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

Introduction

Tobacco smoking continues to represent one of the most influential modifiable determinants of global cardiovascular morbidity and mortality.¹ Chronic exposure to toxicants in cigarette smoke disrupts endothelial homeostasis through biochemical, inflammatory, and autonomic mechanisms that progressively impair vascular structure and function. Alterations in endothelial-related vascular homeostasis are widely recognized as an early pathophysiological hallmark preceding the development of atherosclerosis, hypertension, and major adverse cardiovascular events.^2,3 Among the molecular determinants of endothelial health, nitric oxide (NO) plays a central vasoprotective role by regulating vascular tone, inhibiting platelet aggregation, and preventing adhesion of inflammatory cells to the endothelium.³ Reduced bioavailability of NO is consistently observed in smokers and is considered one of the earliest measurable features of tobacco-induced vascular injury.

A dominant mechanism underlying diminished NO activity in smokers is the heightened oxidative stress triggered by reactive oxygen species (ROS) derived from cigarette smoke.⁴ These oxidants rapidly interact with NO, forming peroxynitrite and other reactive nitrogen species that further damage lipids, proteins, and DNA.⁵ Malondialdehyde (MDA), a terminal product of lipid peroxidation and a widely used biomarker of oxidative stress, reflects cumulative oxidative injury within biological membranes.⁶ Elevated MDA levels among smokers suggest ongoing peroxidative damage that amplifies endothelial dysfunction by compromising membrane integrity, altering receptor function, and disrupting intracellular signaling pathways. Although the independent roles of NO depletion and MDA elevation in vascular impairment are well-documented, the interplay of these two markers as a dynamic oxidative–nitrosative axis in early vascular injury remains insufficiently characterized.

In parallel with oxidative stress, nicotine exposure activates nicotinic acetylcholine receptors (nAChRs), particularly the α7 subtype, which modulate vascular smooth muscle tone, autonomic signaling, inflammation, and ROS-generating enzymatic pathways.^7,8 Chronic activation of nAChRs contributes to endothelial impairment through increased sympathetic outflow, enhanced oxidative enzyme activity, and altered calcium handling within vascular tissues. Given the mechanistic convergence between nicotine-induced receptor activation and oxidative–nitrosative imbalance, nAChR expression offers an important but underexplored biomarker linking smoking exposure with vascular-related biological alterations. Cotinine, the primary and stable metabolite of nicotine, serves as a reliable biochemical indicator of tobacco exposure and enables quantification of smoking severity in conjunction with behavioral indices such as the Brinkman Index.^9,10 Combining cotinine levels with mechanistic biomarkers may therefore improve precision in assessing the biological impact of smoking on vascular injury.

Despite substantial evidence describing isolated pathways of NO depletion, oxidative stress, and nicotine receptor activation, few studies integrate these biomarkers into a unified early pathomechanistic framework of cardiovascular risk. Understanding this multidimensional interaction is essential for characterizing the nascent vascular alterations that precede clinically detectable disease. Such insight holds considerable value not only for early risk stratification but also for developing targeted preventive strategies in at-risk populations. From a public health standpoint, establishing sensitive and biologically relevant biomarkers of early vascular injury is critical in regions with high smoking prevalence, where cardiovascular disease imposes a substantial economic and clinical burden.

In our previously published study, we demonstrated that nicotine exposure, quantified by serum cotinine, was associated with vascular inflammation and oxidative stress through increased interleukin-6 (IL-6) expression and reduced superoxide dismutase (SOD) activity, highlighting an inflammatory–antioxidant imbalance in smokers.¹¹ Building upon these findings, the present study extends the investigation to distinct and previously unexamined mechanistic domains, namely endothelial dysfunction (nitric oxide), lipid peroxidation (malondialdehyde), and nicotinic acetylcholine receptor (nAChR) activation. While cotinine is retained as an exposure-verification biomarker, the primary outcomes, biological pathways, and analytical framework of the current study are fundamentally different, providing novel insight into endothelial, oxidative, and receptor-mediated mechanisms underlying smoking-related cardiovascular risk. By analyzing these interrelated pathways within a single framework, the study aims to generate a more comprehensive biochemical profile of smoking-induced vascular injury. Such an integrative approach may enhance the early detection of endothelial dysfunction, broaden mechanistic understanding, and support improved strategies for cardiovascular risk management.

