Salivary metabolomic biomarkers for non-invasive lung cancer detection.

Identifying novel biomarkers for early detection of lung cancer is crucial. Non-invasively available saliva is an ideal biofluid for biomarker exploration; however, the rationale underlying biomarker detection from organs distal to the oral cavity in saliva requires clarification. Therefore, we analyzed metabolomic profiles of cancer tissues compared with those of adjacent non-cancerous tissues, as well as plasma and saliva samples collected from patients with lung cancer (n = 109 pairs). Additionally, we analyzed plasma and saliva samples collected from control participants (n = 83 and 71, respectively). Capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry were performed to comprehensively quantify hydrophilic metabolites. Paired tissues were compared, revealing 53 significantly different metabolites. Plasma and saliva showed 44 and 40 significantly different metabolites, respectively, between patients and controls. Of these, 12 metabolites exhibited significant differences in all three comparisons and primarily belonged to the polyamine and amino acid pathways; N1-acetylspermidine exhibited the highest discrimination ability. A combination of 12 salivary metabolites was evaluated using a machine learning method to differentiate patients with lung cancer from controls. Salivary data were randomly split into training and validation datasets. Areas under the receiver operating characteristic curve were 0.744 for cross-validation using training data and 0.792 for validation data. This model exhibited a higher discrimination ability for N1-acetylspermidine than that for other metabolites. The probability of lung cancer calculated using this model was independent of most patient characteristics. These results suggest that consistently different salivary biomarkers in both plasma and lung tissues might facilitate non-invasive lung cancer screening.

Salivary metabolites as potential predictive biomarkers for lung surgery complications: a retrospective cross-sectional study.

PURPOSE: Saliva is often used as a tool for identifying systemic diseases because of the noninvasive nature of its collection. Moreover, salivary metabolites can be potential predictive factors for postoperative survival. We conducted the present study to establish whether salivary metabolites can function as predictive biomarkers for lung surgery complications. METHODS: Unstimulated salivary samples were collected from 412 patients before lung surgery. Salivary metabolites were analyzed comprehensively by capillary electrophoresis mass spectrometry. Clinical data with the discriminatory ability of biomarkers were assessed to predict lung surgery complications using multivariate logistic regression analysis. The primary endpoint was the risk factors for postoperative complications of Clavien-Dindo grade ≥ III. RESULTS: Postoperative complications of Clavien-Dindo grade ≥ III developed in 36 patients (8.7%). There was no postoperative 30-day mortality. Male sex (odds ratio [OR], 3.852; 95% confidence interval CI 1.455-10.199; p = 0.007) and salivary gamma-butyrobetaine (OR, 0.809; 95% CI 0.694-0.943; p = 0.007) were identified as significant risk factors for postoperative complications of Clavien-Dindo grade ≥ III. CONCLUSION: Salivary metabolites are potential noninvasive biomarkers for predicting postoperative complications of lung surgery.

Effects of Denture Treatment on Salivary Metabolites: A Pilot Study.

Symptoms of oral discomfort such as dry mouth are common in older people wearing dentures. Such symptoms are mainly treated symptomatically. Many of these symptoms are related to saliva, and associations with salivary volume have been reported. Although denture treatment improves symptoms by increasing the amount of saliva, the effects on salivary components remain unclear. This study aimed to investigate the effects of denture treatment on salivary metabolite changes based on salivary metabolome analyses. We enrolled 21 patients requiring denture treatment. At the first visit, and after completion of denture treatment, saliva outflow was measured under resting and stimulated conditions, samples for salivary metabolite analysis were collected, and masticatory efficiency was tested. In all participants, masticatory efficiency increased after denture treatment. Moreover, the amounts of resting and stimulated saliva were increased. Using salivary metabolome analysis, 61 salivary metabolites were detected. Substantial concentration changes were observed for 4 and 21 metabolites in resting and stimulated saliva, respectively. The four metabolites common to both saliva tests had significantly lower concentrations after treatment. These results suggest that the improvement in masticatory function by dentures is related not only to salivary secretion volume, but also to salivary metabolite composition.

Metabolomic Profiling of Plasma, Urine, and Saliva of Kidney Transplantation Recipients.

