Forensic science plays a crucial role in the administration of justice by providing scientific methods for the identification of individuals, reconstruction of criminal events, and analysis of biological evidence recovered from crime scenes.^1,2 Traditionally, forensic investigations have relied heavily on fingerprints, dental records, and DNA profiling for personal identification.³ Among these techniques, DNA analysis is considered the gold standard because of its high specificity and reliability.⁴ However, DNA evidence may become degraded due to environmental factors such as heat, humidity, ultraviolet radiation, microbial contamination, and prolonged exposure to outdoor conditions.⁵ In such situations, the quality and quantity of genetic material may be insufficient for accurate analysis, highlighting the need for complementary forensic approaches that can provide additional information when conventional methods are limited.

Recent developments in forensic biochemistry have introduced the possibility of using biochemical markers as alternative tools for forensic investigations. Biological fluids such as blood, saliva, sweat, urine, and semen contain numerous metabolites, enzymes, proteins, lipids, and antioxidants that reflect the physiological and metabolic status of an individual^.6 These biochemical constituents often exhibit measurable variations among individuals due to differences in genetics, age, lifestyle, dietary habits, environmental exposure, and health conditions. Such variability creates the potential for developing individualized biochemical profiles that may assist in forensic identification and evidence characterization.

Among the various biological specimens available for forensic analysis, saliva has gained considerable attention because of its non-invasive collection, ease of handling, and widespread presence at crime scenes. Saliva can be deposited through activities such as speaking, coughing, smoking, drinking, eating, or direct contact with objects and surfaces.^7,8 In addition to containing epithelial cells suitable for DNA analysis, saliva is rich in proteins, enzymes, antioxidants, and metabolic compounds that may serve as valuable forensic biomarkers^.9 The biochemical composition of saliva remains relatively stable under controlled conditions and can provide important information regarding both individual characteristics and environmental exposure.¹⁰

Oxidative stress biomarkers have emerged as promising candidates for forensic applications. ¹¹ Oxidative stress results from an imbalance between reactive oxygen species production and antioxidant defense mechanisms within the body. Biomarkers such as malondialdehyde (MDA), a product of lipid peroxidation, and antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT), along with total antioxidant capacity (TAC), provide measurable indicators of oxidative status.¹² These parameters vary among individuals based on genetic background, metabolic activity, nutritional status, and environmental influences. Similarly, metabolic biomarkers such as total protein, glucose, and uric acid contribute additional layers of biochemical information that may enhance individual differentiation.

The concept of biochemical fingerprinting refers to the generation of a unique biological profile based on multiple measurable biochemical parameters.¹³ Unlike traditional fingerprinting, which relies on physical ridge patterns, biochemical fingerprinting utilizes metabolic and molecular signatures that may distinguish one individual from another. Such an approach has the potential to complement DNA profiling by providing supplementary evidence regarding the origin, authenticity, and characteristics of biological samples.¹⁴ Furthermore, biochemical profiling may offer rapid and cost-effective screening methods that can be applied in forensic laboratories with limited resources.¹⁵

Given the increasing interest in integrating biochemical techniques into forensic investigations, there is a need to evaluate the discriminatory power of salivary biomarkers in human populations. Therefore, the present study was designed to investigate the forensic applicability of salivary oxidative stress markers and metabolic biomarkers in a cohort of 90 healthy individuals. By examining variations in MDA, SOD, CAT, TAC, total protein, glucose, and uric acid levels, this study aims to assess whether salivary biochemical profiles can serve as reliable indicators for individual differentiation and contribute to the advancement of forensic biochemistry as a complementary tool in modern forensic science.

Study Design and Setting: This cross-sectional analytical study was conducted to evaluate the potential of salivary oxidative stress and metabolic biomarkers as biochemical fingerprints for individual identification in forensic investigations. The study was carried out in the Department of Biochemistry in collaboration with the Forensic Sciences Unit over a period of six months. Study Population and Sample Size: A total of 100 healthy volunteers aged between 18 and 60 years were recruited through convenience sampling. Both male and female participants were included to ensure variability in the study population. Individuals with systemic diseases, active oral infections, chronic inflammatory disorders, smoking habits, alcohol consumption, antioxidant supplementation, or current medication use known to affect oxidative stress status were excluded from the study. Ethical Considerations Ethical approval was obtained from the Institutional Ethical Review Committee prior to commencement of the study. Written informed consent was obtained from all participants after explaining the objectives and procedures of the study. Participant confidentiality and anonymity were strictly maintained throughout the research process. Saliva Sample Collection Unstimulated whole saliva samples were collected from each participant between 8:00 AM and 10:00 AM to minimize circadian variation. Participants were instructed to abstain from eating, drinking, brushing teeth, or chewing gum for at least two hours before sample collection. Approximately 5 mL of saliva was collected by the passive drooling method into sterile polypropylene tubes. Samples were immediately transported on ice to the laboratory and centrifuged at 3000 rpm for 10 minutes to remove cellular debris. The clear supernatant was aliquoted and stored at −80°C until biochemical analysis. Biochemical Analysis Salivary samples were analyzed using standard spectrophotometric methods. • Malondialdehyde (MDA) levels were determined using the thiobarbituric acid reactive substances (TBARS) assay and expressed as nmol/mL. • Superoxide Dismutase (SOD) activity was measured based on inhibition of nitroblue tetrazolium reduction and expressed as U/mL. • Catalase (CAT) activity was assessed by monitoring hydrogen peroxide decomposition and expressed as U/mL. • Total Antioxidant Capacity (TAC) was determined using the ferric reducing antioxidant power method and expressed as mmol/L. • Total Protein concentration was measured using the Bradford protein assay. • Glucose concentration was estimated using the glucose oxidase-peroxidase method. • Uric Acid levels were determined by the uricase enzymatic method. All analyses were performed in duplicate to ensure analytical accuracy and reproducibility. Statistical Analysis Data were analyzed using SPSS version 26.0. Continuous variables were expressed as mean ± standard deviation (SD). Normality of data distribution was assessed using the Shapiro–Wilk test. Inter-individual variability of salivary biomarkers was evaluated using descriptive statistics and coefficient of variation analysis. Independent sample t-tests and one-way ANOVA were applied where appropriate to compare biomarker levels among demographic groups. Pearson correlation analysis was used to assess relationships among biomarkers. To determine the forensic discriminatory potential of the biomarkers, multivariate discriminant analysis and logistic regression modeling were performed using combined biochemical profiles. Receiver Operating Characteristic (ROC) curve analysis was used to evaluate identification performance. A p-value < 0.05 was considered statistically significant for all analyses.