Materials and Methods

Study Design and Participants

This analytical cross-sectional study included adult smokers and non-smokers recruited using a consecutive sampling approach from the local campus and community health center. Eligible participants were adults aged 18–65 years who were free from clinically diagnosed cardiovascular disease, diabetes mellitus, chronic kidney disease, chronic liver disease, autoimmune disorders, acute infections, and who had not used antioxidant supplements within the preceding month. Women who were pregnant or lactating were excluded. Participants were classified as smokers if they reported daily cigarette consumption for at least one year and had biochemical confirmation of exposure through elevated serum cotinine levels. Smoking intensity was further quantified using the Brinkman Index (cigarettes per day × years of smoking). Non-smokers were defined as individuals who had never smoked and demonstrated serum cotinine levels within the established non-smoking reference range. To minimize misclassification bias, both self-reported smoking status and biochemical verification (cotinine) were applied concurrently. Participants with inconsistent self-report and biochemical results were excluded from the analysis.

A priori sample size estimation was guided by effect sizes reported in recent literature evaluating NO and oxidative stress biomarkers in smokers. Given recruitment constraints, the final sample size was determined adequate for detecting moderate effect sizes. Demographic variables collected included age, sex, employment status, education level, marital status, years of smoking, cigarettes per day, Brinkman Index, blood pressure, BMI, and metabolic laboratory parameters.

Ethical Considerations

The research was carried out in compliance with the Declaration of Helsinki and was approved by the Ethics Committee of Poltekkes Kemenkes Malang, with authorization number No.DP.04.03/F.XXI.31/01104/2024. Written informed consent was obtained from each participant prior to enrollment. Participant identities were anonymized using coded identifiers.

Biochemical Measurements

Sample Collection and Handling

Venous blood samples were collected following an overnight fast of at least 8 hours. Serum was allowed to clot for 15–20 minutes at room temperature and centrifuged at 2000–3000 rpm for 20 minutes, consistent with manufacturer recommendations. Hemolyzed samples were excluded. Samples were stored at –20°C and analyzed within one month to minimize degradation.

To ensure reproducibility, all ELISA assays were performed in duplicate. Measurements with coefficient of variation (CV) exceeding 10% were repeated.

Measurement of Nitric Oxide (NO)

Serum NO concentrations (µmol/L) were analyzed using a sandwich ELISA (Human Nitric Oxide ELISA Kit, Cat. No. E1510Hu). Sensitivity was 1.12 µmol/L, with a detection range of 2–600 µmol/L. Absorbance was measured at 450 nm, and concentrations calculated using four-parameter logistic (4PL) regression.

Measurement of Malondialdehyde (MDA)

Serum MDA (nmol/mL) was measured using the Human MDA ELISA Kit (Cat. No. E1371Hu). The sensitivity was 0.14 nmol/mL with a range of 0.2–70 nmol/mL. Absorbance was measured at 450 nm following manufacturer instructions.

Measurement of Nicotinic Acetylcholine Receptors (nAChRs)

Serum nAChRs (ng/mL) were quantified using the Human N-AChR ELISA Kit (Cat. No. E4501Hu). Sensitivity was 0.053 ng/mL with a range of 0.1–40 ng/mL. ELISA procedures followed the standard sandwich method with HRP detection.

Measurement of Cotinine

Serum cotinine (pg/mL) was measured using the Human Cotinine ELISA Kit (Cat. No. E2043Hu). Sensitivity was 0.019 pg/mL with a standard curve range of 0.5–80 pg/mL. Cotinine levels were used to verify smoking status and quantify exposure intensity.

Clinical and Metabolic Assessments

Blood pressure was measured using a validated automated sphygmomanometer after participants were seated for 5 minutes. Two readings were obtained and averaged. BMI was calculated as weight (kg)/height (m²). Lipid profile, fasting glucose, and C-reactive protein (CRP) were measured using automated analyzers in an accredited laboratory.

Statistical Analysis

Statistical analyses were performed using SPSS version 25. Normality of continuous variables was assessed using the Shapiro–Wilk test. Group differences between smokers and non-smokers were evaluated using independent t-tests for continuous variables and Chi-square tests for categorical variables. Correlation analyses were conducted using Pearson’s correlation coefficients for normally distributed variables and Spearman’s rank correlation when appropriate.

To evaluate the independent association between smoking status and biomarker levels, multivariable linear regression models were constructed. Adjustment was limited to age, BMI, systolic blood pressure, and diastolic blood pressure because these variables differed significantly between groups and are biologically established confounders known to influence vascular biology, oxidative stress, and metabolic biomarker expression. Additional variables such as lipid profile, glucose, and smoking exposure metrics (years of smoking, cigarettes per day, and Brinkman Index) were not included as covariates because they represent potential mediators of the smoking–biomarker pathway or introduced substantial multicollinearity that compromised model stability. Regression coefficients (β), standard errors, and p-values were reported, with statistical significance set at p < 0.05.

