonyl:

The content of protein carbonyl in plasma was evaluated by the method of Reznick and Packer23. The concentration of carbonyl content was expressed as nmol/mg protein by using the molar extinction coefficient of DNPH 22000 M-1 cm-1.

 

Antioxidant enzymes:

Assay of CAT activity:

The activity of catalase (CAT) was measured spectrophotometrically in the erythrocyte hemolysate by the method of Luck24. The enzyme activity was represented as µmoles of H2O2 decomposed/min/mg protein using molar extinction coefficient (71 M-1 cm-1).

 

Assay of SOD activity:

The activity of superoxide dismutase (SOD) was measured spectrophotometrically in the erythrocyte hemolysate according to the method of Kono25. The specific activity of the enzyme was represented as units/mg protein. A single unit of enzyme was defined as the amount of enzyme-inhibiting the rate of reaction by 50% inhibition of NBT.

 

Assay of GST activity:

The activity of glutathione-S-transferase was measured spectrophotometrically in the erythrocyte hemolysate according to the method of Habig et al.26. The GST activity was represented as nmoles of CDNB conjugate formed/min/mg protein using molar extinction coefficient (9.6 mM-1 cm-1).

 

Assay of GPx activity:

The glutathione peroxidase activity was measured spectrophotometrically in the erythrocyte hemolysate by the method of Paglia and Valentine27. Results were expressed as nmoles NADPH oxidized/min/mg protein, using the molar extinction coefficient of NADPH (6.22 x 103 M-1 cm-1) to represent the specific activity of the enzyme.

 

Assay of Reduced Glutathione:

The reduced glutathione content in the whole blood was estimated according to the method of Beutler et al.28. The concentration of erythrocyte glutathione was determined by using standard GSH and concentration was expressed as mg/dl.

 

Statistical Analysis:

Experimental data were evaluated by statistical software of IBM SPSS 25.0.0. Quantitative variable data were expressed as Mean ± Standard deviation (SD). Statistical significance was analyzed by one-way analysis of variance (ANOVA) followed by post hoc Tukey test for normally distributed variables. The variables which were not normally distributed, nonparametric Kruskal Wallis ANOVA followed by Mann-Whitney U-test were performed. Spearman’s correlation coefficient two tailed was analyzed to found the correlation between the pro-oxidants and between pro-oxidants and antioxidants in the groups of obese with and without diabetes. A P value less than and equal to 0.05 considered to be statistically significant.

 

Results:

Biochemical parameters of study subjects:

In the present study demographical characteristics of two obese groups i.e. with and without T2DM were compared with healthy control as shown in Table 1.  BMI was considerably increased in both obese groups as compared to a healthy control group (P < 0.001).  FBG, PPBG, glycated hemoglobin, total cholesterol, Triglycerides, VLDL-cholesterol were significantly increased and HDL-cholesterol significantly decreased in obese T2DM as compared to obese non-diabetic and healthy control (P < 0.001 or P< 0.05).


Table 1. Demographical and clinical details of the subjects in groups.

Parameters

Healthy Control

Obese Non-diabetic

Obese T2DM

BMI ≤ 25

BMI ≥ 30

BMI ≥ 30

Numbers

41

41

41

Age (Years)

39.85 ± 6.67

40.95 ± 5.99

47.66 ± 3.64

 

*P < 0.390

*P < 0.001,**P < 0.001

Gender(M/F)

23/18

15/26

24/17

BMI (Kg/m2)

22.85 ± 1.99

32.56 ± 2.70

33.08 ± 2.80

*P < 0.001

*P < 0.001, **P < 0.295

FBG (mmol/L)

4.48 ± 0.27

4.60 ± 0.38

10.76 ± 1.11

*P < 0.106

*P < 0.001, **P < 0.001

PPBG (mmol/L)

6.49 ± 0.73

6.75 ± 0.63

15.53 ± 2.68

 

*P < 0.753

*P<0.001, **P<0.001

HbA1c (%)

4.53 ± 0.40

4.60 ± 0.35

8.94 ± 1.10

 

*P < 0.904

*P < 0.001, **P < 0.001

Cholesterol (mg/dl)

