Salivary Profiles Research Articles

Time-Course of Salivary Metabolomic Profiles during Radiation Therapy for Head and Neck Cancer.

Oral mucositis (OM) is one of the most frequently observed adverse oral events in radiation therapy for patients with head and neck cancer. Thus, objective evaluation of OM severity is needed for early and timely intervention. Here, we analyzed the time-course of salivary metabolomic profiles during the radiation therapy. The severity of OM (National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events v3.0) of nine patients with head and neck cancer was evaluated. Partial least squares regression-discriminant analysis, using samples collected before radiation therapy, showed that histidine and tyrosine highly discriminated high-grade OM from low-grade OM before the start of radiation therapy (significant difference, p = 0.048 for both metabolites). Further, the pretreatment concentrations of gamma-aminobutyric acid and 2-aminobutyric acids were higher in the high-grade OM group. Although further validations are still necessary, this study showed potentially associated metabolites with worse radiotherapy-related OM among patients with head and neck cancer.

Analysis of Salivary Metabolites by Nuclear Magnetic Resonance Before and After Oral Mucosa Cleaning of Infants in the Pre-dental Period

The aim of the present study was to verify if a protocol for cleaning the oral cavity of infants in the pre-dental period can reduce extrinsic salivary metabolites observed through Nuclear Magnetic Resonance (NMR). A cross-sectional clinical study with a convenience sample was conducted, and infants were recruited at the UFRJ Pediatric Dentistry Clinic. Participants who had used antibiotics and/or antifungals up to 3 months before and whose legal guardians did not consent or sign the Informed Consent Form were excluded. An anamnesis was performed with the guardians and the participants' intraoral clinical examination. Initial collection of unstimulated total saliva was performed using an automatic pipette with sterile plastic tips in the buccal floor region, at least 1 h after the last feeding. Subsequently, the infants' oral mucosa was cleaned with gauze moistened with filtered water, and after 5 min, a new collection was performed, using the same methodology. The obtained samples were immediately transferred on ice to the laboratory, centrifuged (10,000 g), and stored at −80°C. The NMR analyses were performed using a 500-MHz spectrometer Bruker, Germany); evaluations were done via the 1H and 1H-1H TOCSY spectra for metabolite signaling. Eleven pre-dental infants were evaluated, with a mean age of 3.8 months, including six girls (55%). Of these, nine participants (82%) were exclusively breastfed. The higher presence of components such as lactose, glucose, sugars, acetate, alanine, and lactate were observed in the samples before oral mucosa cleaning. Regarding the type of diet, more lactose was observed in the saliva of patients who were exclusively breastfed than those that received mixed feeding. We conclude that the oral mucosa cleaning of infants in the pre-dental period tends to reduce the concentration of extrinsic components from the diet, such as lactose, in the salivary metabolomic profile analyzed by NMR.

Salivary profiles of 11-oxygenated androgens follow a diurnal rhythm in patients with congenital adrenal hyperplasia

CONTEXT Several studies have highlighted the importance of the 11-oxygenated 19-carbon (11oxC19) adrenal-derived steroids as potential biomarkers for monitoring patients with 21-hydroxylase deficiency (21OHD). OBJECTIVE To analyze circadian rhythmicity of 11oxC19 steroids in saliva profiles and evaluate their relevance as potential monitoring parameters in 21OHD. DESIGN, SETTING, AND PARTICIPANTS Cross-sectional single-center study including 59 patients with classic 21OHD (men = 30; women = 29) and 49 body mass index- and age-matched controls (men = 19; women = 30). OUTCOME MEASURES Salivary concentrations of the following steroids were analyzed by liquid chromatography-tandem mass spectrometry: 17-hydroxyprogesterone (17OHP), androstenedione (A4), testosterone (T), 11β-hydroxyandrostenedione (11OHA4), and 11-ketotestosterone (11KT). RESULTS Similar to the previously described rhythmicity of 17OHP, 11OHA4 and 11KT concentrations followed a distinct diurnal rhythm in both patients and controls with highest concentrations in the early morning and declining throughout the day (11-OHA4: mean reduction of hormone concentrations between timepoint 1 and 5 (Δ mean) in male patients = 66%; male controls Δ mean = 83%; female patients Δ mean = 47%; female controls Δ mean = 86%; 11KT: male patients Δ mean = 57%; male controls Δ mean = 63%; female patients Δ mean = 50%; female controls Δ mean = 76%). Significant correlations between the area under the curve for 17OHP and 11KT (rpmale = 0.773<0.0001; rpfemale = 0.737<0.0001), and 11OHA4 (rpmale = 0.6330.0002; rpfemale = 0.5640.0014) were observed in patients but not present or reduced in controls. CONCLUSIONS Adrenal 11oxC19 androgens are secreted following a diurnal pattern. This should be considered when evaluating their utility for monitoring treatment control.

