European Journal of Cardiovascular Medicine

Diabetes Mellitus (DM) is one of the main chronic health condition affecting millions of people worldwide in both developed and developing countries [1,2]. In India more than 77 million have been affected making it the diabetic capital of the world [3]. DM is characterized by hyperglycemia which is also associated with central obesity, dyslipidemia and hypertension [4,5]. However, plenty of population bear unnoticed hyperlipidemia, marked by elevated triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) [6].

Hyperglycemic conditions have harmful effects on the microvasculature of different body parts, especially the eyes. Microvascular abnormalities in the retina lead to many abnormalities, including neovascularization, macular edema, and retinal detachment [7]. The underlying mechanism includes abnormal metabolic pathways, oxidative stress and subclinical inflammation; however the specific mechanism is not yet fully understood. C-reactive protein (CRP) is an important and highly accurate predictor of future heart disease. CRP is a well-known inflammatory marker and acute phase reactant whose relative diagnostic and prognostic role have determined in many musculoskeletal disorders, liver and kidney disorders. Although few attempts have been made to show its role in diabetic patients especially in those with retinopathic changes, yet the role of CRP in the pathogenesis of DR is still unknown [9].

Oxidative stress might also cause damage to the cells which leads to several microvascular complications like diabetic neuropathy, nephropathy and retinopathy and macro vascular complications like stroke and coronary artery disease (CAD). Malondialdehyde (MDA) is a highly reactive lipid peroxidation end product that might be helpful in accessing the risk of DR and its related complications [10]. The activity of antioxidants defence system also change with the regulatory proteins involved in the mechanism of antioxidant defense, making it vital to analyses along with other markers of oxidative stress [11].

The aim of our present study was to estimate oxidative stress and inflammatory markers in type 2 diabetic patients with and without retinopathy.

2.1 Study population

This case–control study was conducted on a total of 100 individuals>40 years of age. Clinical data was obtained from 50 Type 2 Diabetic Retinopathy patients (cases) and 50 age/sex matched Type 2 diabetic patients without retinopathy (controls)who attended Department of Medicine and Department of Ophthalmology, FH Medical College, Agra between December 2022 and May 2023. Diagnosis of Diabetic Retinopathy was based on fundus examination. Personal and medical history such as age, sex, occupation, socio-economic status and associated risk factors etc. was taken based on standardized questionnaire. Body mass index (BMI) was calculated using formula: weight (kg)/height² (m²).Patients having history of Coronary Kidney Disease, Chronic Obstructive Pulmonary Disease, cancer, infectious diseases (TB, HIV and Hepatitis), hypothyroidism, Glaucoma, pre-existing Macular hole/retinal diseases or diabetic patients with <8 years of confirmed cases of Diabetic Retinopathy were excluded.

2.2 Specimen collection and laboratory assays

Under all aseptic blood was collected in the morning following overnight fasting. Venous blood was drawn using a disposable syringe and collected in fluoride, EDTA and plain vial. Blood glucose was estimated using GOD-POD end point colorimetric method. Lipid profile parameters (Total cholesterol, HDL‑C, and triglycerides) were measured using kit method on semi-autoanalyzer. Oxidative stress marker MDA and SOD were estimated using laser and Cushman (1996) method and Pyrogallol method respectively. Inflammatory marker HsCRP were determined by enzyme immunoassay (EIA) method according to manufacturer’s protocol.

2.3 Statistical analysis

The results were analysed by comparing case and control groups. Quantitative data were summarized as mean ± standard error. Unpaired t-test and Pearson’s correlation was performed using SPSS version 25.The significance level was taken as p<0.05.

2.4 Ethics statement

Study was approved by the Institutional Ethics Committee, F.H. Medical College (FHMC/IEC/R.Cell/2022/24). A written informed consent was obtained from each subject.