157.59 ± 20.48

182.24 ± 30.28

197.36 ± 31.02

 

*P < 0.001

*P < 0.001, **P < 0.023

Triglycerides (mg/dl)

113.04 ± 29.55

165.48 ± 45.16

250.18 ± 48.93

 

*P < 0.001

*P < 0.001, **P < 0.001

HDL-cholesterol (mg/dl)

51.27 ± 5.29

48.86 ± 5.09

39.17 ± 4.37

 

*P < 0.073

*P < 0.001, **P < 0.001

LDL-cholesterol (mg/dl)

83.71 ± 17.58

100.73 ± 30.42

100.26 ± 28.20

 

*P < 0.011

*P < 0.008, **P < 0.989        

VLDL-cholesterol (mg/dl)

22.61 ± 5.91

32.65 ± 9.28

50.04 ± 9.79

 

*P < 0.001

*P < 0.001, **P < 0.001

Data are represented as mean ± S.D.           P < 0.05 statistically significant. *P vs. Healthy control **P vs. Obese non-diabetic

BMI: body mass index; FBG: fasting blood glucose; PPBG: postprandial blood glucose; T2DM: type 2 diabetes mellitus;

HDL: high density lipoprotein; LDL: low density lipoprotein; VLDL: very low density lipoprotein.

 

Table 2. Proxidant and antioxidant activity of the subjects in groups.

Parameters

Healthy Control

Obese Non-diabetic

Obese T2DM

Xanthine oxidase (mU/ml)

0.53 ± 0.09

0.77 ± 0.10

1.04 ± 0.14

 

 

*P < 0.001

*P < 0.001, **P < 0.001

MDA( nmol/mg protein)

0.77 ± 0.18

1.25 ± 0.29

1.56 ± 0.26

 

 

*P < 0.001

*P < 0.001, **P < 0.001

AOPP (µmol/l)

168.44 ± 41.51

212.79 ± 43.58

302.09 ± 56.33

 

 

*P < 0.001

*P < 0.001, **P < 0.001

PC (nmol/mg protein)

0.71 ± 0.08

1.00 ± 0.23

1.45 ± 0.26

 

 

*P < 0.001

*P < 0.001, **P < 0.001

GSH(mg/dl)

35.32 ± 3.54

31.65 ± 3.44

24.92 ± 2.30

 

 

*P < 0.001

*P < 0.001, **P < 0.001

SOD (Ua/mg protein)

4.82 ± 1.14

3.60 ± 0.79

2.36 ± 0.67

 

 

*P < 0.001

*P < 0.001, **P < 0.001

CAT (Ub/mg protein)

71.73 ± 9.05

63.01 ± 9.19

58.76 ± 4.32

 

 

*P < 0.001

*P < 0.001, **P < 0.041

GST (Uc/mg protein)

33.75± 3.80

21.77± 1.75

18.80 ± 2.25

 

 

*P < 0.001

*P < 0.001, **P < 0.001

GPx (Ud/mg protein)

27.01 ± 3.90

21.85 ± 3.12

19.40 ± 4.04

 

 

*P < 0.001

*P < 0.001, **P < 0.002

Data are represented as mean ± S.D.           P < 0.05 statistically significant.

*P vs. Healthy Control              **P vs. Obese Non-diabetic

a: one unit of enzyme is amount of enzyme inhibiting the rate of reaction by 50 % inhibition of NBT.

b: µmol of H2O2 decomposed/min               c: nmol of CDNB-GSH conjugate/min        d: nmol of NADPH oxidized/min formed/min

 


Table 3.Correlation among prooxidants in obese non diabetic subjects and obese T2DM patients

Parameters

r value

P value

r value

P value

Obese Non-diabetic

Obese T2DM

XO vs.  MDA

0.424**

0.006

0.676**

0.001

XO vs. AOPP

0.267

0.092

0.348*

0.026

XO vs. PC

0.391*

0.012

0.457**

0.003

MDA vs. AOPP

0.372*

0.017

0.516**

0.001

MDA vs. PC

0.314*

0.046

0.755**

0.001

AOPP vs. PC

0.737**

0.001

0.597**

0.001

Values are expressed as bivariate spearman’s coefficient (r)

*. P is significant at the 0.05 level.