Salivary Proteomic Profiling Identifies Role of Neutrophil Extracellular Traps Formation in Pregnancy Gingivitis

ABSTRACT Pregnancy gingivitis peaks during mid-pregnancy and resolves transiently towards the postpartum period. However, the role of maternal immune response in orchestrating gingival inflammation has not yet been fully understood. Hence, in this study, we examined the salivary protein profile during the three trimesters of pregnancy, in context to pregnancy gingivitis, employing iTRAQ-based quantitative proteomics. Unstimulated saliva was collected from 10 subjects in each trimester of pregnancy and postpartum period. Samples were analysed using iTRAQ analysis and ELISA and SEM was performed to validate results. Neutrophil mediated immune response was overrepresented in all three trimesters of pregnancy, despite the decrease in phagocytic responses during the second and third trimesters. ELISA showed a significantly higher Neutrophil Extracellular Traps (NETs) formation in the third trimester of pregnancy coinciding with the resolution of pregnancy gingivitis. The NETs-associated proteins (neutrophil elastase and myeloperoxidase) showed a positive correlation with estrogen hormones, which was also highest during the third trimester. Sex hormone-driven NETs formation could be the mainstay of defence that contributes to the remission of pregnancy gingivitis. This study has provided a new insight into the role of immune-modulation in pregnancy gingivitis, which will aid development of new therapeutics for managing pregnancy gingivitis in future.

Salivary Protein Profile and Food Intake: A Dietary Pattern Analysis.

Saliva research has gained interest due to its potential as a source of biomarkers. One of the factors inducing changes in saliva, in the short term, is food intake, and evidence exist about changes in salivary proteome induced by some food components. Since this topic of research is in its early stages, it was hypothesized that saliva protein composition could be associated with different levels of adherence to dietary patterns that contain higher amounts of plant products. The aim of the present study was to test this hypothesis, in adults, by comparing salivary protein electrophoretic profiles of individuals with different diet characteristics, particularly dietary patterns (DP) that exhibit different proportions of animal and plant-based products. Dietary habits were assessed in 122 adults (61 from each sex, with ages ranging from 20 to 59 years) using Food Frequency Questionnaires. To identify the dietary patterns, a principal component analysis was used. Individual's non-stimulated saliva was evaluated for flow rate, pH, protein concentration, α-amylase activity, and electrophoretic protein profiles. Seven dietary patterns (DP) were identified. Salivary amylase enzymatic activity was positively associated with animal-based and starchy foods DP, and with plant-based fatty foods without wine DP. At the same time, protein bands containing amylase and type S cystatins were positively associated with the cheese/yoghurt and wine DP. Our results support the association of salivary proteomics and different dietary patterns and highlight the need of considering food consumption habits in studies using saliva, since this is a factor associated with variations in the composition of this fluid.

Cognitive Dysfunction in a Mouse Model of Cerebral Ischemia Influences Salivary Metabolomics.

Vascular dementia, caused by cerebrovascular disease, is associated with cognitive impairment and reduced hippocampal metabolite levels. Specifically, cognitive impairment can be induced by decreased hippocampal brain-derived neurotrophic factor (BDNF) expression. The development of low or non-invasive biomarkers to characterize these diseases is an urgent task. Disturbance of metabolic pathways has been frequently observed in cognitive impairment, and salivary molecules also showed the potentials to reflect cognitive impairment. Therefore, we evaluated salivary metabolic profiles associated with altered hippocampal BDNF expression levels in a cerebral ischemia mouse model using metabolomic analyses. The effect of tacrine (a cholinesterase inhibitor) administration was also examined. The arteries of ICR mice were occluded with aneurysm clips to generate the cerebral ischemia model. Learning and memory performance was assessed using the elevated plus maze (EPM) test. Hippocampal and blood BDNF levels were quantified using an enzyme-linked immunosorbent assay. Glutamate decarboxylase 1 (GAD1) mRNA expression, is associated with cognitive impairment, was quantified by a real-time polymerase chain reaction. The EPM test revealed impaired spatial working memory in the cerebral ischemia mouse model; tacrine administration ameliorated this memory impairment. Cerebral ischemia suppressed GAD1 expression by decreasing hippocampal BDNF expression. In total, seven salivary metabolites, such as trimethylamine N-oxide and putrescine, were changed by cognitive impairment and tacrine administration. Our data suggest that salivary metabolite patterns were associated with cognitive function.

