INTRODUCTION:

Diabetes and obesity concurrence called diabesity has emerged as an epidemic disease of the 21^st 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 disorders^{2, 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 glutathione^10-14. 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 tissue^{4, 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 resistance^{4, 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^-1cm^-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^-1cm^-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 (U^a/mg protein)	4.82 ± 1.14	3.60 ± 0.79	2.36 ± 0.67
		*P < 0.001	P < 0.001, *P < 0.001
CAT (U^b/mg protein)	71.73 ± 9.05	63.01 ± 9.19	58.76 ± 4.32
		*P < 0.001	P < 0.001, *P < 0.041
GST (U^c/mg protein)	33.75± 3.80	21.77± 1.75	18.80 ± 2.25
		*P < 0.001	P < 0.001, *P < 0.001
GPx (U^d/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 H₂O₂decomposed/min c: nmol of CDNB-GSH conjugate/min d: nmol of NADPH oxidized/min formed/min

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 mice^31-32. Our results are consistent with previous reports^31-33. 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 reported^36-37. Increased levels of erythrocyte MDA was reported in obese non-diabetic and obese T2DM patients^38-39. 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 H₂O₂are produced endogenously in normal cellular respiration and also produced by the action of SOD scavenge the superoxide anions^13,31,34,48. 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.

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Received on 05.08.2019 Modified on 23.09.2019

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

DOI: 10.5958/0974-360X.2020.00151.1

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