Kidney biopsy is commonly used to diagnose kidney transplant dysfunction after transplantation. Therefore, the development of minimally invasive and quantitative methods to evaluate kidney function in transplant recipients is necessary. Here, we used capillary electrophoresis-mass spectrometry to analyze the biofluids collected from transplant recipients with impaired (Group I, n = 31) and stable (Group S, n = 19) kidney function and from donors (Group D, n = 9). Metabolomics analyses identified and quantified 97 metabolites in plasma, 133 metabolites in urine, and 108 metabolites in saliva. Multivariate analyses revealed apparent differences in the metabolomic profiles of the three groups. In plasma samples, arginine biosynthesis and purine metabolism between the I and S Groups differed. In addition, considerable differences in metabolomic profiles were observed between samples collected from participants with T cell-mediated rejection (TCR), antibody-mediated rejection, and other kidney disorders (KD). The metabolomic profiles in the three types of biofluids showed different patterns between TCR and KD, wherein 3-indoxyl sulfate showed a significant increase in TCR consistently in both plasma and urine samples. These results suggest that each biofluid has different metabolite features to evaluate kidney function after transplantation and that 3-indoxyl sulfate could predict acute rejection.

Salivary metabolomics for cancer detection.

Introduction: Saliva is an ideal biofluid that can be collected in a noninvasive manner, enabling safe and frequent screening of various diseases. Recent studies have revealed that salivary metabolomics analysis has the potential to detect both oral and systemic cancers. Area covered: We reviewed the technical aspects, as well as applications, of salivary metabolomics for cancer detection. The topics include the effects of preconditioning and the method of sample collection, sample storage, processing, measurement, data analysis, and validation of the results. We also examined the rational relationship between salivary biomarkers and tumors distant from the oral cavity. A strategy to establish standard operating protocols for obtaining reproducible quantification data is also discussed Expert opinion: Salivary metabolomics reflects oral and systematic health status, which potently enables cancer detection. The sensitivity and specificity of each marker and their combinations have been well evaluated, but a validation study is required. Further, the standard operating protocol for each procedure should be established to obtain reproducible data before clinical usage.

Discrimination of oral squamous cell carcinoma from oral lichen planus by salivary metabolomics.

OBJECTIVE: This study was conducted to distinguish salivary metabolites in oral squamous cell carcinoma (OSCC) from those in oral lichen planus (OLP) to identify practical biomarkers for the discrimination of OSCC from OLP. SUBJECTS AND METHODS: Whole unstimulated saliva samples were collected from patients with OSCC (n = 34) and OLP (n = 26). Hydrophilic metabolites in the saliva samples were comprehensively analysed by capillary electrophoresis mass spectrometry. To evaluate the discrimination ability of a combination of multiple markers, a multiple logistic regression (MLR) model was developed to differentiate OSCC from OLP. RESULTS: Fourteen metabolites were found to be significantly different between the OSCC and OLP groups. Among them, indole-3-acetate and ethanolamine phosphate were used to develop the MLR model. The combination of these two metabolites showed a high area under the receiver operating characteristic curve (0.856, 95% confidential interval: 0.762-0.950; p < .001) for discriminating OSCC from OLP. CONCLUSIONS: We identified salivary metabolites for discerning between OSCC and OLP, which is clinically important for detecting the malignant transformation of OLP by both dentists and oral surgery specialists. Our candidate salivary metabolites show potential for non-invasive screening of OSCC versus OLP.

Existence of SARS-CoV-2 Entry Molecules in the Oral Cavity.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor, angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and furin, which promote entry of the virus into the host cell, have been identified as determinants of SARS-CoV-2 infection. Dorsal tongue and gingiva, saliva, and tongue coating samples were examined to determine the presence of these molecules in the oral cavity. Immunohistochemical analyses showed that ACE2 was expressed in the stratified squamous epithelium of the dorsal tongue and gingiva. TMPRSS2 was strongly expressed in stratified squamous epithelium in the keratinized surface layer and detected in the saliva and tongue coating samples via Western blot. Furin was localized mainly in the lower layer of stratified squamous epithelium and detected in the saliva but not tongue coating. ACE2, TMPRSS2, and furin mRNA expression was observed in taste bud-derived cultured cells, which was similar to the immunofluorescence observations. These data showed that essential molecules for SARS-CoV-2 infection were abundant in the oral cavity. However, the database analysis showed that saliva also contains many protease inhibitors. Therefore, although the oral cavity may be the entry route for SARS-CoV-2, other factors including protease inhibitors in the saliva that inhibit viral entry should be considered.

Salivary metabolomics with alternative decision tree-based machine learning methods for breast cancer discrimination.