Multidimensional study of the oral microbiome, metabolite, and immunologic environment in peanut allergy

The oral mucosa is the initial interface between food antigens, microbiota, and mucosal immunity, yet, little is known about oral host-environment dynamics in food allergy. Our aim was to determine oral microbial, metabolic, and immunologic profiles associated with peanut allergy. We recruited 105 subjects (56 with peanut allergy and 49 healthy subjects) for salivary microbiome profiling using 16S ribosomal RNA sequencing, short-chain fatty acid (SCFA) metabolite assays using liquid chromatography/mass spectrometry, and measurement of oral secreted cytokines using multiplex assays. Analyses within and across data types were performed. The oral microbiome of individuals with peanut allergy was characterized by reduced species in the orders Lactobacillales, Bacteroidales (Prevotella spp), and Bacillales, and increased Neisseriales spp. The distinct oral microbiome of subjects with peanut allergy was accompanied by significant reductions in oral SCFA levels, including acetate, butyrate, and propionate, and significant elevation of IL-4 secretion. Decreased abundances of oral Prevotella spp and Veillonella spp in subjects with peanut allergy were significantly correlated with reduced oral SCFA levels (false discovery rate<0.05), and increased oral Neisseria spp was correlated with lower oral SCFA levels (false discovery rate<0.05). Additionally, oral Prevotella spp abundances were correlated with decreased local secretion of TH2-stimulating epithelial factors (IL-33 and thymic stromal lymphopoietin) and TH2 cytokines (IL-4, IL-5, and IL-13), whereas oral Neisseria spp abundance was positively associated with a TH2-skewed oral immune milieu. Our novel multidimensional analysis of the oral environment revealed distinct microbial and metabolic profiles associated with mucosal immune disturbances in peanut allergy. Our findings highlight the oral environment as an anatomic site of interest to examine host-microbiome dynamics in food allergy.

Salivary Microbiota for Gastric Cancer Prediction: An Exploratory Study.

To characterize the salivary microbiota in patients at different progressive histological stages of gastric carcinogenesis and identify microbial markers for detecting gastric cancer, two hundred and ninety-three patients were grouped into superficial gastritis (SG; n = 101), atrophic gastritis (AG; n = 93), and gastric cancer (GC; n = 99) according to their histology. 16S rRNA gene sequencing was used to access the salivary microbiota profile. A random forest model was constructed to classify gastric histological types based on the salivary microbiota compositions. A distinct salivary microbiota was observed in patients with GC when comparing with SG and AG, which was featured by an enrichment of putative proinflammatory taxa including Corynebacterium and Streptococcus. Among the significantly decreased oral bacteria in GC patients including Haemophilus, Neisseria, Parvimonas, Peptostreptococcus, Porphyromonas, and Prevotella, Haemophilus, and Neisseria are known to reduce nitrite, which may consequently result in an accumulation of carcinogenic N-nitroso compounds. We found that GC can be distinguished accurately from patients with AG and SG (AUC = 0.91) by the random forest model based on the salivary microbiota profiles, and taxa belonging to unclassified Streptophyta and Streptococcus have potential as diagnostic biomarkers for GC. Remarkable changes in the salivary microbiota functions were also detected across three histological types, and the upregulation in the isoleucine and valine is in line with a higher level of these amino acids in the gastric tumor tissues that reported by other independent studies. Conclusively, bacteria in the oral cavity may contribute gastric cancer and become new diagnostic biomarkers for GC, but further evaluation against independent clinical cohorts is required. The potential mechanisms of salivary microbiota in participating the pathogenesis of GC may include an accumulation of proinflammatory bacteria and a decline in those reducing carcinogenic N-nitroso compounds.

