The association of oxidative stress biomarkers with type 2 diabetes mellitus: A systematic review and meta‐analysis

Abstract Background and Aims Oxidative stress plays a major role in the development of type 2 diabetes mellitus (T2DM). However, there were controversial outcomes in the literature between the association of oxidative stress biomarkers and T2DM. The purpose of this systematic review and meta‐analysis was to critically examine the association of oxidative stress biomarkers with T2DM. Methods We systematically searched different electronic databases including PubMed/Medline, EMBASE, ScienceDirect, Web of Science, and Cochrane Library to find the relevant studies up to May 2021. The pooled standard mean difference (SMD) with a 95% confidence interval (CI) was used to define the variation between the study groups. Results A total of 22 case‐control studies with 2853 subjects (1667 diabetic patients and 1186 healthy controls) were found to be eligible for this meta‐analysis. The pooled results of meta‐analysis showed a significant difference in the levels malondialdehyde (SMD [95% CI]: 2.27 [1.62, 2.91]), nitric oxide (SMD [95% CI]: 1.40 [0.00, 2.81]), glutathione (SMD [95% CI]: −1.76 [−2.94, −0.59]), and total antioxidant status (SMD [95% CI]: −1.40 [−2.28, −0.51]) between the patient group and healthy subjects, whereas no significant difference was observed in the superoxide dismutase levels (SMD [95% CI]: −1.20 [−2.55, 0.15]) and glutathione peroxidase levels (SMD [95% CI]: 0.07 [−2.80, 2.94]). Conclusion The present analysis suggests that oxidative stress might have a potential role in the pathogenesis of T2DM in humans. However, further studies should be needed to elucidate the possible mechanism and strengthen this evidence.


| INTRODUCTION
Diabetes mellitus (DM) is a group of metabolic disorders characterized by the elevated levels of glucose in the blood and insufficient secretion or action of endogenous insulin. 1 A report by the International Diabetes Federation in 2017 showed the worldwide prevalence of diabetes in the adult population reached 8.8% (424.9 million people). 2 Among them, the majority (87%-91%) of the cases are with type 2 diabetes. Globally, type 2 diabetes mellitus (T2DM) is considered a major public health concern because of its life-threatening complications with the increasing risk of mortality. 3 Although the exact etiologies of T2DM are not well defined, it is believed that autoimmune disease, genetic, and environmental factors play a major role in developing T2DM. 4 Also, recent studies have shown that with the high level of free-radical generation, oxidative stress (OS) initiates a significant role in developing and progressing T2DM. [5][6][7][8] OS can be defined as an imbalance between the production of reactive oxygen species (ROS) and antioxidant defense by which the body can detoxify its harmful effects and inhibit cell damages. The generation of ROS was thought to be a form of pathological cellular stress, but the current investigation is that ROS formed due to the physiological and homeostatic functions of many cells. However, an excess formation of ROS such as superoxide, hydrogen peroxide, and hydroxyl radicals can cause harmful effects on many cellular structures such as protein, lipids, and nucleic acids. 9 Antioxidants such as catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPX) counter the action of ROS by neutralizing their action or by inhibiting their formation. 10 Thus, a balance might be important between ROS and the levels of the antioxidant; otherwise, OS has been implicated in the pathogenesis of a variety of diseases, including cancer, obesity, diabetes mellitus, cardiovascular diseases, and nonalcoholic fatty liver disease. 11,12 Various studies have reported that significant and abnormal increases in the levels of OS biomarkers associated with T2DM. 13 Cochrane Library were used. The search terms included "type 2 diabetes mellitus," "T2DM," "MDA," "malondialdehyde," "oxidative stress," "antioxidants," "total antioxidant capacity," "total antioxidant status," "superoxide dismutase," "glutathione," "glutathione peroxidase," "nitric oxide," "catalase," "vitamin A," "vitamin C," and "vitamin E." The combinations of different search terms were used for identifying the relevant articles, and the search strategies were customized to suit each database. For the present study, ethical approval is not required, as it is a meta-analysis.

| Criteria for inclusion and exclusion
Inclusion criteria for this study were the following: (a) the study must be a case-control study design; (b) it should be published in a peer- (c) the subjects have any history of other diseases.

| Data extraction and management
Both authors independently performed data extraction using standard extraction spreadsheets from the selected articles based on the inclusion criteria and enlisted in a table. The following items were extracted from each study: author's name (first author), year of publication, country, groups, gender distribution, mean age (years), number of participants (male vs female), MDA concentration, SOD concentration, GSH concentration, GPX concentration, CAT concentration, TAS concentration, and NO concentration. After the extraction of the data, the authors cross-checked the data tables and resolved any conflicts and inconsistencies during the data extraction process through discussion with each other.

| Quality assessment
The quality assessment of all included studies was conducted by using a modified Newcastle-Ottawa Quality Assessment scale, and we adopted three main criteria for selecting the studies in this systematic review: (a) an appropriate and clear study objective/research question/aim; (b) a detailed description of the study population with a valid methodology; and (c) applicability of results.

