Specific and robust ion chromatographic determination of hypothiocyanite in saliva samples
Abstract
The enzymatic system in saliva, consisting of salivary peroxidase (SPO), hydrogen peroxide (H2O2), and thiocyanate (SCN−), produces hypothiocyanite (OSCN−) as a high effective antibacterial compound. OSCN− is of great importance for the natural non-specific antibacterial resistance in the oral cavity. However, no analytical method currently exists to selectively quantify OSCN− in saliva samples. A robust and specific analytical method for the determination of OSCN− was developed based on ion chromatography with combined UV and electrochemical detection. Calibration was achieved by calculating a derived calibration factor based on the known ratio of molar extinction coefficients of SCN− and OSCN−. Thus, the specific quantification of OSCN− in saliva samples is possible, as demonstrated here. The median value of 200 saliva samples was determined to be 0.56 mg L−1 (median), with a maximum of 3.9 mg L−1; the minimum value was below the detection limit (< 0.09 mg L−1). The recovery rate in individual saliva samples was 95 ± 8%.
Keywords
Hypothiocyanite Thiocyanate Ion chromatography Saliva peroxidaseNotes
Acknowledgements
The study was supported by the European Regional Development Fund: V-630-S-137-2012/024, V-630-F-137-2012/023, V-630-VB243-2012/022 (Mecklenburg-Western Pomerania Ministry for Economics, Employment and Tourism/Bmp Bulk Medicines & Pharmaceuticals Production GmbH) and by University Medicine Greifswald.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
Supplementary material
References
- 1.Hawkins CL. The role of hypothiocyanous acid (HOSCN) in biological systems. Free Rad Res. 2009;43(12):1147–58.CrossRefGoogle Scholar
- 2.Chandler JD, Day BJ. Thiocyanate: a potentially useful therapeutic agent with host defense and antioxidant properties. Biochem Pharmacol. 2012;84(11):1381–7.CrossRefGoogle Scholar
- 3.Van Haeringen NJ, Ensink FTE, Glasius E. Peroxidase-thiocyanate-hydrogenperoxide system in tear fluid and saliva of different species. Exp Eye Res. 1979;28(3):343–7.CrossRefGoogle Scholar
- 4.Haddadin MS, Ibrahim SA, Robinson RK. Preservation of raw milk by activation of the natural lactoperoxidase systems. Food Control. 1996;7(3):149–52.CrossRefGoogle Scholar
- 5.Gerson C, Sabater J, Scuri M, Torbati A, Coffey R, Abraham JW, et al. The lactoperoxidase system functions in bacterial clearance of airways. Am J Respir Cell Mol Biol. 2000;22(6):665–71.CrossRefGoogle Scholar
- 6.Wijkstrom-Frei C, El-Chemaly S, Ali-Rachedi R, Gerson C, Cobas MA, Forteza R, et al. Lactoperoxidase and human airway host defense. Am J Respir Cell Mol Biol. 2003;29(2):206–12.CrossRefGoogle Scholar
- 7.Reiter B, Härnulv BG. Lactoperoxidase thiocyanate hydrogen-peroxide—a natural antibacterial system. Kieler Milchw Forsch. 1982;34(1):50–3.Google Scholar
- 8.Shin K, Horigome A, Yamauchi K, Takase M, Yaeshima T, Iwatsuki K. Effects of orally administered bovine lactoperoxidase on dextran sulfate sodium-induced colitis in mice. Biosci Biotechnol Biochem. 2008;72(7):1932–5.CrossRefGoogle Scholar
- 9.Bafort F, Parisi O, Perraudin JP, Jijakli MH. Mode of action of lactoperoxidase as related to its antimicrobial activity: a review. Enzyme Res. 2014;2014:1–13.CrossRefGoogle Scholar
- 10.Tenovuo J. Clinical applications of antimicrobial host proteins lactoperoxidase, lysozyme and lactoferrin in xerostomia: efficacy and safety. Oral Dis. 2002;8(1):23–9.CrossRefGoogle Scholar