PURPOSE: The aim of this study is to explore new salivary biomarkers to discriminate breast cancer patients from healthy controls. METHODS: Saliva samples were collected after 9 h fasting and were immediately stored at - 80 °C. Capillary electrophoresis and liquid chromatography with mass spectrometry were used to quantify hundreds of hydrophilic metabolites. Conventional statistical analyses and artificial intelligence-based methods were used to assess the discrimination abilities of the quantified metabolites. A multiple logistic regression (MLR) model and an alternative decision tree (ADTree)-based machine learning method were used. The generalization abilities of these mathematical models were validated in various computational tests, such as cross-validation and resampling methods. RESULTS: One hundred sixty-six unstimulated saliva samples were collected from 101 patients with invasive carcinoma of the breast (IC), 23 patients with ductal carcinoma in situ (DCIS), and 42 healthy controls (C). Of the 260 quantified metabolites, polyamines were significantly elevated in the saliva of patients with breast cancer. Spermine showed the highest area under the receiver operating characteristic curves [0.766; 95% confidence interval (CI) 0.671-0.840, P < 0.0001] to discriminate IC from C. In addition to spermine, polyamines and their acetylated forms were elevated in IC only. Two hundred each of two-fold, five-fold, and ten-fold cross-validation using different random values were conducted and the MLR model had slightly better accuracy. The ADTree with an ensemble approach showed higher accuracy (0.912; 95% CI 0.838-0.961, P < 0.0001). These prediction models also included spermine as a predictive factor. CONCLUSIONS: These data indicated that combinations of salivary metabolomics with the ADTree-based machine learning methods show potential for non-invasive screening of breast cancer.

Identification of salivary metabolites for oral squamous cell carcinoma and oral epithelial dysplasia screening from persistent suspicious oral mucosal lesions.

OBJECTIVE: To identify salivary metabolite biomarkers to differentiate patients with oral squamous cell carcinoma and oral epithelial dysplasia (OSCC/OED) from those with persistent suspicious oral mucosal lesions (PSOML). SUBJECTS AND METHODS: Whole unstimulated saliva samples were collected from age-, sex-, and race-matched patients who had a lesion in the oral cavity and for whom open biopsies were performed. The patients included OSCC (n = 6), OED (n = 10), and PSOML (n = 32). Hydrophilic metabolites in saliva samples were comprehensively analyzed using capillary electrophoresis mass spectrometry. To evaluate the discrimination ability of a combination of multiple markers, a multiple logistic regression (MLR) model was developed to differentiate OSCC/OED from PSOML. RESULTS: Six metabolites were significantly different in OSCC/OED compared with PSOML. From these six metabolites, ornithine, o-hydroxybenzoate, and ribose 5-phosphate (R5P) were used to develop the MLR model, which resulted in a high value for the area under receiver operating characteristic curve (AUC 0.871, 95% confidential interval (CI) 0.760-0.982; p < 0.001) to discriminate OSCC/OED from PSOML. CONCLUSIONS: This is the first study to identify salivary metabolites that discriminate OSCC/OED from PSOML rather than from healthy controls. The profiles of salivary metabolites were significantly different between OSCC/OED and PSOML. The ability to discriminate OSCC/OED from PSOML is important for dentists who are not oral surgery specialists. These salivary metabolites showed potential for non-invasive screening to discriminate OSCC/OED from PSOML. CLINICAL RELEVANCE: Salivary metabolites in this study showed potential for non-invasive screening to discriminate OSCC/OED from PSOML.

Effect of oral functional training on immunological abilities of older people: a case control study.

BACKGROUND: Oral functional ability decreases with age, and systemic immunological ability and quality of life can also deteriorate. Continuous moderate whole-body exercise for older people is known to improve oral functional and their immunological abilities. Here, we evaluated the effect of oral exercise as an alternative training method for highly older people who cannot perform whole-body exercises. METHODS: Unstimulated whole saliva samples had been collected for three times before training as baseline data and one time after 3 and 6 weeks of training each. Participants were instructed to conduct self-massage; their tongues were used to press their orbicularis oris muscle and buccinators, and instructed to perform bilateral massage of three major glands for facilitating saliva secretion. Medical histories, daily life habits and characteristics were also collected. RESULTS: Totally 30 participants (84.2 ± 8.5 years) were enrolled. In contrast to previous researches, increase in salivary Immunoglobulin A (IgA) after the training was not observed. Interestingly, hierarchical clustering analyses revealed clear individual variations as two prominent clusters and a strong positive correlation between stimulated saliva flow rate and IgA flow rate, regardless of the continuous oral functional exercise. Only body mass index (BMI) showed significant differences between the two groups (Z = 2.06, P = 0.039, Wilcoxon rank-sum test) among all collected parameters. CONCLUSION: Oral functional training limitedly effects on salivary parameters of highly older people. On the other hand, BMI characterized salivary features more than any other parameters, such as the presence of diseases or medication use in these people. TRIAL REGISTRATION: UMIN-CTR Clinical Trial UMIN000028394 on 27/July 2017, retrospectively registered.