**. P is significant at the 0.01 level.

Comparison of Pro-oxidant and antioxidant status in different study groups:

Serum XO activity, oxidant mediated erythrocyte MDA level, plasma AOPP, and PC levels were significantly increased in obese T2DM and obese non-diabetic groups as compared to healthy controls (P <  0.001). Whereas antioxidant activities such as GSH, SOD, CAT, GST, and GPx significantly decreased in obese T2DM and obese non-diabetic groups as compared to healthy controls (P < 0.001 or P < 0.05) and results are shown in Table 2. 


 


Correlation among prooxidants in obese non-diabetic subjects and obese T2DM patients:

To assess the combined role of XO with other oxidative stress parameters in both the obese groups, correlation indices were obtained as shown in Table 3. A positive relationship was observed between XO and MDA, XO and PC, MDA, and AOPP, MDA and PC, AOPP and PC in obese non-diabetic subjects (Fig. 1). Moreover, a positive relationship was also observed in XO and MDA, XO and AOPP, XO and PC, MDA and AOPP, MDA and PC, AOPP and PC in obese T2DM patients (Fig. 2).

 

Spearman’s correlation coefficient between oxidant and antioxidants parameters:

In table 4. Obese non-diabetic subjects, a negative correlation were found among in MDA with GSH (r = -0.359, P = 0.021), SOD (r = -0.345, P = 0.027), GPx (r = -0.558, P < 0.0001) between AOPP and SOD (r = -0.328, P = 0.036); between PC and SOD (r = -0.332, P = 0.034). In obese T2DM patients, a negative correlation were found among  XO with SOD ( r = -0.607, P = 0.0001), CAT (r = -0.353, P = 0.023), MDA with GSH (r= -0.549, P < 0.0001), SOD( r= -0.616, P < 0.0001), CAT (r = -0.414, P = 0.007), GPx (r = -0.351, P = 0.025), and PC with GSH ( r = -0.426, P = 0.006), SOD ( r = -0.503, P < 0.0001), CAT ( r = -0.404, P = 0.009).

 

 


Table 4. Correlation among prooxidants and antioxidants in obese non diabetic subjects and obese T2DM patients

Obese non-diabetic

Obese T2DM

Parameters

r value

P value

r value

P value

XO vs.  GSH

0.001

0.993

-0.225

0.158

XO vs.  SOD

-0.259

0.102

-0.607**

0.0001

XO vs. CAT

0.026

0.870

-0.353*

0.023

XO vs. GST

-0.061

0.705

-0.152

0.342

XO vs. GPx

0.038

0.815

-0.171

0.286

MDA vs. GSH

-0.359*

0.021

-0.549**

0.0001

MDA vs.  SOD

-0.345*

0.027

-0.616**

0.0001

MDA vs. CAT

0.038

0.814

-0.414**

0.007

MDA vs. GST

-0.300

0.057

-0.192

0.228

MDA vs. GPx

-0.558**

0.0001

-0.351*

0.025

AOPP vs.  GSH

-0.075

0.643

-0.288

0.068

AOPP vs.  SOD

-0.328*

0.036

-0.098

0.541

AOPP vs. CAT

-0.081

0.615

-0.142

0.376

AOPP vs. GST

-0.046

0.775

-0.088

0.586

AOPP vs. GPx

-0.117

0.468

-0.301

0.056

PC vs.  GSH

-0.263

0.097

-0.426**

0.006

PC vs.  SOD

-0.332*

0.034

-0.503**

0.001

PC vs. CAT

0.107

0.504

-0.404**

0.009

PC vs. GST

-0.170

0.287

-0.165

0.304

PC vs. GPx

-0.240

0.130

-0.262

0.097

Values are expressed as bivariate spearman’s coefficient (r)

*. P is significant at the 0.05 level.              **. P is significant at the 0.01 level.


