Biochemical Alterations Related to Vascular Injury in Smokers: Evidence from Nitric Oxide, Malondialdehyde, Nicotinic Acetylcholine Receptors, and Cotinine

Purpose: To examine simultaneous alterations in nitric oxide (NO), malondialdehyde (MDA), nicotinic acetylcholine receptors (nAChRs), and cotinine levels in smokers compared with non-smokers, and to clarify early mechanistic pathways linking tobacco exposure with vascular-related and oxidative stress. Patients and Methods: A cross-sectional study was conducted among 200 adults (100 smokers, 100 non-smokers) meeting predefined eligibility criteria and free from major cardiometabolic disease. Venous blood samples were analyzed for NO, MDA, nAChRs, and cotinine using standardized ELISA methods. Anthropometric and hemodynamic measurements were obtained using validated procedures. Group differences were assessed using independent t-tests and chi-square tests. Correlations were evaluated using Pearson or Spearman coefficients. Multivariable linear regression models adjusted for age, BMI, systolic and diastolic blood pressure to determine the independent association of smoking with biomarker profiles. Results: Smokers demonstrated significantly lower NO levels (70.22 ± 9.54 vs 198.27 ± 13.85 µmol/L; p < 0.001) and markedly higher concentrations of MDA (39.48 ± 4.54 vs 19.72 ± 5.32 nmol/mL; p < 0.001), nAChRs (20.19 ± 3.25 vs 12.05 ± 2.13 ng/mL; p < 0.001), and cotinine (61.07 ± 8.84 vs 4.99 ± 1.95 pg/mL; p < 0.001). After adjustment, smoking remained a strong independent predictor of reduced NO (β = −128.29; p < 0.001) and elevated MDA (β = 19.89; p < 0.001), nAChRs (β = 8.03; p < 0.001), and cotinine (β = 55.50; p < 0.001). These findings indicate persistent oxidative, nitrosative, and receptor-mediated biochemical disturbances associated with smoking, irrespective of baseline physiological differences. Conclusion: Smokers exhibit a distinct biochemical pattern characterized by reduced NO bioavailability, heightened oxidative stress, increased nAChR activation, and elevated cotinine levels. These alterations reflect early vascular-related biochemical disturbances associated with smoking and support the utility of these biomarkers for early cardiovascular risk detection.