Effect of timing of collection of salivary metabolomic biomarkers on oral cancer detection.

The aim of this study is to evaluate the effect of duration after meals for saliva collections for oral cancer detection using metabolomics. Saliva samples were collected from oral cancer patients (n = 22) and controls (n = 44). Saliva from cancer patients was collected 12 h after dinner, and 1.5 and 3.5 h after breakfast. Control subjects fasted >1.5 h prior to saliva collection. Hydrophilic metabolites were analyzed using capillary electrophoresis mass spectrometry. Levels of 51 metabolites differed significantly in controls vs. oral cancer patients at the 12-h fasting time point (P < 0.05). Fifteen and ten metabolites differed significantly at the 1.5- and 3.5-h time points, respectively. The area of under receiver operating characteristic curve for discriminating oral cancer patients from controls was greatest at the 12-h fasting time point. The collection time after meals affects levels of salivary metabolites for oral cancer screening. The 12-h fasting after dinner time point is optimal. This study contributes to design of saliva collection protocols for metabolomics-based biomarker discovery.

Relationship between salivary immunoglobulin a, lactoferrin and lysozyme flow rates and lifestyle factors in Japanese children: a cross-sectional study.

OBJECTIVE: The antimicrobial substances in saliva contribute to the maintenance of both oral health and overall health of the body. Therefore, the associations among immunoglobulin A (IgA), lactoferrin and lysozyme flow rates in the saliva of children, and their relationships with the physical attributes and lifestyle factors of children, were examined. MATERIALS AND METHODS: Saliva was collected from 90 children who visited the Kanagawa Dental University Hospital Pediatric Dentistry, and questionnaires were completed by guardians. IgA, lactoferrin and lysozyme concentrations were measured in the saliva samples using enzyme-linked immunosorbent assays (ELISAs). RESULTS: The IgA flow rate in saliva increased as age, height and weight increased. A correlation was found between lactoferrin and lysozyme flow rates. When the antimicrobial substance flow rates in the saliva were divided into two groups of 22 children each based on the highest and lowest quartiles, children with either a low or high IgA flow rate also had a high or low lactoferrin flow rate, respectively. The same pattern was observed for lactoferrin and lysozyme flow rates. CONCLUSIONS: There is a high probability that the IgA flow rate in the saliva of children reflects and corresponds to the developmental status of immune function as the child ages and increases in height and weight. The flow rates of lactoferrin and lysozyme were correlated in children. In addition, regarding lifestyle factors, the duration of sleep and lactoferrin flow rate were also related.

Capillary electrophoresis-mass spectrometry-based metabolome analysis of serum and saliva from neurodegenerative dementia patients.

Despite increasing global prevalence, the precise pathogenesis and terms for objective diagnosis of neurodegenerative dementias remain controversial, and comprehensive understanding of the disease remains lacking. Here, we conducted metabolomic analysis of serum and saliva obtained from patients with neurodegenerative dementias (n = 10), including Alzheimer's disease, frontotemporal lobe dementia, and Lewy body disease, as well as from age-matched healthy controls (n = 9). Using CE-TOF-MS, six metabolites in serum (ß-alanine, creatinine, hydroxyproline, glutamine, iso-citrate, and cytidine) and two in saliva (arginine and tyrosine) were significantly different between dementias and controls. Using multivariate analysis, serum was confirmed as a more efficient biological fluid for diagnosis compared to saliva; additionally, 45 metabolites in total were identified as candidate markers that could discriminate at least one pair of diagnostic groups from the healthy control group. These metabolites possibly provide an objective method for diagnosing dementia-type by multiphase screening. Moreover, diagnostic-type-dependent differences were observed in several tricarboxylic acid cycle compounds detected in serum, indicating that some pathways in glucose metabolism may be altered in dementia patients. This pilot study revealed novel alterations in metabolomic profiles between various neurodegenerative dementias, which would contribute to etiological investigations.

Metabolomics Analysis of Blood, Urine, and Saliva Samples Based on Capillary Electrophoresis-Mass Spectrometry.