Potential confounding variables were carefully considered during the analytical phase. Age, body mass index (BMI), systolic blood pressure, and diastolic blood pressure were included as covariates in multivariable regression models because they differed significantly between groups and are well-established determinants of endothelial function and oxidative stress. Other potential confounders, including dietary patterns, alcohol consumption, and physical activity, were not included in the final models due to the absence of standardized quantitative measurements and to avoid model overfitting. However, major comorbid conditions were controlled through strict exclusion criteria at the recruitment stage, thereby reducing their potential confounding effect.

Results

Table 1 presents the baseline characteristics of participants in the smoker and non-smoker groups, including demographic, anthropometric, and blood pressure parameters. The objective of this section is to assess group comparability and identify statistically significant differences that may influence the interpretation of subsequent biomarker analyses.

Table 1 Baseline Participant Features Stratified by Smoking Status

Significant differences were observed between groups, with smokers being younger and having higher BMI compared with non-smokers. Systolic and diastolic blood pressures were also significantly elevated among smokers, indicating a less favorable cardiovascular profile. Gender distribution differed markedly, with smokers dominated by males. Educational attainment also varied significantly, with non-smokers more frequently achieving university education levels. These findings highlight important baseline disparities that must be considered when interpreting biomarker outcomes.

Significant differences in all measured biomarkers were observed between smokers and non-smokers (Table 2). Smokers demonstrated markedly lower NO concentrations compared with non-smokers (p < 0.001), reflecting reduced nitric oxide bioavailability suggestive of impaired endothelial-related vascular homeostasis. Conversely, MDA levels were substantially higher in smokers (p < 0.001), consistent with increased lipid peroxidation and heightened oxidative stress. Levels of nAChRs were also significantly elevated among smokers (p < 0.001), supporting nicotine-induced receptor activation as a relevant mechanistic pathway. Cotinine concentrations exhibited the most pronounced difference between groups (p < 0.001), reinforcing its reliability as a biochemical indicator of tobacco exposure. Together, these findings reflect a coherent biological profile in smokers characterized by altered endothelial-related signaling, enhanced oxidative stress, and activation of nicotinic receptor pathways.

Table 2 Comparison of Biomarker Levels Between Smokers and Non-Smokers

Given these substantial unadjusted differences in biomarker profiles, it was essential to evaluate whether these associations persisted after accounting for baseline imbalances between groups. Adjusted analysis was necessary to address baseline differences between smokers and non-smokers that could confound the associations observed in unadjusted comparisons. Variables such as age, BMI, systolic blood pressure, and diastolic blood pressure differed between groups and are known determinants of vascular regulation, oxidative stress, and metabolic biomarker levels. Without adjustment, these imbalances could bias the estimation of smoking effects, either inflating or masking true biological differences. Incorporating these covariates into multivariable regression models allowed for a clearer interpretation of the independent impact of smoking on nitric oxide, oxidative stress markers, and nicotinic receptor activation.

The multivariable adjusted analysis evaluating the independent association between smoking status and biomarker levels after controlling for age, BMI, systolic blood pressure, and diastolic blood pressure. Adjustment was limited to age, BMI, and blood pressure because these variables differed significantly between groups and are biologically established confounders of endothelial function and oxidative stress, whereas additional variables acted as mediators or introduced multicollinearity that compromised model stability. Adjusted effects were estimated using multivariable linear regression models (Table 3).

Table 3 Adjusted Linear Regression Models Evaluating the Impact of Smoking on Biomarker Profiles

Discussion

This study demonstrates a coherent pattern of biochemical alterations in smokers characterized by markedly reduced nitric oxide (NO) levels, elevated malondialdehyde (MDA), increased nicotinic acetylcholine receptor (nAChR) expression, and substantially higher cotinine concentrations. These findings persisted even after adjustment for age, body mass index, and blood pressure, underscoring smoking as an independent determinant of early vascular-related biochemical alteration, oxidative stress, and nicotine-related receptor activation.¹² Collectively, the results support the presence of a multifaceted pathomechanistic pathway linking tobacco exposure with nascent vascular injury.

Smokers exhibited significantly lower NO concentrations compared with non-smokers, a finding consistent with the inhibitory effects of cigarette-derived oxidants on endothelial nitric oxide synthase (eNOS) activity.^13,14 Reduced NO bioavailability represents a central hallmark of endothelial-related vascular regulation and has been implicated in the early development of atherosclerosis, hypertension, and impaired vascular reactivity.^2,15–17 The magnitude of NO reduction in this study, even after multivariable adjustment, suggests that diminished NO results directly from biochemical pathways triggered by chronic smoking.