Discussion:

The present studies on the level of oxidative stress-related parameters in obese with and without diabetes were conducted in the northern region of India. The results in the three groups clearly showed that there is an increase of pro-oxidants and decrease of antioxidants in obese and obese diabetic subjects in contrast to healthy controls. Our results are consistent with existing literature that obese subjects without diabetes mellitus also exhibit an increase in systemic oxidative stress by ROS and free radical generation. In obesity, an excess of free fatty acid leads to its oxidation through the mitochondrial ROS production29.Also in these subjects, there is hyperglycemia, hyperleptinemia, insufficient antioxidant defenses, increased tissue lipid levels, increased rates of free ROS formation and chronic inflammation30.

 

Assay of xanthine oxidase activity in the groups showed a two-fold increase of XO activity in obese T2DM subjects as compared to a healthy control group. Moreover, XO activity was positively correlated with MDA, AOPP, and PC in obese T2DM and with MDA and PC in obese non-diabetic. Previous studies reported XO activity as the major source of ROS in diabetes mellitus. XO activity is observed to be an independent predictor of obesity in humans and mice31-32. Our results are consistent with previous reports31-33. In this study, we find a negative correlation between XO and CAT, XO and SOD in obese T2DM only, which shows a strong association between oxidative stress-related molecules and enzymes. These findings support the previous report of Klisic et al.31. and Feoli et al.34.Though no correlation could be observed between XO and antioxidants in obese non-diabetics,  a noteworthy rise in the activity of XO and a diminish in the concentration of antioxidants was seen in obese non-diabetics in contrast to healthy controls. These results suggest that a decrease in antioxidant molecules and enzymes might play a vital role in the development of obesity (even without diabetes). The results obtained are in agreement with previous reports35.

 

In various pathological conditions and in increased oxidative stress, ROS are involved in the peroxidation of polyunsaturated fatty acids (PUFA) and oxidative modification of some specific proteins has been reported36-37. Increased levels of erythrocyte MDA was reported in obese non-diabetic and obese T2DM patients38-39. In the present study, erythrocyte MDA levels were considerably high in obese T2DM and obese non-diabetic as compared to healthy control subjects. The increase in MDA was more in obese T2DM patients as compared to that in obese non-diabetic. Previous studies have shown that obesity itself can promote the formation of high levels of lipid peroxide and diminish in the activity of protective enzymes40. The results of the current study are in concurrence with the results of Cazzola et al.41, who showed that in obese subjects, the ratio of reduced to oxidized glutathione was decreased with the onset of free radical-induced hemolysis, also membrane fluidity was decreased and there was an increase in the ratio of cholesterol and phospholipids.

 

In the present study, there was a noteworthy increase in all parameters of the lipid profile (except HDL cholesterol) in obese T2DM as compared to healthy control. This study was in line with the previous study of Mawhoob et al.42, Sutrakar et al. and Baghel et al.43. These derangements probably could be due to a significantly enhanced rate of lipid peroxidation in T2DM. Inhibition in the activity of SOD enzyme leads to accumulation of superoxide radicals which can further elevate MDA levels44.

 

Our results also indicated that there is an increase in protein oxidation products as shown by an increase in plasma levels of PC and AOPP in obese with and without T2DM (table 2).The results were in accordance with the previous report of Singh et al.45. It is well studied that protein carbonyl is the marker of tissue injury and protein damage by oxidative stress with the observation of elevated levels of carbonylated proteins. In this study we observed MDA positively correlated with PC in obese T2DM and obese non-diabetic groups, such relation of oxidative stress was also seen in subjects with familial hypercholesterolemia36.

 

In our previous study, we reported that an increased level of AOPP is associated with obesity and diabetes that correlate with insulin resistance45. Also, we found a significant correlation between AOPP and PC; a similar relation is also seen in this study. Oxidative stress affects both lipids and proteins which can be observed with a significant correlation between MDA-AOPP in this study. Similar results were also observed in an experimental animal study of the rat with streptozotocin-induced diabetes mellitus46. As reported by Miric et al.33 no significant relation between XO and AOPP could be observed in T2DM patients, however, in our current study, we found a significant relationship between XO activity and AOPP in obese T2DM.