Capillary electrophoresis-mass spectrometry (CE-MS) is an ideal method for analyzing various metabolites in biological samples. CE-MS can simultaneously identify and quantify hundreds of charged metabolites using only two acquisition methods for positively and negatively charged metabolites. Furthermore, CE-MS is commonly used for analyzing biological samples to understand the pathology of diseases at the metabolic level and biofluid samples, such as blood and urine, to explore biomarkers. Here, we introduce a protocol that delineates the handling of clinical samples to ensure that the CE-MS analysis yields reproducible quantified data. We have focused on sample collection, storage, processing, and measurement. Although the implementation of rigorous standard operating protocols is preferred for enhancing the quality of the samples, various limitations in an actual clinical setting make it difficult to adhere to strict rules. Therefore, the effect of each process on the quantified metabolites needs to be evaluated to design a protocol with acceptable tolerances. Furthermore, quality controls and assessments to handle clinical samples are introduced.

Differential diagnosis of lung cancer and benign lung lesion using salivary metabolites: A preliminary study.

BACKGROUND: Saliva is often used as a biomarker for the diagnosis of some oral and systematic diseases, owing to the non-invasive attribute of the fluid. In this study, we aimed to identify salivary biomarkers for distinguishing lung cancer (LC) from benign lung lesion (BLL). MATERIALS AND METHODS: Unstimulated saliva samples were collected from 41 patients with LC and 21 with BLL. Salivary metabolites were comprehensively analyzed using capillary electrophoresis mass spectrometry. To differentiate between patients with LCs and BLLs, the discriminatory ability of each biomarker was assessed. Furthermore, a multiple logistic regression (MLR) model was developed for evaluating discriminatory ability of each salivary metabolite. RESULTS: The profiles of 10 salivary metabolites were remarkably different between the LC and BLL samples. Among them, the concentration of salivary tryptophan was significantly lower in the samples from patients with LC than in those from patients with BLL, and the area under the curve (AUC) for discriminating patients with LC from those with BLL was 0.663 (95% confidence interval [CI] = 0.516-0.810, p = 0.036). Furthermore, from the MLR model developed using these metabolites, diethanolamine, cytosine, lysine, and tyrosine, were selected using the back-selection regression method. The MLR model based on these four metabolites had a high discriminatory ability for patients with LC and those with BLL (AUC = 0.729, 95% CI = 0.598-0.861, p = 0.003). CONCLUSION: The four salivary metabolites can serve as potential non-invasive biomarkers for distinguishing LC from BLL.

Effect of Underwater Exercise on Salivary Metabolites of Older Persons With Disability.

BACKGROUND/AIM: Underwater exercise is aimed at preventing aging, maintaining, and improving motor function, and improving physical function. However, its rehabilitation effects have not been well evaluated. In order to gain insight into the molecular basis of its rehabilitation effects, possible changes in the salivary metabolites of four older persons with disability (mean age: 72.5 years) during underwater exercise were investigated. MATERIALS AND METHODS: Halitosis was measured by Breathtron; salivary bacterial number by bacterial counter; amino acids by amino acid analyzer; 8-oxoguanine by ELISA; and intracellular metabolites by capillary electrophoresis, time-of-flight mass spectrometry, liquid chromatography, and triode quadrupole mass spectrometry. RESULTS: Underwater exercise induced apparent declines in two major salivary amino acids (glycine and proline) and bacterial numbers in the cheek mucosa and salivary, without apparent changes in the halitosis and urine 8-oxoguanine concentration. Older subjects showed higher concentrations of most of 166 metabolites compared to young volunteers (mean age: 38.8 years old). Fifteen compounds were significantly reduced with the progression of underwater exercise. CONCLUSION: Improvement of upright balance function with underwater exercise is correlated with several salivary components.

Multi-Omics Analysis of Anti-Inflammatory Action of Alkaline Extract of the Leaves of Sasa sp.