Analysis of oral health and salivary biochemical parameters of women with anorexia and bulimia nervosa

Análisis de parámetros bioquímicos de salud bucal y salival de mujeres con anorexia y bulimia nerviosa Objective: The aim of this study was to analyze the association between caries, dental erosion and salivary biochemical profile of women with anorexia and bulimia, comparing them with a control group. Methods: The participants were women from the Eating Disorders Program of the Mental Health Clinic. We evaluated caries and dental erosion and collected stimulated saliva samples for the analyses of salivary flow, phosphor, calcium, acid and alkaline phosphatase, amylase, FRAP and uric acid. Data were expressed as means and standard deviations and were analysed using Mann-Whitney test with p&lt;0.05 regarded as significant. Results: 13 women with anorexia and bulimia participated in the study, with an mean age of 28.9 years, and 15 comprised the control group. The DMFT index was 15.5 for the case group and 4.0 for the control group. Erosion was found mostly on the palatal/lingual surface and on incisor teeth of case group. Most biochemical analyzes showed a significant difference between the two groups. Conclusion: We concluded that, in patients with anorexia and bulimia, due to a decreased salivary flow and self-induced vomiting habits, there is an increase in the caries and dental erosion index, as well as salivary changes.

Comparison of lipid profile in tobacco and non-tobacco abusers – A comparative study

Aims and Objectives: To evaluate and correlate the salivary and serum lipid profile in healthy individuals, tobacco chewers and smokers. Also, to compare the salivary lipid profile within each group statistically. Materials and Methods: A total of 60 samples were taken, 20 in each group. Fasting blood and unstimulated saliva sample collected and the lipid analysis (Total Cholesterol - TCHL, Triglycerides - TGL, High density lipid cholesterol - HDL, Low density lipid cholesterol - LDL, very low-density lipid cholesterol - VLDL) was done on an autoanalyzer based on spectrophotometric principle. Statistical analysis: Data are evaluated and statistical analysis was done using unpaired student “t” test and Karl Pearson’s correlation. Results: A moderate correlation was found between salivary and serum lipid profile of the study group and control group with exception to LDL. Low lipid profile was observed in the study group in comparison with control group. Conclusion: Saliva can be used as a non-invasive diagnostic tool for assessing lipid profile, however diagnostic value of saliva has to be determined in terms of sensitivity, specificity and reproducibility in larger samples and different disease setting. Keywords: Tobacco, Saliva, Serum, Lipids.

Elderly Subjects Supplemented with L-Glutamine Shows an Improvement of Mucosal Immunity in the Upper Airways in Response to Influenza Virus Vaccination.

Background: Although glutamine is able to improve the immune response, its action in the upper airway immunity against the influenza virus vaccine remains unclear. Therefore, we aimed to evaluate the L-glutamine supplementation effect on the mucosal immune/inflammatory response of elderly subjects vaccinated against the influenza virus. Methods: Saliva sampling from 83 physically active elderly volunteers were collected pre- and 30 days after influenza virus vaccination and supplementation with L-glutamine (Gln, n = 42) or placebo (PL, n = 41). Results: Gln group showed higher salivary levels of interleukin (IL)-17, total secretory immunoglobulin A (SIgA), and specific-SIgA post-vaccination than values found pre-vaccination and in the PL group post-vaccination. Whereas higher salivary levels of IL-6 and IL-10 were observed post-vaccination in the Gln group, IL-37 levels were lower post-vaccination in both groups than the values pre-vaccination. Tumor necrosis factor (TNF)-α levels were unchanged. Positive correlations between IL-6 and IL-10 were found in all volunteer groups pre- and post-vaccination and also between IL-17 and IL-6 or IL-10 in the Gln group post-vaccination. A negative correlation between IL-37 and IL-10 was found pre- and post-vaccination in the PL group. Conclusion: Gln supplementation was able to modulate salivary cytokine profile and increase SIgA levels, both total and specific to the influenza virus vaccine, in physically active elderly subjects.

Evaluation of salivary VOC profile composition directed towards oral cancer and oral lesion assessment.