 

In the current study SOD, CAT, GST, GPx, and GSH were observed to be notably decreased in obese non-diabetic and obese T2DM patients as compared with healthy control subjects. SOD act as the first line of defense for radical scavenging enzyme against the superoxide radicals produced during hyperglycemia and obesity mediated oxidative stress. SOD scavenges the O2- radicals to hydrogen peroxide (H2O2) which in turn gets scavenged by GPx and catalase by converting into water and molecular oxygen. The decrease in erythrocyte SOD activity in obese subjects and obese T2DM patients was in concordant with the study of Albuali et al.38 and Picu et al.47 ROS and H2O2 are produced endogenously in normal cellular respiration and also produced by the action of SOD scavenge the superoxide anions13,31,34,48. Erythrocyte catalase is the main regulator for the removal of H2O2. In this study, a considerable decrease in erythrocyte catalase activity was found in obese T2DM and obese non-diabetic this may result due to accumulation of H2O2 and O2- radicals.

 

In the present study, we found a substantial decrease in reduced glutathione level in obese with and without T2DM, which may be due to its faster utilization during stress conditions leading to the formation of oxidized glutathione (GSSG). The activity of erythrocyte GST and GPx was observed to be reduced suggesting higher ROS formation in obese with and without diabetes. Parallel results have been obtained by Albuali et al.38 and Pasupathi et al.48. Moreover, the present study showed that there is an inverse relationship between oxidant XO, MDA, and PC levels with enzymes activities of antioxidants in obese T2DM. Furthermore, obese non-diabetic also found a negative correlation among MDA, AOPP, and PC with few indices of antioxidants. This correlation suggested that there is significant depletion of antioxidants with excess in the formation of ROS resulting in increased of XO, MDA, AOPP and PC levels in obese subjects and obese T2DM patients. Therefore, this study supports the relationship between oxidative stress-related molecules and enzymes in obese with and without diabetes.   

 

CONCLUSION:

The present study revealed that the activity of XO and levels of MDA, AOPP, and PC get significantly raised in obese group and in obese diabetic group as compared to healthy control group. Antioxidant molecules and enzymes such as GSH, CAT, SOD, GST and GPx significantly decreased in obese group and obese diabetic group. The increased levels of oxidative stress related molecules and decrease in enzyme activity in obese without diabetes are highly likely to progress to diabetes or other secondary diseases. These indicated new studies are needed to explore better management, early specific therapies for obesity that may be useful in the prevention of diabetes and other oxidative stress related diseases such as atherosclerosis, dyslipidemia, hyperinsulinemia, and cardiovascular diseases.

 

References:

1.      Cho NH, Whiting D, Forouhi N, Guariguata L, Hambleton I, Li R, et al. IDF Diabetes Atlas, International Diabetes Federation (IDF), Brussels, Belgium, 7th edition, 2015.Available from URL: https://www.idf.org/e-library/epidemiology-research/diabetes-atlas/13-diabetes-atlas-seventh-edition.

2.      Verma S, Hussain ME. Obesity and diabetes: An update. Diabetes Metab Synd 2017; 11(1):73-7.

3.      World Health organization, Geneva. World Obesity Federation.Available from URL: https://www.who.int/en/news-room/fact-sheets/detail/obesity-and-overweight.

4.      Matsuda M, Shimomura I. Increased oxidative stress in obesity: Implications for metabolic syndrome, diabetes, hypertension, dyslipidemia, atherosclerosis, and cancer. Obes Res Clin Pract 2013; 7(5):e330-341.

5.      Ruskovska T, Bernlohr DA. Oxidative stress and protein carbonylation in adipose tissue - Implications for insulin resistance and diabetes mellitus. J proteomics 2013; 92:323-334.

6.      Marrocco I, Altieri F, Peluso I. Measurement and Clinical Significance of Biomarkers of oxidative stress in humans.Oxid Med Cell Longev 2017; 6501046.

7.      Vadivelan R, Dhanabal SP, Raja Rajeswari, Shanish A, Elango K, Suresh B. Oxidative stress in diabetes- a key therapeutic agent. Research J. Pharmacology and Pharmacodynamics. 2010; 2(3): 221-227.

8.      Buvana C, Sumathy A, Sukumar M. In silico identification of potential xanthine oxidase inhibitors for the treatment of gout and cardiovascular disease. Asian J. Research Chem 2013; 6(11):1049-1053.

