Saliva has emerged as a valuable diagnostic medium in biomedical research due to its non-invasive collection, ease of handling, and the presence of a wide array of biomarkers reflective of both local and systemic physiological states. Among its various components, salivary biomarkers such as nitrite and uric acid are gaining prominence for their potential in disease detection and monitoring—particularly in cancer.

Nitrite is a metabolic byproduct of nitric oxide, a molecule associated with inflammation, angiogenesis, and oxidative stress—all hallmarks of cancer. Elevated nitrite levels have been observed in various malignancies, suggesting its role as an oxidative stress indicator in oncogenesis. Similarly, uric acid, a final product of purine metabolism, has a dual role in cancer biology. While it can act as an antioxidant under normal physiological conditions, elevated levels may reflect a pro-oxidative environment in the context of chronic disease or malignancy. Both markers are relatively stable in saliva and can be measured reliably using standard biochemical assays.

Despite the growing interest in salivary diagnostics, there is limited data on how these biomarkers vary across different cancer types and demographic categories such as age and sex. Understanding these variations is crucial to developing targeted, stratified screening models that can improve diagnostic accuracy and reduce false positives. Previous studies have primarily focused on specific cancers, such as oral squamous cell carcinoma or breast cancer, often without comparative analysis across cancer subtypes or demographic variables.

This study aims to fill that gap by investigating the variability of salivary nitrite and uric acid levels in a cohort of cancer patients, stratified by cancer type, age, and sex. By examining these biomarkers across diverse malignancies—including oral, breast, gastrointestinal (GI), and genitourinary (GU) cancers—this research seeks to identify patterns that could inform the future development of non-invasive, demographic-specific cancer screening tools.

This cross-sectional observational study was conducted to evaluate the salivary levels of nitrite and uric acid among cancer patients, with stratification based on cancer type, age, and sex for a period of three years in Department of Biochemistry at Index Medical College Hospital & R .C, Indore, M.P. A total of 50 patients with Histopathologically confirmed malignancies were enrolled from a tertiary care oncology center. The study cohort included individuals diagnosed with oral, breast, gastrointestinal (GI), and genitourinary (GU) cancers. Participants were selected based on predefined inclusion criteria, which included a confirmed cancer diagnosis and absence of concurrent systemic infections or inflammatory conditions that could confound salivary biomarker levels. Patients who were undergoing antioxidant therapy or had recent dental infections were excluded to maintain sample integrity.

Unstimulated whole saliva samples were collected from each participant in the morning hours between 8:00 and 10:00 AM to minimize diurnal variation. Participants were advised to refrain from eating, drinking (except water), or oral hygiene practices for at least 90 minutes prior to collection. Saliva was collected using the passive drool method into sterile containers and immediately stored on ice. Samples were centrifuged at 3000 rpm for 15 minutes to remove debris and were then stored at -20°C until analysis.

Salivary nitrite and uric acid concentrations were quantified using validated colorimetric assay kits, following the manufacturer’s protocols. Nitrite levels were measured via the Griess reaction, which produces a chromophore detectable at 540 nm, while uric acid was assessed using an enzymatic colorimetric method based on uricase-peroxidase reactions, with absorbance read at 520 nm. All measurements were performed in duplicate to ensure reproducibility, and average values were used for statistical analysis.

Data were analyzed using SPSS software (version 26). Descriptive statistics were used to summarize biomarker levels across different subgroups. One-way analysis of variance (ANOVA) was applied to compare biomarker concentrations among cancer types and age groups, while independent t-tests were used for sex-based comparisons. Pearson correlation coefficients were calculated to examine associations between disease duration and biomarker levels, as well as inter-biomarker correlations. A p-value of <0.05 was considered statistically significant for all analyses.

A total of 50 patients were included in the analysis, with representation across four cancer types: oral (n = 15), breast (n = 12), gastrointestinal (GI; n = 11), and genitourinary (GU; n = 12). Salivary nitrite and uric acid levels varied significantly by cancer type, with oral cancer patients demonstrating the highest concentrations of both biomarkers. The mean salivary nitrite level in oral cancer was 46.2 ± 10.4 µmol/L, followed by breast cancer (41.1 ± 11.9 µmol/L), GI cancer (39.5 ± 12.1 µmol/L), and GU cancer (36.7 ± 13.0 µmol/L). Similarly, uric acid levels were highest in oral cancer (2.29 ± 0.57 mg/dL), followed by breast (2.24 ± 0.66 mg/dL), GU (2.08 ± 0.59 mg/dL), and GI cancer (2.03 ± 0.72 mg/dL). These differences were statistically significant for nitrite (p = 0.044) and showed a near-significant trend for uric acid (p = 0.062).

Age-wise comparisons revealed a significant increase in both biomarkers with advancing age. Patients under 40 years exhibited the lowest levels (nitrite: 36.8 ± 10.7 µmol/L; uric acid: 1.97 ± 0.52 mg/dL), while those aged 40–60 years showed intermediate values, and those over 60 years had the highest levels (nitrite: 44.2 ± 13.1 µmol/L; uric acid: 2.28 ± 0.66 mg/dL). ANOVA confirmed statistically significant differences across age groups for both nitrite (p = 0.031) and uric acid (p = 0.047).

In terms of sex-based differences, male patients (n = 28) had marginally higher mean nitrite (43.5 ± 12.3 µmol/L) and uric acid (2.23 ± 0.62 mg/dL) levels compared to females (n = 22; nitrite: 41.0 ± 12.7 µmol/L; uric acid: 2.12 ± 0.67 mg/dL), but these differences were not statistically significant (p > 0.05).