- 11.Aune TM, Thomas EL. Accumulation of hypothiocyanate ion during peroxidase-catalysed oxidation of thiocyanate ion. Eur J Biochem. 1977;80:209–14.CrossRefGoogle Scholar
- 12.Aune TM, Thomas EL. Oxidation of protein sulfhydryls by products of peroxidase-catalyzed oxidation of thiocyanate ion. Biochemist. 1978;17(6):1005–10.CrossRefGoogle Scholar
- 13.Pruitt KM, Adamson M, Arnold R. Lactoperoxidase binding to streptococci. Infect Immun. 1979;25(1):304–9.Google Scholar
- 14.Thomas EL, Bates KP, Jefferson MM. Hypothiocyanate ion: detection of the antimicrobial agent in human saliva. J Dent Res. 1980;59:1466–72.CrossRefGoogle Scholar
- 15.Carlsson J. Catalytic activity of lactoperoxidase in the presence of SCN. Biochem Biophys Res Commun. 1983;116(2):568–73.CrossRefGoogle Scholar
- 16.Tenovuo J, Pruitt KM, Mansson-Rahemtulla B, Harrington P, Baldone DC. Products of thiocyanate peroxidation: properties and reaction mechanisms. Biochim Biophys Acta. 1986;870:377–84.CrossRefGoogle Scholar
- 17.Gau J, Furtmüller P-G, Obinger C, Arnhold J, Flemmig J. Enhancing hypothiocyanite production by lactoperoxidase—mechanism and chemical properties of promotors. Biochem Biophys Rep. 2015;4:257–67.Google Scholar
- 18.Hoogendoorn H, Piessens JP, Scholtes W, Stoddard LA. Hypothiocyanite ion; the inhibitor formed by the system lactoperoxidase-thiocyanate-hydrogen peroxide. I. Identification of the inhibiting compound. Caries Res. 1977;11(2):77–84.CrossRefGoogle Scholar
- 19.Hoogendoorn H, Scholtes W. Effect of inhibitor of lactoperoxidase system on glycolysis of different microorganisms. Caries Res. 1977;11(2):123.CrossRefGoogle Scholar
- 20.Pruitt KM, Tenovuo J. Kinetics of hypothiocyanite production during peroxidase-catalyzed oxidation of thiocyanate. Biochim Biophys Acta. 1982;704(2):204–14.CrossRefGoogle Scholar
- 21.Hogg DM, Jago GR. The antibacterial action of lactoperoxidase. The nature of the bacterial inhibitor. Biochem J. 1970;117(4):779–90.CrossRefGoogle Scholar
- 22.Nagy P, Wang X, Lemma K, Ashby MT. Reactive sulfur species: hydrolysis of hypothiocyanite to give thiocarbamate-S-oxide. J Am Chem Soc. 2007;129(51):15756–7.CrossRefGoogle Scholar
- 23.Kalmar J, Woldegiorgis KL, Biri B, Ashby MT. Mechanism of decomposition of the human defense factor hypothiocyanite near physiological pH. J Am Chem Soc. 2011;133(49):19911–21.CrossRefGoogle Scholar
- 24.Seifu E, Buys EM, Donkin EF. Significance of the lactoperoxidase system in the dairy industry and its potential applications: a review. Trend Food Sci Tech. 2005;16(4):137–54.CrossRefGoogle Scholar
- 25.Thomas EL. Lactoperoxidase-catalysed oxidation of thiocyanate: equilibria between oxidized forms of thiocyanate. Biochemist. 1981;20:3273–80.CrossRefGoogle Scholar
- 26.Nagy P, Alguindigue SS, Ashby MT. Lactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide: a reinvestigation of hypothiocyanite by nuclear magnetic resonance and optical spectroscopy. Biochemist. 2006;45(41):12610–6.CrossRefGoogle Scholar
- 27.Hegde S, Chatterjee E, Rajesh KS, Kumar MS. Estimation and correlation of salivary thiocyanate levels in periodontally healthy subjects, smokers, nonsmokers, and gutka-chewers with chronic periodontitis. Indian J Dent Res. 2016;27(1):12–4.CrossRefGoogle Scholar
- 28.Pruitt KM, Tenovuo J, Fleming W, Adamson M. Limiting factors for the generation of hypothiocyanite ion, an antimicrobial agent, in human saliva. Caries Res. 1982;16(4):315–23.CrossRefGoogle Scholar
- 29.Yang BC, Zhang FF, Liang XM. Recent development in capillary ion chromatography technology. Cent Eur J Chem. 2012;10(3):472–9.Google Scholar
