Research on Biomedical Engineering
Research on Biomedical Engineering
Original Article

Analysis of saliva by Fourier transform infrared spectroscopy for diagnosis of physiological stress in athletes

Caetano Júnior, Paulo Cesar; Strixino, Juliana Ferreira; Raniero, Leandro

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Introduction: Saliva is the most promising biofluid to monitor the physiological state of athletes, because this method is not invasive and has low contamination risks. The characterization of saliva by Fourier transform infrared spectroscopy (FT-IR) has been studied as an alternative technique to the standard clinical analysis. However, methodological procedures for saliva analysis are not completely clear, especially in terms of influence of storage conditions and sample preparations for infrared analysis. Thawed saliva includes a precipitate, which may influence the infrared spectral analysis. Thus, the purpose of this study was to show the spectral differences of the precipitate, supernatant, and a combo, as well as the best way to classify the physiological state of the athletes by FT-IR. Methods: The saliva collection was performed before, immediately after, and two hours after a handball match. After the storage of samples at –20 ○C, it was possible to identify two phases (precipitate and supernatant) and to determine the biochemical differences between the spectra of each phase, which were distinctly analyzed by the second derivative and deconvolution bands. Results: The precipitate and supernatant results showed characteristic bands, especially in the protein regions. All FT-IR spectra were also statistically classified by linear discriminant analysis (LDA), using principal component analysis (PCA). The LDA precipitate and supernatant had lower value when compared to combo spectra (Combination of precipitate and supernatant) with 82%, showing that this combination is the best way to discriminate spectra of saliva samples collected before, immediately after, and 2 h after physical effort. Discussion: The results showed that it is possible to differentiate biochemically the two salivary phases, as well as the importance of the homogenization process of saliva samples to classify the physiological status of athletes using FT-IR.


Infrared spectroscopy, Saliva, Sports.


Al-Shehri S, Henman M, Charles BG, Cowley D, Shaw PN, Liley H, Tomarchio A, Punyadeera C, Duley JA. Collection and determination of nucleotide metabolites in neonatal and adult saliva by high performance liquid chromatography with tandem mass spectrometry. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. 2013; 931:140-7. PMid:23792366

Beekes M, Lasch P, Naumann D. Analytical applications of Fourier transform-infrared (FT-IR) spectroscopy in microbiology and prion research. Veterinary Microbiology. 2007; 123(4):305-19. PMid:17540519

Bonne NJ, Wong DT. Salivary biomarker development using genomic, proteomic and metabolomic approaches. Genome Medicine. 2012; 4(10):82. PMid:23114182

Cai S, Singh BR. A distinct utility of the amide III infrared band for secondary structure estimation of aqueous protein solutions using partial least squares methods. Biochemistry. 2004; 43(9):2541-9. PMid:14992591

Carvalho CS, Andrade, LEC, Keusseyan SP, Rangel JL, Ferreira-Strixino J, Martin AA, Raniero LJ. Study of advanced rheumatoid arthritis. Brazilian Journal of Biomedical Engineering. 2014; 30 (1) 54-63

Cheheltani R, Rosano JM, Wang B, Sabri AK, Pleshko N, Kiani MF. Fourier transform infrared spectroscopic imaging of cardiac tissue to detect collagen deposition after myocardial infarction. Journal of Biomedical Optics. 2012; 17(5):056014. PMid:22612137

Chiappin S, Antonelli G, Gatti R, De Palo EF. Saliva specimen: a new laboratory tool for diagnostic and basic investigation. Clinica Chimica Acta; International Journal of Clinical Chemistry. 2007; 383(1-2):30-40. PMid:17512510

DeCaro JA. Methodological considerations in the use of salivary alpha-amylase as a stress marker in field research. American Journal of Human Biology: The Official Journal of the Human Biology Council. 2008; 20(5):617-9. PMid:18491409

Diaz MM, Bocanegra OL, Teixeira RR, Soares SS, Espindola FS. Salivary nitric oxide and alpha-amylase as indexes of training intensity and load. International Journal of Sports Medicine. 2013; 34(1):8-13. PMid:22960992

Edgar WM. Saliva and dental health. Clinical implications of saliva: report of a consensus meeting. British Dental Journal. 1990; 169(3-4):96-8. PMid:2206655

Ellis DI, Goodacre R. Metabolic fingerprinting in disease diagnosis: biomedical applications of infrared and Raman spectroscopy. Analyst (London). 2006; 131(8):875-85. PMid:17028718

Everitt BS. Statistical methods for medical investigations. 2nd ed. London: Edward Arnold; 1994

Fox PC, van der Ven PF, Baum BJ, Mandel ID. Pilocarpine for the treatment of xerostomia associated with salivary gland dysfunction. Oral Surgery, Oral Medicine, and Oral Pathology. 1986; 61(3):243-8. PMid:3517744

Francis CA, Hector MP, Proctor GB. Precipitation of specific proteins by freeze-thawing of human saliva. Archives of Oral Biology. 2000; 45(7):601-6. PMid:10785524

Franck P, Nabet P, Dousset B. Applications of infrared spectroscopy to medical biology. Cell Mol Biol (Noisy-le-grand). 1998; 44(2):273-5. PMid:9593577

Gonnissen HK, Rutters F, Mazuy C, Martens EA, Adam TC, Westerterp-Plantenga MS. Effect of a phase advance and phase delay of the 24-h cycle on energy metabolism, appetite, and related hormones. The American Journal of Clinical Nutrition. 2012; 96(4):689-97. PMid:22914550

Hansen AM, Garde AH, Persson R. Sources of biological and methodological variation in salivary cortisol and their impact on measurement among healthy adults: a review. Scandinavian Journal of Clinical and Laboratory Investigation. 2008; 68(6):448-58. PMid:18609093