Endogenous substances have been analyzed in biological samples in order to be related with metabolic dysfunctions and diseases. The study aimed to investigate profiles of volatile organic compounds (VOCs) from fresh and incubated saliva donated by healthy controls, individuals with oral tissue lesions and with oral cancer, in order to assess case-specific biomarkers of oxidative stress. VOCs were pre-concentrated using headspace-solid phase microextraction and analyzed using gas chromatography-mass spectrometry. Then, VOCs positively modulated by incubation process were subtracted, yielding profiles with selected features. Principal component analysis and hierarchical cluster analysis were used to inspect data distribution, while univariate statistics was applied to indicate potential markers of oral cancer. Machine learning algorithm was implemented, aiming multiclass prediction. The removal of bacterial contribution to VOC profiles allowed the obtaining of more specific case-related patterns. Artificial neural network model included 9 most relevant compounds (1-octen-3-ol, hexanoic acid, E-2-octenal, heptanoic acid, octanoic acid, E-2-nonenal, nonanoic acid, 2,4-decadienal and 9-undecenoic acid). Model performance was assessed using 10-fold cross validation and receiver operating characteristic curves. Obtained overall accuracy was 90%. Oral cancer cases were predicted with 100% of sensitivity and specificity. The selected VOCs were ascribed to lipid oxidation mechanism and presented potential to differentiate oral cancer from other inflammatory conditions. These results highlight the importance of interpretation of saliva composition and the clinical value of salivary VOCs. Elucidated metabolic alterations have the potential to aid the early detection of oral cancer and the monitoring of oral lesions.

Assessment of Salivary Lipid Profile Parameters in Healthy and Type 2 Diabetes Mellitus Patients

Introduction: Recent studies suggest that saliva could be functionally equivalent to serum in reflecting the physiological state of the body. Studies are being conducted to develop saliva as a tool for the screening and monitoring of chronic systemic diseases like Diabetes mellitus. Dyslipidaemia is most commonly assessed by laboratory lipid profile analysis in serum sample. This study is a step towards validation of saliva as a tool for the screening and monitoring of dyslipidaemia. Aim: To measure, compare and correlate the serum and salivary lipid profile parameters {Total Cholesterol (TC), High Density Lipoprotein (HDL) and Low Density Lipoprotein (LDL)} in Type 2 Diabetes mellitus patients and healthy individuals. Materials and Methods: The case control study was carried out for a period of one year i.e., from March 2019 to February 2020. The study comprised of 200 participants between the ages 31-70 years. The participants included 100 diabetic subjects with a history of diabetes for at least three years and 100 age matched subjects as controls. TC, HDL and LDL was estimated by using kits based on spectrophotometric principle. Statistical analysis was done on Statistical Package for Social Sciences (SPSS) Version 16.0. Mean, Standard deviation, Pearson’s correlation coefficient, along with regression analysis was carried out and compared between the groups. Results: There is a significant difference between the salivary TC, HDL and LDL levels in healthy individuals and diabetics. A strong and significant positive correlation of serum TC (Pearson correlation coefficient ‘r’=0.87 for controls, 0.66 for diabetics, p&lt;0.01), HDL (Pearson correlation coefficient ‘r’=0.78 for controls, 0.88 for diabetics, p-value &lt;0.01) and LDL (Pearson correlation coefficient ‘r’= 0.81 for controls, 0.87 for diabetics, p-value &lt;0.01) levels with its respective salivary levels was obtained in all the participants (controls and diabetics) of the study. The regression coefficient was calculated and a formula was derived for prediction of serum lipid profile parameters using their respective salivary levels. Conclusion: Standardisation of the techniques for the estimation of salivary lipid profile parameters, including triglycerides and Very LDL (VLDL) and setting up a reference range will also make it useful in clinical practice.

Salivary Metabolic Profiling of Systemic Disorders and Oral Neoplastic and Preneoplastic Conditions- A Narrative Review

Salivary metabolic profiling has emerged as an important mode of analysing the metabolic markers that aids in early disease detection in various systemic diseases. The metabolomics study states that, the transitional and the end products of interactions that take place between genes, proteins, and the environment are found to be involved in various disease processes. Salivary metabolomics stands as a highly specific and sensitive method in diagnosis of various conditions making it a better alternative to the conventional serum and tissue-based methods well. These metabolomics studies incorporate various analytical technologies for identifying each component that could be used as a biomarker. Hence, we reviewed the current state of salivary metabolomics, diagnostic efficiency and its associated technologies and its future role in identification and monitoring the disease prognosis. The study selection was done by locating those research papers that provided information on salivary diagnostics using metabolic markers for early diagnosis in systemic disorders, neoplastic and preneoplastic conditions with help of search engines like Pubmed, Google Scholar, Web of Science and Cochrane library. The results of each study were critically evaluated to accentuate the principal role played by these biomarkers in the field of salivary diagnostics.