A total of 100 healthy volunteers participated in the study, including 54 males (54%) and 46 females (46%), with a mean age of 36.8 ± 11.2 years. Analysis of salivary samples revealed substantial inter-individual variability in oxidative stress and metabolic biomarkers, supporting their potential utility as biochemical fingerprints for forensic identification.

Table 1. Salivary Oxidative Stress Biomarkers Among Study Participants (n = 100)

The oxidative stress markers demonstrated marked variability among individuals. Catalase activity showed the highest absolute variation, while MDA and SOD exhibited substantial dispersion, indicating individual-specific oxidative profiles.

Table 2. Salivary Metabolic Biomarkers Among Study Participants (n = 100)

Among the metabolic markers, total protein and uric acid demonstrated wide inter-individual variation. Glucose levels also showed considerable variability, contributing to the uniqueness of salivary biochemical profiles.

Table 3. Forensic Discriminatory Performance of Salivary Biomarkers

The present study investigated the forensic applicability of salivary oxidative stress and metabolic biomarkers as a novel biochemical fingerprinting approach for individual identification. The findings demonstrated significant inter-individual variation in all measured salivary biomarkers, including malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (TAC), total protein, glucose, and uric acid. These variations generated distinctive biochemical profiles capable of differentiating individuals with a high identification accuracy of 92.0%, supporting the hypothesis that saliva contains unique biochemical signatures with potential forensic value. Oxidative stress markers showed considerable variability among participants. MDA, a marker of lipid peroxidation, exhibited substantial differences between individuals, reflecting variations in oxidative metabolism, environmental exposure, and physiological status. Similarly, antioxidant enzymes such as SOD and CAT demonstrated wide ranges of activity, indicating differences in individual antioxidant defense mechanisms. These findings suggest that oxidative stress biomarkers contribute significantly to the uniqueness of salivary biochemical profiles and may serve as valuable indicators in forensic investigations. Total antioxidant capacity also displayed marked inter-individual differences. Since TAC represents the cumulative effect of enzymatic and non-enzymatic antioxidants, its variability likely reflects differences in dietary habits, metabolic activity, genetic background, and environmental influences. The inclusion of TAC alongside individual oxidative stress markers enhanced the overall discriminatory power of the biochemical fingerprinting model. Among the metabolic biomarkers, total protein and uric acid emerged as the strongest contributors to individual differentiation. Salivary protein composition is influenced by genetic factors, glandular function, and physiological conditions, resulting in characteristic patterns that vary among individuals. Uric acid, one of the major antioxidants present in saliva, also demonstrated substantial variability and provided additional discriminatory information. The strong performance of these biomarkers suggests that metabolic characteristics may play a crucial role in developing personalized biochemical profiles for forensic applications. The multivariate analysis revealed that combining multiple biomarkers significantly improved identification performance compared with the use of single markers alone. The observed identification accuracy of 92.0%, together with high sensitivity and specificity, indicates that integrated biochemical profiling can effectively distinguish individuals within a population. This finding supports the concept of biochemical fingerprinting, where a combination of molecular and metabolic characteristics creates a unique biological signature analogous to traditional fingerprints. The forensic relevance of saliva further strengthens the practical implications of this approach. Saliva is frequently encountered at crime scenes on cigarette butts, drinking containers, food remnants, bite marks, envelopes, and personal objects. Collection is non-invasive, inexpensive, and relatively simple. Therefore, biochemical analysis of salivary evidence may provide supplementary information when DNA samples are degraded, insufficient, or unavailable for analysis. Additionally, biochemical fingerprinting may serve as a rapid screening tool in resource-limited forensic laboratories. Despite these promising findings, several limitations should be considered. The study included only healthy individuals and evaluated a limited number of biomarkers. Factors such as disease status, medication use, dietary variations, psychological stress, and environmental conditions may influence salivary biochemical composition and require further investigation. Moreover, longitudinal studies are needed to assess the temporal stability of these biomarkers and their reliability under different storage and environmental conditions commonly encountered in forensic settings.

The present study demonstrates that salivary oxidative stress and metabolic biomarkers possess significant potential as a novel biochemical fingerprinting tool for forensic identification. The combined analysis of MDA, SOD, CAT, TAC, total protein, glucose, and uric acid revealed substantial inter-individual variability and achieved high identification accuracy. These findings suggest that salivary biochemical profiling can serve as a valuable complementary approach to conventional forensic methods, particularly when DNA evidence is degraded or unavailable. Further large-scale studies are warranted to validate its reliability and facilitate its integration into modern forensic investigations.

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