Humphrey SP, Williamson RT. A review of saliva: normal composition, flow, and function. The Journal of Prosthetic Dentistry. 2001; 85(2):162-9. PMid:11208206

Jagannathan N, Thiruvengadam C, Ramani P, Premkumar P, Natesan A, Sherlin HJ. Salivary ferritin as a predictive marker of iron deficiency anemia in children. The Journal of Clinical Pediatric Dentistry. 2012; 37(1):25-30. PMid:23342563

Jerônimo DP, Souza RA, Silva FF, Camargo GL, Miranda HL, Xavier M, Sakane KK, Ribeiro W. Detection of creatine in rat muscle by FTIR spectroscopy. Annals of Biomedical Engineering. 2012; 40(9):2069-77. PMid:22419197

Khaskheli AR, Sirajuddin, Sherazi ST, Mahesar SA, Kandhro AA, Kalwar NH, Mallah MA. Estimation of ibuprofen in urine and tablet formulations by transmission Fourier transform infrared spectroscopy by partial least square. Spectrochimica Acta. Part A: Molecular and Biomolecular Spectroscopy. 2013; 102:403-7. PMid:23237846

Khaustova SA, Shkurnikov MU, Grebenyuk ES, Artyushenko VG, Tonevitsky AG. Assessment of biochemical characteristics of the saliva using Fourier transform mid-infrared spectroscopy. Bulletin of Experimental Biology and Medicine. 2009; 148(5):841-4. PMid:20396807

Khaustova S, Shkurnikov M, Tonevitsky E, Artyushenko V, Tonevitsky A. Noninvasive biochemical monitoring of physiological stress by Fourier transform infrared saliva spectroscopy. Analyst (London). 2010; 135(12):3183-92. PMid:20953513

Krimm S, Bandekar J. Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. Advances in Protein Chemistry. 1986; 38:181-364. PMid:3541539

Meeusen R, Duclos M, Foster C, Fry A, Gleeson M, Nieman D, Raglin J, Rietjens G, Steinacker J, Urhausen A, European College of Sport Science, American College of Sports Medicine. Prevention, diagnosis, and treatment of the overtraining syndrome: joint consensus statement of the European College of Sport Science and the American College of Sports Medicine. Medicine and Science in Sports and Exercise. 2013; 45(1):186-205. PMid:23247672

Moreira A, Freitas CG, Nakamura FY, Drago G, Drago M, Aoki MS. Effect of match importance on salivary cortisol and immunoglobulin A responses in elite young volleyball players. Journal of Strength and Conditioning Research / National Strength & Conditioning Association. 2013; 27(1):202-7. PMid:22395269

Nater UM, Rohleder N. Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: current state of research. Psychoneuroendocrinology. 2009; 34(4):486-96. PMid:19249160

Novakovic N, Cakic S, Todorovic T, Raicevic BA, Dozic I, Petrovic V, Perunovic N, Gostovic SS, Sretenovic JK, Colak E. Antioxidative status of saliva before and after non-surgical periodontal treatment. Srpski Arhiv za Celokupno Lekarstvo. 2013; 141(3-4):163-8. PMid:23745337

Oberg KA, Ruysschaert JM, Goormaghtigh E. The optimization of protein secondary structure determination with infrared and circular dichroism spectra. European Journal of Biochemistry / FEBS. 2004; 271(14):2937-48. PMid:15233789

Omelia EJ, Uchimoto ML, Williams G. Quantitative PCR analysis of blood- and saliva-specific microRNA markers following solid-phase DNA extraction. Analytical Biochemistry. 2013; 435(2):120-2. PMid:23333269

Perez-Guaita D, Ventura-Gayete J, Pérez-Rambla C, Sancho-Andreu M, Garrigues S, de la Guardia M. Protein determination in serum and whole blood by attenuated total reflectance infrared spectroscopy. Analytical and Bioanalytical Chemistry. 2012; 404(3):649-56. PMid:22547354

Petibois C, Déléris G, Cazorla G. Perspectives in the utilisation of Fourier-transform infrared spectroscopy of serum in sports medicine: health monitoring of athletes and prevention of doping. Sports Medicine (Auckland, N.Z.). 2000; 29(6):387-96. PMid:10870865

Rohleder N, Nater UM. Determinants of salivary alpha-amylase in humans and methodological considerations. Psychoneuroendocrinology. 2009; 34(4):469-85. PMid:19155141

Salazar MG, Jehmlich N, Murr A, Dhople VM, Holtfreter B, Hammer E, Völker U, Kocher T. Identification of periodontitis associated changes in the proteome of whole human saliva by mass spectrometric analysis. Journal of Clinical Periodontology. 2013; 40(9):825-32. PMid:23790309

Schultz CP, Ahmed MK, Dawes C, Mantsch HH. Thiocyanate levels in human saliva: quantitation by Fourier transform infrared spectroscopy. Analytical Biochemistry. 1996; 240(1):7-12. PMid:8811872

Stuart B. Biological applications of infrared spectroscopy. Chichester: John Wiley & Sons; 1997

Thieme D. Potential and limitations of alternative specimens in doping control. Bioanalysis. 2012; 4(13):1613-22. PMid:22831477

Toone RJ, Peacock OJ, Smith AA, Thompson D, Drawer S, Cook C, Stokes KA. Measurement of steroid hormones in saliva: Effects of sample storage condition. Scandinavian Journal of Clinical and Laboratory Investigation. 2013; 73(8):615-21. PMid:24033227

Ward JH Jr. Hierarchical grouping to optimize an objective function. Journal of the American Statistical Association. 1963; 58(301):236-44.
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