Figure 8. Boxplots of the most important variables (VOMS) for discriminating between (a) COVID-CTRL and (b) COVID - RECOV. MTA - 3-methoxy-5-(trifluoromethyl)aniline; TND - 1,1,6-Trimethyl-dihydronaphthalene.
Some VOMs, such as D-carvone, MTA, TDN, and α-terpinene, are associated with diet [32-35], so their interpretation as potential biomarkers of COVID-19 infection and progression is not straightforward.
3. DISCUSSION
This study focused on the analysis of the volatile composition of the urine of patients infected with SARS-CoV-2 upon recovery. Overall, 101 different VOMs were identified in the urine of the recruited subjects, and statistically significant differences were found between these groups and control subjects. Accordingly, a volatile signature composed of D-carvone, MTA, TDN, 2-heptanone, and TONEA and nonanoic acid, α-terpinene, β-damascenone, α-isophorone, and t-furan linalool were defined for COVID-19 patients and patients recovered from the disease, respectively. Correspondent boxplots show sharp variations in the levels of the referred VOMs between analysed groups. The interpretation of these variations in urine composition is hindered by the fact that they undergo modifications due to various factors, including metabolic processes, pH fluctuations, bacterial activity, and the degradation of urine components. Additionally, external factors such as diet, lifestyle habits, health conditions, physical stress, and the environment also affect the urine composition [16]. Terpenes are often associated with exogenous sources such as beverages, foods, and flavouring ingredients, although they can also be produced through the mevalonic acid pathway [28,35]. Carotenoid-rich foods are a source of volatile norisoprenoids, which are produced through enzymatic degradation and have been reported in other studies involving volatile urinary fingerprinting [36]. Phenolic compounds are often found in urine as by-products of metabolic processes, but they can also be produced through the consumption of food, beverages, industrial chemicals, and environmental pollutants [37]. Ketones are a key subgroup of chemicals detected in urine, and multiple metabolic pathways are involved in their production, including carbohydrate metabolism, decarboxylation of oxo-acids, and lipid peroxidation [38,39]. Some studies have suggested that a considerable portion of ketones in urine may stem from gut bacterial activity, external sources such as foodstuffs, beverages, flavouring ingredients, or pollution [31,40]. To our knowledge, this is the first study to reveal changes in the urinary volatilomic profile following SARS-CoV-2 infection and recovery from COVID-19. Such changes define volatile signatures with the potential to be used in noninvasive COVID-19 diagnosis and management. In this context, the number of samples constitutes a limitation of this study, which can be circumvented by future disease outbreaks. In this scenario, the experimental conditions, safety protocols, and collaboration between the research entities involved in this study can be promptly activated, allowing the recruitment of more subjects and relevant clinical information. Age, diet, previous clinical condition, sex, and gender, for instance, were interferents that could not be included in the present study.
4. CONCLUSION
A novel, fast, and sensitive analytical approach was developed and successfully applied in diagnosis of COVID-19 infection. This exploratory study using HS-SPME/GC-MS, unveiled significant differences in the volatilomic patterns of COVID-19 patients and recovered patients compared to control subjects, evidencing that SARS-CoV-2 infection triggered metabolic changes that also affect the urinary volatile composition of the infected patients. This constitutes a signature with potential for COVID-19 diagnosing and monitoring of the disease progression with potential to be successfully used in clinical applications. Overall, the results show the feasibility of using urine samples for the non-invasive COVID-19 diagnosis and further studies with larger cohorts are desirable, envisaging the developing of this complementary tool for COVID-19 diagnosis.