Untargeted Metabolomic Profiling in Saliva of Smokers and Nonsmokers by a Validated GC-TOF-MS Method

Abstract

A GC-TOF-MS method was developed and validated for a metabolic fingerprinting in saliva of smokers and nonsmokers. We validated the method by spiking 37 different metabolites and 6 internal standards to saliva between 0.1 μM and 2 mM. Intraday coefficients of variation (CVs) (accuracies) were on average, 11.9% (85.8%), 8.2% (88.9%), and 10.0% (106.7%) for the spiked levels 25, 50, and 200 μM, respectively (N = 5). Interday CVs (accuracies) were 12.4% (97%), 18.8% (95.5%), and 17.2% (105.9%) for the respective levels of 25, 50, and 200 μM (N = 5). The method was applied to saliva of smokers and nonsmokers, obtained from a 24 h diet-controlled clinical study, in order to identify biomarkers of endogenous origin, which could be linked to smoking related diseases. Automated peak picking, integration, and statistical analysis were conducted by the software tools MZmine, Metaboanalyst, and PSPP. We could identify 13 significantly altered metabolites in smokers (p < 0.05) by matching them against MS libraries and authentic standard compounds. Most of the identified metabolites, including tyramine, adenosine, and glucose-6-phosphate, could be linked to smoking-related perturbations and may be associated with established detrimental effects of smoking.

Global metabolomic profiles reveal differences in oxidative stress and inflammation pathways in smokers and moist snuff consumers

“…Several studies have shown adenosine and other purines to be elevated in COPD. Further, purine degradation products such as adenosine, hypoxanthine, and inosine were present at higher levels in gingival crevicular fluid from patients with periodontal disease [19,26] and in saliva of smokers [15]. Data presented herein showed elevated levels of hypoxanthine (plasma) and a corresponding lower urinary hypoxanthine, as well as elevated levels of adenosine and inosine in saliva of SMK relative to the MSC (Figure 3B).…”

“…Untargeted metabolomic profiling is a powerful tool employed in biomarker research [28] and to gain a better understanding of the effects of nutritional and environmental exposure [29,30], critical path [31], and for other purposes. Several researchers have utilized metabolomic profiling to evaluate the effects of smoking through NMR-based [14] and MS-based technologies [15,32]. In pilot experiments, the MSbased metabolomics yielded higher numbers of metabolites (this study) than NMR-based methods (unpublished data).…”

“…Several researchers have shown that smokers and nonsmokers could be readily distinguished based on their metabolomic profiles [14,15], and those analyses included nicotine and its metabolites. We were able to replicate those results in our study as well.…”

“…Although a relatively newer approach compared to other "omic" technologies, metabolomics is a powerful analytical tool. It has been utilized to evaluate the effect of exposure to smoke constituents [13] and to assess the effect of smoking and smoking cessation [14][15][16][17][18]. In an effort to elucidate biochemical changes in healthy consumers of moist snuff and cigarettes, and to identify potential candidate biomarkers of tobacco effect, we conducted a global metabolomic profiling of plasma, urine, and saliva from the same study cohorts described in the candidate biomarker investigation [53].…”

Influence of glutathione-S-transferase (GST) inhibition on lung epithelial cell injury: role of oxidative stress and metabolism

“…For instance, glutamate acts as a neurotransmitter in various oxidative stress-related neurodegenerative conditions and has been linked to the development of lung injury in animal models of hyperoxia and sepsis (10,30,36). In addition, threonine levels increase in response to hypoxic injury (42), and elevated levels of tyramine have been reported following myocardial infarction, acute cystitis, and smoking (22,27,43). Further experimental work and bioinformatics analysis is required to determine the precise role of these metabolites in ischemia, oxidative stress, and inflammation.…”