Dysbiosis in the Salivary Microbiome Associated with IgA Nephropathy-‍A‍ ‍Japanese Cohort Study.

IgA nephropathy is one of the leading causes of chronic kidney disease in Japan. Since the origin and mechanisms by which IgA nephropathy develops currently remain unclear, a confirmed disease diagnosis is currently only possible by highly invasive renal biopsy. With the background of the salivary microbiome as a rich source of biomarkers for systemic diseases, we herein primarily aimed to investigate the salivary microbiome as a tool for the non-invasive diagnosis of IgA nephropathy. In a comparison of salivary microbiome profiles using 16S rRNA amplicon sequencing, significant differences were observed in microbial diversity and richness between IgA nephropathy patients and healthy controls. Furthermore, recent studies reported that patients with IgA nephropathy are more likely to develop inflammatory bowel diseases and that chronic inflammation of the tonsils triggered the recurrence of IgA nephropathy. Therefore, we compared the salivary microbiome of IgA nephropathy patients with chronic tonsillitis and ulcerative colitis patients. By combining the genera selected by the random forest algorithm, we were able to distinguish IgA nephropathy from healthy controls with an area under the curve (AUC) of 0.90, from the ulcerative colitis group with AUC of 0.88, and from the chronic tonsillitis group with AUC of 0.70. Additionally, the genus Neisseria was common among the selected genera that facilitated the separation of the IgA nephropathy group from healthy controls and the chronic tonsillitis group. The present results indicate the potential of the salivary microbiome as a biomarker for the non-invasive diagnosis of IgA nephropathy.

Salivary profile of matrix metalloproteinase-8, tissue inhibitors of matrix metalloproteinase-1, myeloperoxidase, and nitrous oxide in smokers versus nonsmokers with chronic periodontitis

Salivary profile of matrix metalloproteinase-8, tissue inhibitors of matrix metalloproteinase-1, myeloperoxidase, and nitrous oxide in smokers versus nonsmokers with chronic periodontitis

Salivary characteristics may be associated with burning mouth syndrome?

BackgroundBurning mouth syndrome (BMS) it is characterized by burning and uncomfortable sensations with no clinical alterations or laboratory findings. The evaluation of the salivary characteristics of people with BMS can help the understanding of the pathogenesis of this condition. This case-control study aimed to trace the salivary profile of women with burning mouth syndrome (BMS). Material and Methods40 women with BMS and 40 control women were recruited. Unstimulated salivary flow rate (uSFR), pH, salivary cortisol levels, salivary viscosity, and oral health impact profile (OHIP-14 questioner) were determined. P< 0.05 was considered statistically significant. ResultsFor uSFR, mean values obtained for BMS and for control group respectively were 0.35 and 0.61 mL/min; for pH, 7.23 and 7.34; for cortisol levels, 0.36 and 0.15 μg/dL; for viscosity values, 31.1 and 45.01 mPas and for OHIP-14 scores, 21.7 and 5.7. To uSFR, cortisol levels, viscosity values and OHIP-14 scores, differences were statistically significant. Salivary cortisol levels and OHIP-14 scores were correlated positively (rho = 0.624; p< 0.05). ConclusionsBMS women have lower uSFR and salivary viscosity and higher salivary cortisol levels that were associated with worse quality of life, compared with the control group. Key words:Xerostomia, Burning mouth syndrome, Viscosity.

Evaluation of Saliva Stability for NMR Metabolomics: Collection and Handling Protocols.