Efficient utilization of alkaline extracts of several plants for the treatment of oral diseases has been reported. To investigate the mechanism of anti-inflammatory activity of alkaline extract of the leaves of Sasa sp. (SE), multi-omics analysis using metabolomics and DNA array was performed. Human gingival fibroblasts (HGFs) were treated for IL-1ß to induce inflammation (detected by PGE2 production in culture medium) in the presence or absence of SE. Both IL-1ß and SE showed slight hormetic growth stimulation against HGF. SE inhibited PGE2 production dose- and time-dependently. Its inhibitory action was more pronounced by first treating the cells with SE, rather than with IL-1ß. At 3 h after IL-1ß treatment, 18 amino acids (except cysteine and glutamic acid), total glutathione (GSH, GSSG, Cys-GSH disulfide), Met-sulfoxide, 5-oxoproline, and SAM declined, whereas DNA expressions of AKT, CASP3, and CXCL3 were elevated. These changes were reversed by simultaneous treatment with SE. The present study suggests that the anti-inflammatory action of SE is mediated via various metabolic pathways for cell survival, apoptosis, and leukocyte recruitment.

High-throughput screening of salivary polyamine markers for discrimination of colorectal cancer by multisegment injection capillary electrophoresis tandem mass spectrometry.

Polyamine metabolites provide pathophysiological information on disease or therapeutic efficacy, yet rapid screening methods for these biomarkers are lacking. Here, we developed high-throughput polyamine metabolite profiling based on multisegment injection capillary electrophoresis triple quadrupole tandem mass spectrometry (MSI-CE-MS/MS), which allows sequential 40-sample injection followed by electrophoretic separation and specific mass detection. To achieve consecutive analysis of polyamine samples, 1 M formic acid was used as the background electrolyte (BGE). The BGE spacer volume had an apparent effect on peak resolution among samples, and 20 nL was selected as the optimal volume. The use of polyamine isotopomers as the internal standard enabled the correction of matrix effects in MS detection. This method is sensitive, selective and quantitative, and its utility was demonstrated by screening polyamines in 359 salivary samples within 360 min, resulting in discrimination of colorectal cancer patients from noncancer controls.

Metabolomic profiling reveals salivary hypotaurine as a potential early detection marker for medication-related osteonecrosis of the jaw.

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but serious adverse event of bone-modifying agents used to prevent bone complications in cancer patients with bone metastasis. Currently, early treatment is the only way to prevent further progression, as the pathogenesis of MRONJ has not yet been elucidated, and a standard treatment has not been established. The aim of this study was to identify a marker for early detection marker of MRONJ by exploring substances in saliva specific to MRONJ at an early stage. We collected salivary samples from 17 patients with MRONJ and conducted metabolomic analyses using capillary electrophoresis mass spectrometry for non-targeted analysis of hydrophilic metabolites. In the screening cohort, we compared the saliva of patients with stage ≥1 advanced MRONJ (n = 9) with that of controls without MRONJ before chemotherapy (n = 9). The top 5 most elevated salivary metabolites were histamine, 3-(4-hydroxyphenyl)propionate, malonate, carnosine, and hypotaurine. In the validation cohort, we analyzed additional patients with stage ≥1 advanced MRONJ (n = 8) and controls without MRONJ after chemotherapy (n = 9), confirming a significant 2.28-fold elevation in the salivary concentration of hypotaurine. These results revealed elevated salivary hypotaurine concentration as a potential marker for the early detection of MRONJ.

Changes in Metabolic Profiles of Human Oral Cells by Benzylidene Ascorbates and Eugenol.

Sodium-5,6-benzylidene-L-ascorbate (SBA), and its component units, benzaldehyde (BA) and sodium ascorbate (SA), are known to exert antitumor activity, while eugenol exerts anti-inflammatory activity. To narrow down their intracellular targets, metabolomic analysis was performed. Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Fine cell structures were observed under transmission electron microscope. Cellular metabolites were extracted with methanol and subjected to capillary electrophoresis-mass spectrometry (CE-MS) for quantification of intracellular metabolites. Results showed that SBA was cleaved into BA and SA under acidic condition. Among these three compounds, BA showed the highest-tumor specificity in vitro against human oral squamous cell carcinoma (OSCC) cell line. BA did not induce the vacuolization in HSC-2 OSCC cells, and its cytotoxicity was not inhibited by catalase, in contrast to SBA and SA. Only BA suppressed the tricarboxylic acid (TCA) cycle at early stage of cytotoxicity induction. Eugenol more rapidly induced the vacuolization and suppressed the TCA cycle in three human normal oral cells (gingival fibroblast, periodontal ligament fibroblast, pulp cell). Neither BA nor eugenol affected the ATP utilization, further supporting that they do not induce apoptosis. The present study demonstrated for the first time that both BA and eugenol suppressed the TCA cycle in tumor cells and normal cells, respectively. It is crucial to design methodology that enhances the antitumor potential of BA and reduces the cytotoxicity of eugenol to allow for safe clinical application.