9.      Batteli MG, Polito L, Bolognesi A.  Xanthine oxidoreductase in atherosclerosis pathogenesis: Not only oxidative stress. Atherosclerosis 2014; 237:562-567.

10.   Pathade PA, Ahire YS, Bairagi VA , Abhang DR. Antioxidants therapy in cognitive dysfunction associated with diabetes mellitus: An overview. Research J. Pharmacology and Pharmacodynamics. 2011; 3(2): 39-44.

11.   Prithviraj Chakraborty P, Kumar S, Dutta D, Gupta V. Role of antioxidants in common health diseases. Research J. Pharm. and Tech 2009; 2(2):238-244.

12.   Menon R. Antioxidants and their therapeutic potential- a review. Research J. Pharm. and Tech 2013; 6(12):1426-1429.

13.   Vaishali M. Antioxidants in Health and Diseases. Research J. Pharm. and Tech 2014; 7(4): 489-493.

14.   Tandra Das .T. Role of antioxidants in health and diseases-a review; Research J. Pharm. and Tech2015; 8(8):1033-1037.

15.   Furukawa S, et al. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest. 2004; 114(12): 1752-61.

16.   Manna P, Jain SK.Obesity, Oxidative Stress, Adipose Tissue Dysfunction, and the Associated Health Risks: Causes and Therapeutic Strategies. Metab Syndr Relat Disord 2015; 13(10): 423 - 44.

17.   Mohsen H, Shaden H, Quobili Faiza AL,Taghrid H.Correlation of serum leptin levels with insulin resistance in Syrian obese patients with type 2 diabetes mellitus. Research J. Pharm. and Tech 2013; 10(6):1149-51.

18.   Lohr GW, Waller HD. Glucose-6-phosphate dehydrogenase. Meth Enzy Anal 1974; 2:636-643.

19.   Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18: 499–502.

20.   Lowry OH, Rosebrough NJ, Farr, AL, Randall RJ. Protein measurement with the Folin phenol reagent. J  Bio Chem 1951; 193(1): 265-75.

21.   Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978; 52: 302-310.

22.   Witko-Sarsat V, Friedlander M, Nguyen Khoa T, Capeillère-Blandin C, Nguyen AT, Canteloup S. et al. Advanced oxidation protein products as novel mediators of inflammation and monocyte activation in chronic renal failure. J Immunol 1998; 161(5):2524-2532.

23.   Reznick AZ, Packer L. Oxygen Radicals in Biological Systems Part C. Methods Enzymol 1994; 233(1991): 357-363.

24.   Luck H. Catalase. In: Bergmeyer HU, Editor. Methods of Enzymatic Analysis 1971; vol. III. Academic press, New York, pp. 885-894.

25.   Kono Y. Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Arch Biochem Biophys 1978; 186(1):189-95.

26.   Habig WH, Pabst MJ and Jakoby WB. Glutathione S –Transferases: the first enzymatic step in mercapturic acid formation. J Biol Chem 1974; 249:7130-7139.

27.   Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70(1):158-69.

28.   Beutler  E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1963; 61: 882-8.

29.   Chattopadhyay M, Khemka VK, Chatterjee G, Ganguly A, Mukhopadhyay S, Chakarbarti S. Enhanced ROS production and oxidative damage in subcutaneous white adipose tissue mitochondria in obese and type 2 diabetes subjects. Mol Cell Biochem 2015; 399:95-103

30.   Vincent HK, Taylor AG: Biomarkers and potential mechanisms of obesity induced oxidant stress in humans. Int J Obes 2006; 30:400-400.

31.   Klisic A, Kocic G, Kavaric N, Jovanovic M, Stanisic V, Ninic A. Body mass index is independently associated with xanthine oxidase activity in overweight/obese population. Eat weight Disord 2018.

32.   Chiney MS, Schwarzenberg SJ, Johnson LA. Altered xanthine oxidase and N-acetyltransferase activity in obese children. Br J Clin Pharmacol 2011; 72(1):109–115.