3.1 Baseline characteristics and biochemical parameters

Mean age among type 2 diabetes with retinopathy and without retinopathy group were 71.66 ± 13.07 years and 50 ± 8.01 years respectively and showed highly significant statistical difference. Majority of diabetic retinopathy patients (66%) were in age group >70 years whereas, majority of diabetic patients without retinopathy (60%) were in age group 30-50 years.The mean Weight, height and BMI levels showed no statistically significant difference between the two groups (Fig. 1 and 2). Fasting and post-prandial blood glucose as well as HbA1c were significantly elevated in diabetic retinopathy group compared without retinopathy (Fig 3).

Serum lipids (TG and VLDL) were also significantly elevated in diabetic retinopathy group compared without retinopathy. However, the difference was non-significant for LDL and total cholesterol. HDL was significantly deceased in diabetic retinopathy group compared without retinopathy (p-value <0.001) (Fig 4).

Oxidative stress markers (MDA and SOD) and inflammatory marker (HsCRP) were also found to be elevated in diabetic retinopathy group compared to diabetic patients without retinopathy(Fig 5 and 6).All baseline characteristics and biochemical parameters of the cases and controls included in this study are presented in Table 1.

Table-1: Baseline characteristics and biochemical parameters of the enrolled cases and controls

Figure 1: Graph showing age wise distribution of subjects among case and control group

Figure 2: Graph showing mean±SD of age, height, weight and BMI between case and control group

Figure 3: Graph showing mean±SD of HbA1c, fasting and post prandial blood sugar between case and control group

Figure 4: Graph showing mean±SD of total cholesterol, triglycerides, HDL, LDL and VLDL between case and control group

Figure 5: Graph showing mean±SD of Oxidative stress marker MDA and SOD between case and control group

Figure 6: Graph showing mean±SD of Inflammatory marker HsCRP between case and control group

3.2Correlation of biochemical parameters among themselves

Serum MDA levels show positive correlation with SOD in both case and control group. However the correlation was found to be strong and significant for control group (p-value 2.56R-06) compared to case group which showed non-significant positive correlation of MDA with SOD(Table 2, Fig 7)

Table-2: Pearson’s correlation between MDA sand SOD in enrolled cases and controls

Figure 7: Scatter diagram showing correlation between SOD and MDA in (A) control and (B) case group

3.3 Visual A and sign of DR on Fundus Examination in Cases

Values of Visual A showed highly significant increase in diabetic retinopathy group compared to diabetic patients without retinopathy (Table 3, Fig 8).

On fundus examination 1/50 (2%) showed non proliferative diabetic retinopathy (NPDR), MILD DR, 16/50 (32%) showed NPDR, mod DR to severe DR, 11/50 (22%) showed NPDR, severe DR to very severe DR, 2/50 (4%) showed proliferative diabetic retinopathy (PDR), mild to mod DR, 7/50 (14%) showed PDR, High risk DR and 13/50 (26%) showed PDR, Advanced DR (Table 4, Fig 9)

Table-3: Mean±SD of Visual A in enrolled cases and controls

Visual A

Figure 8: Graph showing mean±SD of Visual A between case and control group

Table-4: Sign of DR on Fundus Examination in cases

Figure 9: Pie chart showing No. of cases with sign of DR on Fundus Examination.

Investigating the oxidative stress and inflammatory markers in serum of diabetic patients with and without retinopathy might reveal possible markers for disease control and monitoring its progressions they cause mitochondrial dysfunction and cell death leading to damaged retina and blood vessels.

In this study we observed age to be a partially contributing factor in the disease progression as majority of DR patients are in the age group >70 years and majority of patients without retinopathy were in the age group of 30-50years.Similar prevalence of DR with increasing age was reported in other epidemiological study by Rema M et al [12].However a non-significant decrease was observed in BMI of DR group compared to patients without retinopathy.