Beyond the simple reduction in circulating NO concentrations, impaired NO dynamics in smokers reflect deeper molecular consequences. Chronic exposure to tobacco toxicants induces eNOS uncoupling, a dysfunctional state wherein the enzyme produces superoxide rather than NO.^4,18 This phenomenon further amplifies oxidative stress and perpetuates a vicious biochemical cycle. Emerging studies demonstrate that smokers show reduced tetrahydrobiopterin (BH4) availability, a key eNOS cofactor, thereby impairing NO synthesis and exacerbating vascular stiffness.^19–21 Such changes may occur long before overt clinical symptoms emerge, suggesting that NO depletion is a highly sensitive early marker of vascular injury.

Mechanistically, reactive oxygen species (ROS) from cigarette smoke rapidly scavenge NO to form peroxynitrite, thereby reducing vasodilatory capacity and promoting nitrative stress.^22,23 This interaction not only lowers circulating NO but also impairs endothelial signaling pathways required for vascular homeostasis, platelet inhibition, and anti-inflammatory regulation.^24,25 The marked decline in NO observed in the smoker group suggests substantial disruption of endothelial redox balance at a subclinical stage.¹⁴

The downstream consequences of chronic NO depletion are far-reaching. Reduced NO levels shift the vascular environment toward a more vasoconstrictive, pro-inflammatory, and pro-thrombotic phenotype. Leukocyte adhesion, platelet activation, and smooth muscle cell migration are all enhanced when NO bioavailability is impaired.^26,27 These processes converge to create a vascular milieu that predisposes individuals to early atherogenesis. Furthermore, diminished NO signaling increases sympathetic vascular tone, which may partly explain the higher systolic and diastolic pressures observed among smokers in this study.^28,29

MDA levels were significantly higher among smokers, reflecting amplified lipid peroxidation induced by ROS and aldehyde-reactive compounds contained in cigarette smoke.³⁰ MDA is widely recognized as a stable end-product of polyunsaturated fatty acid oxidation and serves as an indicator of cumulative oxidative membrane injury.³¹ The substantial increase in MDA observed in smokers indicates continuous oxidative stress burden, which likely contributes to endothelial damage, eNOS uncoupling, and impaired vascular signaling.³²

Importantly, elevated MDA levels provide insight not only into oxidative injury but also into the structural consequences of chronic lipid peroxidation.³⁰ Lipid oxidation products such as MDA can form adducts with membrane proteins, altering receptor function, impairing signal transduction, and damaging cellular organelles.^31,32 These events diminish the resilience of endothelial cells and amplify susceptibility to apoptosis. Recent studies suggest that peroxidation products may also modulate gene expression via redox-sensitive transcription factors such as NF-κB, thereby enhancing inflammatory cascades that contribute to vascular remodeling.^33,34 Thus, elevated MDA should be viewed not simply as a passive byproduct of oxidation but as an active mediator of vascular oxidative injury.

The strong independent association between smoking status and MDA levels after adjustment supports the role of oxidative stress as a direct mechanistic pathway rather than a consequence of demographic differences. Chronic smokers demonstrate upregulation of oxidative enzymatic systems such as NADPH oxidase and mitochondrial ROS production.³⁵ Persistent oxidative stress not only reduces NO but also modifies endothelial lipids and proteins, thereby accelerating vascular dysfunction.³⁶

The elevated nAChRs concentrations observed in smokers highlight the role of chronic nicotine exposure in modulating vascular autonomic and inflammatory pathways. Nicotine binds predominantly to the α7-nAChR subtype, triggering downstream signaling that influences calcium influx, oxidative enzyme activation, sympathetic stimulation, and inflammatory cytokine release.³⁷ These effects collectively may contribute to endothelial dysfunction, vascular stiffness, and increased cardiovascular risk.

nAChRs have historically been studied in the context of neuronal signaling, but their expression in endothelial and vascular smooth muscle cells has gained increasing attention. Activation of α7-nAChR in vascular tissue has been shown to increase intracellular Ca²⁺ levels, activate MAPK pathways, and potentiate ROS generation via mitochondrial mechanisms.³⁸ Evidence also suggests that nAChRs stimulation can impair endothelial barrier integrity by modulating cytoskeletal dynamics, thereby increasing vascular permeability.³⁹ These mechanisms present a compelling rationale for considering nAChRs expression as a prognostic biomarker of smoking-induced vascular injury, complementing the information provided by NO and MDA.