- 30.Christy AA, Egeberg PK. Oxidation of thiocyanate by hydrogen peroxide—a reaction kinetic study by capillary electrophoresis. Talanta. 2000;51(6):1049–58.CrossRefGoogle Scholar
- 31.DIN 32645. Chemical analysis: decision limit, detection limit and determination limit: estimation in case of repeatability, terms, methods, evaluation. Berlin: Beuth Verlag; 2011.Google Scholar
- 32.Collier HB. Note on molar absorptivity of reduced Ellmans reagent, 3-carboxylato-4-nitrothiophenolate. Anal Biochem. 1973;56(1):310–1.CrossRefGoogle Scholar
- 33.Riddles PW, Blakeley RL, Zerner B. Reassessment of Ellman reagent. Method Enzymol. 1983;91:49–60.CrossRefGoogle Scholar
- 34.Kgesa T, Choonara YE, Tyagi C, Tomar LK, Kumar P, du Toit LC, et al. Disulphide-thiol chemistry: a multi-faceted tool for macromolecular design and synthesis of polyfunctional materials for specialized drug delivery. Curr Drug Deliv. 2015;12(3):282–98.CrossRefGoogle Scholar
- 35.Hansen RE, Winther JR. An introduction to methods for analyzing thiols and disulfides: reactions, reagents, and practical considerations. Anal Biochem. 2009;394(2):147–58.CrossRefGoogle Scholar
- 36.Winther JR, Thorpe C. Quantification of thiols and disulfides. Bba-Gen Subjects. 2014;1840(2):838–46.CrossRefGoogle Scholar
- 37.Flemmig J, Rusch D, Czerwinska ME, Rauwald HW, Arnhold J. Components of a standardised olive leaf dry extract (Ph. Eur.) promote hypothiocyanite production by lactoperoxidase. Arch Biochem Biophys. 2014;549:17–25.CrossRefGoogle Scholar
- 38.Thürkow B, Jess G, Weuffen W. Comparative studies on the determination of thiocyanate in biological-materials. Pharmazie. 1982;37(4):264–9.Google Scholar
- 39.Valdes MG, Diaz-Garcia ME. Determination of thiocyanate within physiological fluids and environmental samples: current practice and future trends. Crit Rev Anal Chem. 2004;34(1):9–23.CrossRefGoogle Scholar
- 40.De Brabander HF, Verbeke R. Determination of thiocyanate in tissues and body-fluids of animals by gas-chromatography with electron-capture detection. J Chromatogr. 1977;138(1):131–42.CrossRefGoogle Scholar
- 41.Vesey CJ, Kirk CJC. Two automated methods for measuring plasma thiocyanate compared. Clin Chem. 1985;31(2):270–4.Google Scholar
- 42.Singh RP, Smesko SA, Abbas NM. Ion chromatographic characterization of toxic solutions: analysis and ion chemistry of biological liquids. J Chromatogr A. 1997;774(1–2):21–35.CrossRefGoogle Scholar
- 43.Chen ZF, Darvell BW, Leung VWH. Validation of ion chromatography for human salivary anionic analysis. Arch Oral Biol. 2004;49(11):855–62.CrossRefGoogle Scholar
- 44.Tenovuo J, Pruitt KM. Relationship of the human salivary peroxidase system to oral health. J Oral Pathol. 1984;13(6):573–84.CrossRefGoogle Scholar
- 45.Courtois P, Pourtois M. Purification of NADH: hypothiocyanite oxidoreductase in Streptococcus sanguis. Biochem Mol Med. 1996;57(2):134–8.CrossRefGoogle Scholar
- 46.Lenander-Lumikari M, Tenovuo J, Mikola H. Effects of lactoperoxidase system-containing toothpaste on levels of hypothiocyanite and bacteria in saliva. Caries Res. 1993;27:285–91.CrossRefGoogle Scholar
- 47.Kirstila V, Lenander-Lumikari M, Tenovuo J. Effects of a lactoperoxidase-system-containing toothpaste on dental plaque and whole saliva in vivo. Acta Odontol Scand. 1994;52(6):346–53.CrossRefGoogle Scholar
- 48.Tenovuo J, Anttonen T. Peroxidase-catalyzed hypothiocyanite production in human salivary sediment in relation to oral health. Caries Res. 1980;14(5):269–75.CrossRefGoogle Scholar