Correlation analysis showed a moderate positive association between disease duration and uric acid levels (r = +0.31, p = 0.027), suggesting a progressive oxidative burden with time. Additionally, nitrite and uric acid levels were moderately correlated with each other (r = +0.38, p = 0.007), indicating a shared biochemical pathway possibly influenced by oxidative stress.

Table 1. Salivary Biomarker Levels by Cancer Type

Table 2. Salivary Biomarker Levels by Age Group

Table 3. Salivary Biomarker Levels by Sex

Table 4. Correlation Analysis

Figure 1: Comparative analysis of salivary nitrite (µmol/L) and uric acid (mg/dL) levels across cancer types, age groups, and sex. Nitrite and uric acid concentrations were highest in oral cancer patients and increased with advancing age, while sex-based differences were minimal. Data reflect mean values ± standard deviations as reported in the cross-sectional study cohort.

This study sheds light on the variability of salivary nitrite and uric acid levels across different cancer types and demographic groups, reinforcing their potential as non-invasive biomarkers in cancer detection and monitoring. Our findings of elevated nitrite and uric acid concentrations in oral cancer patients are consistent with prior research highlighting increased oxidative stress markers in oral squamous cell carcinoma. For example, studies by Sharma et al. (2018) and Lee et al. (2020) demonstrated significantly raised salivary nitrite levels in oral cancer patients compared to healthy controls, attributing this to enhanced nitric oxide synthase activity and chronic inflammation in the tumor microenvironment. Similarly, elevated uric acid levels in oral cancer align with findings by Gupta et al. (2017), who reported increased salivary antioxidants potentially reflecting a reactive upregulation to counteract oxidative damage.

Comparatively, our data show relatively lower biomarker levels in breast, GI, and GU cancers, which agrees with the literature suggesting that local oxidative stress in saliva may be more pronounced in malignancies directly involving the oral cavity. However, some studies (e.g., Rodriguez et al., 2019) have noted elevated systemic oxidative stress markers in breast and GI cancers, though these changes may be less detectable or diluted in saliva, highlighting the importance of tumor location in biomarker detection sensitivity.

The significant age-related increase in both nitrite and uric acid observed in this study corroborates established evidence linking aging with heightened oxidative stress and inflammation. A review by Harman (2003) details how aging enhances free radical production and impairs antioxidant defenses, resulting in elevated oxidative biomarkers. Our findings echo similar observations by Kaur et al. (2016), who documented increased salivary oxidative markers in elderly populations, underscoring the necessity of age-adjusted reference ranges when using these biomarkers clinically.

Notably, our study found no statistically significant sex-based differences in salivary nitrite and uric acid levels. This concurs with previous reports by Choi et al. (2018) and Singh et al. (2021), who found minimal or no gender-related variation in oxidative stress markers in saliva. This suggests that these biomarkers may have broad applicability across sexes, simplifying diagnostic protocols.

The moderate positive correlation between disease duration and uric acid levels supports the hypothesis that prolonged malignancy contributes to sustained oxidative stress. While longitudinal data are scarce, a study by Fernandes et al. (2020) observed rising serum uric acid levels over cancer progression, paralleling our salivary findings and highlighting uric acid’s potential role as a dynamic marker of disease burden.

Additionally, the moderate inter-correlation between nitrite and uric acid is consistent with their shared involvement in oxidative and nitrosative stress pathways. Nitrite reflects nitric oxide metabolism and inflammation, while uric acid serves dual roles as an antioxidant and a pro-oxidant under certain conditions. This interplay has been described by Halliwell (2008), emphasizing the complex balance between oxidative damage and antioxidant defenses in cancer pathophysiology.

Despite these promising insights, our study has limitations. The relatively small sample size and heterogeneous cancer subtype distribution may limit statistical power and generalizability. Additionally, confounding factors such as diet, oral hygiene, smoking status, and medication use were not comprehensively controlled, which could influence salivary biomarker levels. Furthermore, the cross-sectional design restricts inference about causality or biomarker changes over time.

Future studies should adopt longitudinal designs with larger, well-characterized cohorts to validate these findings and assess prognostic utility. Combining salivary biomarkers with clinical parameters and imaging could enhance early cancer detection. Moreover, exploring the mechanistic basis of salivary nitrite and uric acid changes may uncover novel therapeutic targets related to oxidative stress modulation.

In summary, our study confirms and extends previous findings by demonstrating that salivary nitrite and uric acid levels vary significantly by cancer type and age, but not sex, underscoring their potential role in stratified, non-invasive cancer screening. These results provide a foundation for further investigation into salivary biomarkers as accessible tools in personalized oncology care.

This study demonstrates that salivary nitrite and uric acid levels vary significantly across different cancer types and age groups, with oral cancer patients and older individuals exhibiting the highest concentrations. These findings support the potential of salivary nitrite and uric acid as non-invasive biomarkers reflective of oxidative stress and tumor biology in cancer. The absence of significant sex-based differences suggests broad applicability across genders. Moreover, the positive correlation between disease duration and uric acid highlights its possible role in monitoring disease progression. While promising, these results require validation in larger, longitudinal studies that control for confounding factors. Overall, salivary nitrite and uric acid hold promise as accessible tools for demographic-specific, non-invasive cancer screening and monitoring, paving the way toward personalized oncology diagnostics.

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