The persistence of elevated nAChRs after adjustment indicates that receptor activation reflects a direct biological response to nicotine rather than confounding factors. Their upregulation thus represents a mechanistic bridge linking nicotine exposure with altered vascular endothelial-related signaling.³⁹

As expected, cotinine levels in smokers were markedly higher than in non-smokers, reinforcing its status as the most reliable biomarker for validating smoking status and quantifying exposure intensity. The large effect size in the adjusted models confirms that cotinine strongly discriminates smokers from non-smokers and provides a stable index of systemic nicotine exposure.^40,41 When interpreted alongside NO, MDA, and nAChR alterations, cotinine enhances the biological plausibility of the mechanistic pathways identified in this study.

The combined alterations in NO, MDA, and nAChRs support a unified model in which smoking disrupts vascular homeostasis through synergistic oxidative, inflammatory, and receptor-mediated mechanisms.⁴² Reduced NO bioavailability appears to be driven by both increased ROS-mediated scavenging and potential eNOS dysfunction.⁴³ Elevated MDA reflects chronic oxidative lipid damage that compromises endothelial stability.^30,32 Concurrently, nAChRs activation augments oxidative stress and autonomic imbalance.

This constellation of biomarker changes underscores a multifaceted early vascular injury profile in smokers, even in the absence of clinically manifest cardiovascular disease. These findings expand current understanding by demonstrating that the interplay among nitric oxide depletion, oxidative stress, and receptor activation can be detected at the subclinical stage. This supports their relevance as early indicators of cardiovascular risk.

The biochemical pattern identified in this study has important implications for cardiovascular risk stratification, particularly in populations with high smoking prevalence. A mechanistic biomarker panel combining NO, MDA, nAChRs, and cotinine may improve sensitivity for detecting early vascular impairment compared with conventional risk factors alone. The integration of biochemical and hemodynamic predictors may be especially useful in younger adults, in whom traditional risk algorithms often underestimate cardiovascular risk. Additionally, these biomarkers may serve as intermediate endpoints for evaluating the efficacy of smoking cessation therapies, antioxidant supplementation, or lifestyle interventions targeting endothelial health.^44,45

Our previous study demonstrated that nicotine exposure, quantified by serum cotinine, was associated with vascular inflammation and antioxidant imbalance through increased interleukin-6 expression and reduced superoxide dismutase activity, highlighting an inflammatory–oxidative stress pathway in smokers.¹¹ In contrast, the present study advances this work by examining distinct mechanistic endpoints that were not evaluated previously, namely endothelial dysfunction as reflected by nitric oxide bioavailability, lipid peroxidation assessed by malondialdehyde levels, and nicotinic acetylcholine receptor activation. By integrating these biomarkers, the current study extends beyond inflammatory–antioxidant interactions to capture endothelial, oxidative, and receptor-mediated mechanisms of smoking-related cardiovascular injury. Importantly, although cotinine is retained as an exposure-verification biomarker, the primary outcomes, biological pathways, and analytical framework differ substantially from the prior study, underscoring the novel and complementary contribution of the present findings to the understanding of early cardiovascular risk associated with smoking.

Research Limitations

Several limitations should be acknowledged. The cross-sectional design precludes causal inference and limits the ability to establish temporal relationships between smoking exposure and biomarker alterations. The relatively moderate sample size, although sufficient to detect significant group differences, may limit statistical power for subgroup analyses and reduce generalizability to broader populations. The absence of longitudinal follow-up restricts the ability to evaluate dynamic changes in biomarkers over time or to assess progression toward clinically manifest cardiovascular disease. Smoking exposure was quantified using both self-report and cotinine levels, yet interindividual differences in nicotine metabolism may introduce variability. Potential mediators such as lipid profile and glucose were excluded from regression models to avoid overadjustment, which may limit the evaluation of downstream metabolic pathways. In addition, other lifestyle-related confounding factors, including diet, alcohol intake, and physical activity, were not quantitatively assessed and therefore could not be included in the regression models, which may influence oxidative stress and endothelial function. Because no direct functional vascular assessments were performed, findings should be interpreted cautiously as biochemical evidence of vascular-related alterations rather than direct proof of endothelial dysfunction. Finally, the sample was community-based, and findings may not generalize to populations with comorbid conditions or heavier smoking intensity.

Conclusion

This study demonstrates that smokers exhibit a distinct biochemical signature characterized by reduced NO, elevated MDA, increased nAChRs activation, and markedly higher cotinine levels. These alterations persist after adjusting for demographic and physiological confounders, indicating that smoking independently contributes to early vascular-related biochemical disturbance, oxidative stress, and receptor-mediated dysregulation. Collectively, these findings provide mechanistic insight into smoking-related vascular injury and support the potential utility of these biomarkers in early cardiovascular risk detection and preventive strategies.