Atherosclerosis is a progressive inflammatory disorder of the arterial wall and the primary pathological substrate underlying cardiovascular diseases, which remain the leading cause of morbidity and mortality worldwide. While dyslipidemia has long been recognized as a major risk factor, increasing evidence indicates that oxidative stress and endothelial dysfunction play central roles in both the initiation and progression of atherosclerotic lesions. The vascular endothelium is essential for maintaining vascular homeostasis through regulation of vasomotor tone, platelet activity, leukocyte adhesion, and smooth muscle proliferation. One of the most critical mediators of endothelial function is nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS). NO exerts potent vasodilatory, anti-inflammatory, anti-thrombotic, and anti-proliferative effects. Reduced NO bioavailability is considered a hallmark of endothelial dysfunction and an early indicator of atherosclerosis. A major mechanism leading to NO depletion is oxidative stress, defined as an imbalance between reactive oxygen species (ROS) generation and antioxidant defenses. Excess ROS rapidly inactivate NO, forming peroxynitrite and other reactive intermediates that further damage endothelial cells and impair eNOS activity. Oxidative stress also promotes oxidation of low-density lipoprotein (LDL), enhancing its uptake by macrophages and accelerating foam cell formation. Malondialdehyde (MDA) is a stable and widely used biomarker of lipid peroxidation and oxidative injury. Elevated circulating MDA levels reflect increased oxidative degradation of polyunsaturated fatty acids in cell membranes and lipoproteins. Clinical studies have demonstrated increased MDA levels in cardiovascular diseases; however, the relationship between MDA, NO depletion, and inflammatory signaling in established atherosclerosis requires further elucidation. Inflammation and oxidative stress are closely interlinked processes in atherogenesis. Pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) enhance ROS production and suppress eNOS expression, while oxidative stress can activate inflammatory transcription factors such as NF-κB, creating a vicious cycle that perpetuates vascular injury. Despite growing recognition of this interplay, clinical studies simultaneously evaluating oxidative stress markers, NO bioavailability, inflammatory mediators, and lipid profile in atherosclerosis remain limited, particularly in the Indian population. Therefore, the present study was designed to investigate oxidative stress-induced endothelial dysfunction in atherosclerosis by assessing serum MDA and NO levels and their associations with inflammatory biomarkers and dyslipidemia.

This was a comparative, cross-sectional observational study conducted in the Department of Biochemistry, Index Medical College, from January 2022 to December 2024. Study Population A total of 220 participants were enrolled and divided into two groups: • Group I (Atherosclerosis): 110 patients aged 40–60 years with clinically, radiologically, or biochemically confirmed atherosclerosis • Group II (Controls): 110 age- and sex-matched healthy individuals with no history of cardiovascular, metabolic, or inflammatory disease Inclusion and Exclusion Criteria Participants with acute infections, autoimmune disorders, malignancy, chronic renal or hepatic disease, recent myocardial infarction, or those on antioxidant or anti-inflammatory therapy were excluded. Smokers, chronic alcoholics, pregnant women, and lactating women were also excluded. Sample Collection After overnight fasting, 5 mL of venous blood was collected aseptically. Serum and plasma were separated by centrifugation and stored at −80 °C until analysis. Biochemical Analysis • Malondialdehyde (MDA): Measured as thiobarbituric acid reactive substances (TBARS) using spectrophotometric method • Nitric Oxide (NO): Estimated by Griess reaction through measurement of nitrite/nitrate levels • Inflammatory Markers (IL-6, TNF-α, NF-κB): Measured using ELISA • Adhesion Molecules (ICAM-1, VCAM-1): Quantified by sandwich ELISA • Lipid Profile: Total cholesterol, triglycerides, HDL-C measured enzymatically; LDL-C calculated using Friedewald’s formula Statistical Analysis Data were analyzed using SPSS version 25. Results were expressed as mean ± standard deviation (SD). Independent Student’s t-test was used to compare groups. Pearson’s correlation coefficient assessed associations between variables. Multiple linear regression identified independent predictors of endothelial dysfunction. A p-value < 0.05 was considered statistically significant.