Maintaining a salivary metabolic profile upon sample collection and preparation is determinant in metabolomics. Nuclear magnetic resonance (NMR) spectroscopy was used to identify metabolite changes during short-term storage, at room temperature (RT)/4 °C/−20 °C, and after sample preparation, at RT/4 °C (mimicking typical clinical/laboratory settings). Interestingly, significant metabolic inter-individual and inter-day variability were noted, probably determining sample stability to some extent. After collection, no changes were noted at −20 °C (at least for 4 weeks). RT storage induced decreases in methylated macromolecules (6 h); lactate (8 h); alanine (12 h); galactose, hypoxanthine, pyruvate (24 h); sarcosine, betaine, choline, N-acetyl-glycoproteins (48 h), while acetate increased (48 h). Less, but different, changes were observed at 4 °C, suggesting different oral and microbial status at different temperatures (with a possible contribution from inter-individual and inter-day variability), and identifying galactose, hypoxanthine, and possibly, choline esters, as potential general stability indicators. After preparation, addition of NaN3 did not impact significantly on saliva stabilization, neither at RT nor at 4 °C, although its absence was accompanied by slight increases in fucose (6.5 h) and proline (8 h) at RT, and in xylose (24 h) at 4 °C. The putative metabolic origins of the above variations are discussed, with basis on the salivary microbiome. In summary, after collection, saliva can be stored at RT/4 °C for up to 6 h and at −20 °C for at least 4 weeks. Upon preparation for NMR analysis, samples are highly stable at 25 °C up to 8 h and at 4 °C up to 48 h, with NaN3 addition preventing possible early changes in fucose, proline (6–8 h), and xylose (24 h) levels.

Perfluorinated polymer modified vertical silicon nanowires as ultra low noise laser desorption ionization substrate for salivary metabolites profiling

Metabolites in the body fluid are becoming a rich source of disease biomarkers. Developing an effective and high throughput detection and analysis platform of metabolites is of great importance for potential biomarker discovery and validation. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been successfully applied in rapid biomolecules detection in large scale. However, non-negligible background interference in low molecule-weight region still constitutes a main challenge even though various nanomaterials have been developed as an alternative to traditional organic matrix. In this work, a novel composite chip, silicon nanowires loaded with fluorinated ethylene propylene (FEP@SiNWs) was fabricated. It can serve as an excellent substrate for nanostructure-initiator mass spectrometry (NIMS) detection with ultra-low background noise in low molecular weight region (<500 Da). Ion desorption efficiency and internal energy transfer of FEP@SiNWs were studied using benzylpyridinium salt and tetraphenylboron salt as thermometer chemicals. The results indicated that a non-thermal desorption mechanism might be involved in the LDI process on FEP@SiNWs. Owing to the higher LDI efficiency and low background interference of this novel substrate, the metabolic fingerprint of complex bio-fluids, such as human saliva, can be sensitively and stably acquired. As a proof of concept, FEP@SiNWs chip was successfully used in the detection of salivary metabolites. With the assistance of multivariate analysis, 22 metabolic candidates (p < 0.05) which can discriminate type 2 diabetes mellitus (2-DM) and healthy volunteers were found and identified. The role of these feature metabolites in the metabolic pathway involved in 2-DM was confirmed by literature mining. This work demonstrates that FEP@SiNWs-based NIMS might be served as an efficient and high throughput platform for metabolic biomarker exploration and clinical diagnosis.

Salivary Metabolic Profile of Patients with Lung Cancer, Chronic Obstructive Pulmonary Disease of Varying Severity and Their Comorbidity: A Preliminary Study.

The aim of the work was to study the features of the salivary biochemical composition in the combined pathology of lung cancer and chronic obstructive pulmonary disease (COPD) of varying severity (COPD I, COPD II). The study group included patients with lung cancer (n = 392), non-malignant lung pathologies (n = 168) and healthy volunteers (n = 500). Before treatment, the salivary biochemical composition was determined according to 34 indicators. Survival analysis performed by the Kaplan-Meier method. Biochemical parameters (catalase, imidazole compounds ICs, sialic acids, lactate dehydrogenase (LDH)) that can be used to monitor patients at risk (COPD I) for timely diagnosis of lung cancer are determined. A complex of salivary biochemical indicators with prognostic value in lung cancer was revealed. For patients with lung cancer without COPD, a group of patients with a favorable prognosis can be distinguished with a combination of ICs < 0.478 mmol/L and LDH >1248 U/L (HR = 1.56, 95% CI 0.40–6.07, p = 0.03891). For COPD I, a level of ICs < 0.182 mmol/L are prognostically favorable (HR = 1.74, 95% CI 0.71–4.21, p = 0.07270). For COPD II, combinations of pH < 6.74 and LDH >1006 U/L are prognostically favorable. In general, for patients with lung cancer in combination with COPD I, the prognosis is more favorable than without COPD.