33.   Miric DJ, Kisic BM, Fillipovic-Danic S, Grbic Rade, Dragojevic I, Miric MB, Puhalo-Sladoje D. Xanthine oxidase activity in type 2 diabetes mellitus patients with and without diabetic peripheral neuropathy. Journal of Diabetes Research 2016; 4370490.

34.   Feoli AMP, Macagnan FE, Piovesan CH, Bodanese LC, Siqueira IR. Xanthine oxidase activity is associated with risk factors for cardiovascular disease and inflammatory and oxidative status markers in metabolic syndrome: effects of a single exercise session. Oxid Med Cell Longev 2014; 587083.

35.   Singh K, Singh S. Comparative study on malondialdehyde and certain antioxidants in north west obese Indians. J cardiovasc Dis Res 2015; 6(3), 138-144.

36.   Pirinccioglu AG, Gökalp D, Pirinccioglu M, Kizil G, Kizil M. Malondialdehyde (MDA) and protein carbonyl (PCO) levels as biomarkers of oxidative stress in subjects with familial hypercholesterolemia.Clin Biochem 2010;43(15):1220-4. 

37.   Gupta NK, Srivastva N, Bubber P, Puri S. The antioxidant potential of azadirachta indica ameliorates cardioprotection following diabetic mellitus-induced microangiopathy. Pharmacognosy Magazine 2016; 12(46):371-378.

38.   Albuali WH. Evaluation of oxidant-antioxidant status in overweight and morbidly obese Saudi children.World J clin pediatr 2014; 8; 3(1):6-13.

39.   Kocić R, Pavlović D, Kocić G, Pešić M. Susceptibility to oxidative stress, insulin resistance, and insulin secretory response in the development of diabetes from obesity. Vojnosanit Pregl. 2007; 64(6):391-7.

40.   Olusi SO. Obesity is an independent risk factor for plasma lipid peroxidation and depletion of erythrocyte cytoprotectic enzymes in humans. Int J Obes Relat Metab Disord. 2002; 26:1159–1164.

41.   Cazzola R, Rondanelli M, Russo-Volpe S, Ferrari E, Castaro B. Decreased membrane fluidity and altered susceptibility to peroxidation and lipid composition in overweight and obese female erythrocytes. J Lipid Res. 2004; 45(10):1846-51.

42.   Alkadasi MN, Alshami AM, Alhabal HY. Study the relation of serum lipids with body mass index among students in zabeed education collage, Hudaiadah University, Yemen. Asian J. Pharm. Ana2015; 5(1): 31-35.

43.   Sutrakar SK, Baghel DS. Biochemical parameters variations in type–II  diabetes mellitus: special reference in rewa region. Asian J. Research Chem 2014; 7(10):877-881.

44.   Suryawanshi NP, Bhutey AK, Nagdeote AN, Jadhav AA, Manoorkar GS. Study of lipid peroxide and lipid profile in diabetes mellitus. Indian J Clin Biochem 2006; 21:126–30.

45.   Singh S, Mahajan A, Kaur J. Study of relationship between the protein oxidation markers and adipokines in obese type 2 diabetic patients. Asian J Pharm Clin Res 2019;12(6):204-209.

46.   Vural P, Kabaca G, Firat RD, Degirmecioglu S. Administration of selenium decreases lipid peroxidation and increases vascular endothelial growth factor in streptozotocin induced diabetes mellitus. Cell J 2017; 19(3):452-460.

47.   Picu A, Petcu L, Ştefan S, Mitu M, Lixandru D, Ionescu-Tîrgovişte C. et al. Markers of Oxidative Stress and Antioxidant Defense in Romanian Patients with Type 2 Diabetes Mellitus and Obesity. Molecules 2017; 22(5):714.

48.   Pasupathi P, Chandrasekar V, Kumar US. Evaluation of oxidative stress, enzymatic and non-enzymatic antioxidants and metabolic thyroid hormone status in patients with diabetes mellitus. Diabetes & Metabolic Syndrome: Clinical Research and Reviews 2009; 3, 160-165.

 

 

 

Received on 05.08.2019            Modified on 23.09.2019

Accepted on 31.10.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(2):801-809.

DOI: 10.5958/0974-360X.2020.00151.1