18


Figure 3. Effect of ionic strength, sample volume, pH and stirring on the extraction efficiency of VOMs


Characterisation and Comparative Analysis of Salivary VOMs


Following the optimisation of the best extraction conditions, salivary volatile metabolites were analysed using dHS-SPME in combination with GC coupled to mass spectrometry with the aim to distinguish individuals with BC from healthy subjects. Different profiles for healthy subjects and BC patients were recognised, with significant differences in the fatty acids, higher alcohols and phenols (Figure 4).


The major metabolites identified in both groups were acetic acid, 2-ethyl-1-hexanol, propanoic acid, butanoic acid, methoxyphenyl- oxime and phenol (see Figure 5).


Overall, phenol and 2-ethyl-1-hexanol were the compounds that presented the greater difference between the healthy individuals and BC patients studied.


Figure 6. Variation of the main VOMs identified in healthy individuals and BC patients (major compounds: 1. Acetic acid; 2. 2-Ethyl-1- hexanol; 3. Propanoic acid; 4. Butanoic acid; 5. Methoxyphenyl-oxime; 6. Phenol).


Acknowledgments


Figure 4. Distribution of major chemical families by healthy individuals (Control) and BC patients. FA- Fatty Acids; HAl- Higher Alcohols; PH- Phenols


Conclusions


Saliva presents significant advantages as a diagnostic tool when compared with blood or urine. Saliva sampling is easy, safe, and non-invasive and may be collected repetitively without discomfort to the patient. Here we optimised the characterisation of the volatile composition of saliva using dHS-SPME followed by GC-qMS analysis. Different experimental factors were evaluated and the most effective extraction was achieved using 2 ml of acidified saliva sample containing 10% NaCl (w/v), stirred and exposed to the CAR/ PDMS fibre for 45 min, at 38°C. Preliminary results point to higher abundances of fatty acids, higher alcohols and phenols in saliva samples from healthy subjects than BC patients. Ongoing studies are focusing in individual volatiles that could be used as cancer biomarkers. This work reveals the potential of saliva as a promising biological fluid for cancer biomarkers research, particularly its suitability for the early disease diagnosis.


The authors acknowledge the financial support of the FP7 New INDIGO Partnership Programme through the HCV project (New- INDIGO/0003/2012 project, ERA- NET) and Fundação para a Ciência e a Tecnologia (FCT) with funds from the Portuguese Government (Project PEst-OE/QUI/UI0674/2011), the MS Portuguese Network (Rede/1508/RNEM/2005) and the fellowship SFRH/BPD/66177/2009 given to Jorge Pereira. Chromaleont is also acknowledged for the travel grant awarded to Jorge Pereira.


References


1. Ullah MF, Aatif M. The footprints of cancer development: Cancer biomarkers. Cancer Treatment Reviews. 2009;35(3):193-200. 2. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA: a cancer journal for clinicians. 2010;60(5):277-300.


3. Silva CL, Passos M, Camara JS. Investigation of urinary volatile organic metabolites as potential cancer biomarkers by solid-phase microextraction in combination with gas chromatography-mass spectrometry. Br J Cancer. 2011;105(12):1894-904.


4. Matsumura K, Opiekun M, Oka H, Vachani A, Albelda SM, Yamazaki K, et al. Urinary volatile compounds as biomarkers for lung cancer: a proof of principle study using odor signatures in mouse models of lung cancer. PloS one. 2010;5(1):e8819. 5. Liu J, Duan Y. Saliva: a potential media for disease diagnostics and monitoring. Oral oncology. 2012;48(7):569-77.


Figure 5. GC-qMS chromatograms from BC patients and healthy subjects (control) (major compounds: 1.Acetic acid; 2. 2-Ethyl-1- hexanol; 3. Propanoic acid; 4. Butanoic acid; 5.Methoxyphenyl-oxime; 6.Phenol).


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