We also observed significantly elevated fasting and post-prandial blood glucose and HbA1c levels in DR group compared to controls. The elevation of TG and VLDL and decrease in the levels of HDL was significantly noticeable in the DR group. However, TC and LDL showed no difference in the mean of both groups.

We also observed significant increase in mean levels of MDA and SOD and a positive association among them in both the study groups. However, the association was significant in the control group. Previous studies also reported similar findings [10].Anuradha P et al. studied oxidative stress in DR patients compared to controls and reported higher serum MDA levels in DR patients[13]. Another case-control study by Brzović-Šarić V et al. on analysis of oxidative stress markers in vitreous and serum of DR patients and reported 3-4 times higher activity of overall serum SOD and MDA levels in PDR than NPDR, suggesting MDA monitoring in preventing diabetic complications[11]. The reason that oxidative stress is increased in DR patients might be due to enhanced influx of glucose via polyol and hexosamine pathways activates protein kinase C and contributes to enhanced production of advanced glycation end products which generates Reactive Oxygen Species (ROS) and exaggerate oxidative stress, however, the exact underlying mechanism still remain elusive [14]. The fact that retina has increased content of polyunsaturated fatty acid (PUFA) and elevated oxygen uptake/glucose oxidation that makes it more sensitive to oxidative stress [15].Mitochondria are major source of SOD under normal condition and increased lipid peroxidation due to ROS also damages retinal mitochondria making it leaky with increased duration and severity of diabetes, thus, making its level elevated in pathologic conditions [16].

We also observed increased inflammatory marker HsCRP in DR group compared to diabetic group without retinopathy. Several studies also reported similar findings. Song J et al. reported positive association of elevated HsCRP levels with increased severity of DR and reported higher HsCRP levels in the PDR group compared to NPDR group [8].Qui F et al. showed that overexpression of HsCRP deteriorated retinal neurodegeneration, also inducing cell death and ROS overproduction in retinal cell lines, thus, playing a pathogenic role in DR[17].Nada WM et al. also reported significantly increased HsCRP levels in PDR group compared to normal controls and diabetic patients without retinopathy, however, they concluded, CRP to be considered as biomarker for PDR only as it is not a good indicator for follow up patients [18].In contrast, study performed by Lim LS et al to determine the relationship between HsCRP and DR reported that persons with higher levels ofHsCRPand BMI were less likely to have DR. Possible explanation could be proangiogenic properties of CRP or DR patients could have undergone  behavioural modifications leading to lower CRP and BMI levels[19],whereas Hernández-Da Mota SE et al. reported no significant difference in HsCRP levels among diabetic patients with and without retinopathy and control group[20].

Our study may have some limitations like bias from lifestyle habits like diet, smoking and alcohol status etc. may exist and could possibly affect severity of DR.Information regarding co-morbidities was also limited. Our study was also conducted on a small sample size, thus may not be generalized to all regions/ethnicities.

In conclusion, the result of this study have shown significant elevation in the levels of MDA, SOD and HsCRP in DR group which suggests role of oxidative stress and inflammation in the pathogenesis of DR and its severity. Lifestyle modifications that could help in maintaining blood glucose levels and improving insulin sensitivity that could further help in reducing oxidative stress and inflammation. Hence, reducing the chances of progression to diabetic complications.

1. AUTHOR’S CONTRIBUTION

Anas M. contributed in sample collection and performing laboratory investigations and manuscript writing. Batham V. contributed in diagnosis of diabetic retinopathy and proving clinical samples as well as reviewing manuscript. Iqubal F. performed and Amir AH supervised in designing, data analysis and review of the manuscript.

2. ACKNOWLEDGMENTS

All the authors would like to thank all the study participants recruited in this study and staff members of department of Ophthalmology and Department of Biochemistry, FHMC for their co-operation.

3. FUNDING

This research received no funding from any government or private institutions.

4. CONFLICT OF INTERESTS

The authors declare no conflict of interests.

5.  DATA STATEMENT

Data used in this study are available at request from the corresponding author.

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