| Statistical analysis
A statistical software named Review Manager V5.3 (Cochrane Collaboration, Copenhagen, Denmark) was used for the meta-analysis. We calculated the standard mean difference (SMD) with a corresponding 95% confidence interval (CI) for each parameter using the randomeffects model. The SMD was calculated as the ratio of the mean difference to the pooled SD by the z-test. The existence of heterogeneity among studies was evaluated using I 2 and its resultant P-value using chi-squared tests. I 2 values of 25%, 50%, and 75% define the heterogeneity as low, medium, and high heterogeneity, respectively.
The random-effect model and forest plots were adopted as the pooling method, and funnel plots were used to investigate the publication bias. A P-value <.05 was considered a statistically significant difference between groups.

| Search results
As shown in Figure 1, a total of 483 studies initially were identified through different database searching. After the removal of duplication, additional screening, and analysis of the titles and abstracts, 33 articles were included as eligible for this study. Of 33 articles, finally, 22 studies were included in the qualitative and quantitative review and meta-analysis in the study.   [24][25][26]30 reported the association between GPX levels and T2DM (306 cases and 288 controls) ( Table 2), 5. A total of five studies 19,20,29,33 reported the association between TAS levels and T2DM (376 cases and 359 controls) ( Table 2), and 6. A total of four studies 20,21,31,36 reported the association between NO levels and T2DM (217 cases and 176 controls) ( Table 2).

| Association between MDA and T2DM
There were 21 studies to be included in the meta-analysis to evaluate

| Association between SOD and T2DM
The meta-analysis of eight included studies in this systematic review revealed a lower level of SOD in patients with T2DM compared to the controls, but the difference was not statistically significant (SMD  we observed a significant level of heterogeneity among the included studies for SOD (I 2 = 98%, P < .00001) ( Figure 3A).

| Association between GPX and T2DM
We found four papers that reported the GPX activity in diabetic patients. The meta-analysis revealed that there was no significant difference observed in the level of GPX in the patient group when

| Association between TAS and T2DM
The meta-analysis of five included studies reported on TAS activity revealed that the TAS level was significantly lowered in the patient group compared to the control group (SMD [95% CI]: À1.40 [À2.28,

| Association between NO and T2DM
Based on a random-effects meta-analysis, comparing the NO level between the patient group and the control group, a significant differ-  Figure 3E).

| Publication bias
A funnel plot was used to analyze the publication bias in this systematic review and meta-analysis. The visual inspection of funnel plots of OS biomarkers in DM did not suggest potential publication bias except for GPX (Data S2).

| DISCUSSION
Many studies have reported that OS is involved in the pathogenesis of multiple disorders including type 1 and type 2 diabetes. 1,4 To our best knowledge, this is the first systematic review and meta-analysis to find out the evidence on the association of OS biomarkers such as MDA, SOD, GSH, GPX, TAS, and NO levels in patients with T2DM.
From our meta-analysis, we found significantly higher levels of MDA and NO (considered as oxidants), and significantly lower levels of GSH and TAS (known as antioxidants) in patients compared to control subjects. On the other hand, we observed there was no significant difference in the levels of SOD and GPX between patients and control subjects. The overall results revealed that the impaired oxidants and antioxidants balance play a vital role in the pathogenesis of T2DM.
In this meta-analysis, we observed a significantly increased level of MDA in patients with T2DM in almost all studies. Both experimental and clinical studies revealed that free radicals are formed in T2DM by glucose degradation, nonenzymatic glycation of proteins, and subsequent oxidative degradation. [37][38][39] The increased levels of free radicals may lead to lipid peroxidation and the level of MDA is usually measured as a well-known marker of lipid peroxidation. 40,41 As a marker of oxidants, the level of NO in diabetic patients was reported in this study, and the analysis revealed a higher level in patients compared to that of control groups.
It has been reported that the increase in lipid peroxidation is strongly associated with a decline in enzymatic and nonenzymatic antioxidant defense mechanisms. 42 This meta-analysis revealed that the levels of TAS and GSH were significantly lower in diabetic patients compared with control subjects. However, there was no association Although our study followed a standard search strategy in the current meta-analysis, this study has some limitations. The major limitation of this study is that firstly, significant heterogeneity was encountered perhaps due to various regimens, doses, duration, study settings, population enrolled, etc for the analysis of OS biomarkers in the included studies. Secondly, we did not analyze any correlation between the complications of T2DM with OS biomarkers due to inadequate data and also sensitivity analysis of the included studies. Finally, subgroup analysis and meta-regression could not be performed in this meta-analysis due to the limited studies in the literature.

| CONCLUSION
This systematic review and meta-analysis provide evidence that the increased OS has a major role in the pathogenesis and progression of T2DM. Therefore, further studies are needed to strengthen this evidence, especially on the association of SOD and GPX levels with T2DM.

FUNDING
This study did not receive any specific grant from any organizations like the public or commercial.

CONFLICT OF INTEREST
We have declared that we have no competing interests.
All authors have read and approved the final version of the manuscript.
Sujan Banik had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.

TRANSPARENCY STATEMENT
Sujan Banik affirms that this manuscript is an honest, accurate, and transparent account of the study being reported, that no important aspects of the study have been omitted, and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.