Baseline Characteristics The two groups were comparable in age and sex distribution. However, body mass index and blood pressure were significantly higher in the atherosclerosis group, reflecting increased cardiometabolic risk. Oxidative Stress Marker (MDA) Serum MDA levels were significantly elevated in patients with atherosclerosis (4.85 ± 1.12 nmol/mL) compared to controls (2.03 ± 0.76 nmol/mL; p < 0.001). This indicates a marked increase in lipid peroxidation and oxidative stress in atherosclerotic patients. Nitric Oxide Levels Mean serum NO levels were significantly lower in the atherosclerosis group (14.2 ± 3.6 µmol/L) compared to healthy controls (27.8 ± 4.1 µmol/L; p < 0.001), reflecting impaired endothelial NO production and bioavailability. Association with Inflammatory Biomarkers Correlation analysis revealed a strong positive correlation between MDA and TNF-α (r = 0.71, p < 0.001), indicating close interaction between oxidative stress and inflammation. NO levels showed a significant inverse correlation with IL-6 (r = −0.60, p < 0.001), suggesting inflammation-mediated suppression of endothelial NO. Multivariate Regression Analysis Multiple linear regression identified MDA as an independent positive predictor (β = 0.34, p < 0.001) and NO as an independent negative predictor (β = −0.36, p < 0.001) of endothelial dysfunction, even after adjusting for inflammatory variables and lipid parameters. Table 1. Baseline Clinical Characteristics of Study Participants Parameter Atherosclerosis Group (n = 110) Control Group (n = 110) p-value Age (years) 52.3 ± 6.1 51.7 ± 5.8 0.48 Sex (Male : Female) 68 : 42 66 : 44 0.72 Body Mass Index (kg/m²) 26.5 ± 3.2 24.8 ± 2.9 0.001* Systolic BP (mmHg) 138 ± 12 124 ± 10 <0.001* Diastolic BP (mmHg) 86 ± 8 78 ± 7 <0.001* Values expressed as mean ± SD. *Statistically significant (p < 0.05) Table 2. Oxidative Stress and Endothelial Function Markers Biomarker Atherosclerosis Group Control Group p-value Malondialdehyde (nmol/mL) 4.85 ± 1.12 2.03 ± 0.76 <0.001* Nitric Oxide (µmol/L) 14.2 ± 3.6 27.8 ± 4.1 <0.001* Elevated oxidative stress with significant nitric oxide depletion in atherosclerosis. Table 3. Inflammatory Biomarkers Associated with Oxidative Stress Biomarker Atherosclerosis Group Control Group p-value NF-κB (ng/mL) 6.72 ± 1.34 3.01 ± 1.12 <0.001* IL-6 (pg/mL) 5.98 ± 1.22 2.53 ± 0.98 <0.001* TNF-α (pg/mL) 7.10 ± 1.45 3.62 ± 1.05 <0.001* Enhanced inflammatory milieu accompanying oxidative injury. Table 4. Endothelial Adhesion Molecules Adhesion Molecule Atherosclerosis Group (ng/mL) Control Group (ng/mL) p-value ICAM-1 320.4 ± 55.3 178.9 ± 40.2 <0.001* VCAM-1 512.7 ± 60.8 296.1 ± 45.5 <0.001* Oxidative–inflammatory endothelial activation in atherosclerosis. Table 5. Lipid Profile and Oxidative Stress Association Lipid Parameter Atherosclerosis Group (mg/dL) Control Group (mg/dL) p-value Total Cholesterol 234.6 ± 32.5 178.2 ± 28.6 <0.001* LDL-C 148.9 ± 28.1 97.4 ± 20.3 <0.001* HDL-C 34.5 ± 6.8 49.3 ± 7.1 <0.001* Triglycerides 186.2 ± 44.6 126.9 ± 36.2 <0.001* Atherogenic dyslipidemia contributing to lipid peroxidation. Table 6. Pearson Correlation Between Oxidative Stress, NO, and Inflammation Correlation Pair r-value p-value MDA vs TNF-α +0.71 <0.001* MDA vs NF-κB +0.66 <0.001* NO vs IL-6 −0.60 <0.001* NO vs TNF-α −0.58 <0.001* Strong oxidative–inflammatory coupling and NO suppression. Table 7. Multiple Linear Regression Analysis: Predictors of Endothelial Dysfunction Variable β-Coefficient Standard Error p-value Malondialdehyde (MDA) 0.34 0.07 <0.001* Nitric Oxide (NO) −0.36 0.09 <0.001* TNF-α 0.29 0.08 <0.01* IL-6 0.27 0.07 <0.01* Oxidative stress and NO depletion independently drive endothelial dysfunction

The present study provides strong clinical evidence that oxidative stress-induced lipid peroxidation plays a critical role in endothelial dysfunction in atherosclerosis. The significantly elevated MDA levels observed in patients reflect enhanced oxidative degradation of lipids, which contributes directly to endothelial injury and plaque progression. Oxidative stress is a major determinant of NO depletion in atherosclerosis. Excess ROS rapidly inactivate NO and impair eNOS function, leading to reduced vasodilation and a pro-inflammatory, pro-thrombotic endothelial phenotype. The markedly reduced NO levels observed in this study are consistent with previous reports identifying NO deficiency as an early and persistent feature of atherosclerosis. The strong inverse relationship between NO and IL-6 suggests that inflammatory cytokines further exacerbate endothelial dysfunction by suppressing NO synthesis. Conversely, the positive association between MDA and TNF-α highlights the bidirectional relationship between oxidative stress and inflammation, wherein each process amplifies the other. Dyslipidemia observed in the atherosclerosis group likely contributes to oxidative stress through increased availability of oxidizable lipoproteins, particularly LDL. Oxidized LDL further enhances ROS production and inflammatory signaling, accelerating vascular damage. Importantly, regression analysis confirmed that MDA and NO independently predict endothelial dysfunction, emphasizing their clinical relevance beyond traditional lipid markers and inflammatory cytokines.

Oxidative stress-induced lipid peroxidation, reflected by elevated malondialdehyde levels, plays a pivotal role in endothelial dysfunction in atherosclerosis by impairing nitric oxide bioavailability and amplifying inflammatory signaling. Assessment of MDA and NO may provide valuable insights into disease severity and progression and represent potential targets for therapeutic intervention. Acknowledgments The authors acknowledge the Department of Biochemistry, Index Medical College, for technical and laboratory support.

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