Oxidative Consumption of Oral Biomolecules by Therapeutically-Relevant Doses of Ozone

Hubert Chang¹, Edward Lynch², Martin Grootveld^2,3*

¹83 Chambers Lane, Willesden Green, London, UK.
²Warwick Dentistry, Warwick Medical School, University of Warwick, Warwick, UK.
³Institute for Materials Research and Innovation (IMRI), University of Bolton, Bolton, UK.

DOI: 10.4236/aces.2012.22028                

Abstract

In view of its potent microbicidal actions, ozone (O₃) offers much potential for application as a therapeutic agent in oral health, e.g. in the treatment of dental caries. This oxidant is extremely reactive towards biomolecules present in the oral environment, and in this study we have employed high-resolution proton (¹H) nuclear magnetic resonance (NMR) spectroscopy to determine the nature and extent of the oxidation of biomolecules known to be present in carious dentin, plaque and saliva. Phosphate-buffered (pH 7.00) aqueous solutions containing sodium pyruvate, α-D-glucose, L-cys teine and L-methionine (5.00 mM) were treated with gaseous O₃ (4.48 mmol.) delivered by a therapeutic O₃ generating device. Attack of O₃ on methionine and cysteine generated the corresponding primary oxidation products of these substrates, specifically methionine sulphoxide [98% ± 4% (mean ± SEM) yield] and cystine (95% ± 6% yield) respectively, and treatment of pyruvate with this oxidant produced acetate and CO₂ via an oxidative decarboxylation process (93% ± 4% yield). Reaction of O₃ with α-D-glucose gave rise to formate as a major product (24% ± 2% yield). In conclusion, multicomponent ¹H NMR analysis of appropriate chemical model systems provides valuable molecular information regarding the reactivity of O₃ towards biomolecules present in the oral environment, information which is of much relevance to its therapeutic mechanisms of action. Moreover, in view of the much higher concentrations of these O₃-scavenging biomolecules in oral fluid and/or soft tissue environments than that of O₃ applied, they may also serve to offer protection against putative adverse effects inducible by any of this oxidant which escapes from its site of therapeutic application (e.g., at primary root carious lesions).

Keywords

Ozone Therapy; Ozone Scavengers; Oral Biomolecules; Oral Fluids and Tissues; 1H NMR Analysis

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	D. Beighton, E. Lynch and M. R. Heath, “A Microbiological Study of Primary Root Caries Lesions with Different Treatment Needs,” Journal of Dental Research, Vol. 72, No. 3, 1993, pp. 623-629. doi:10.1177/00220345930720031201
[2]	C. J. L. Silwood, M. C. Grootveld, A. W. D. Claxson and E. Lynch, “1H and 13C NMR Spectroscopic Analysis of Human Saliva,” Journal of Dental Research, Vol. 81, No. 6, 2002, pp. 422-427. doi:10.1177/154405910208100613
[3]	E. Lynch, “Kariesbehandlung Mit Ozon,” Die Quintessenz, Vol. 54, 2003, pp. 608-610.
[4]	A. Baysan, R. Whiley and E. Lynch, “Anti-Microbial Effects of a Novel Ozone Generating Device on Micro-Organisms Associated with Primary Root Carious Lesions,” Caries Research, Vol. 34, 2000, pp. 498-501. doi:10.1159/000016630
[5]	A. Baysan, E. Lynch and M. Grootveld, “The Use of Ozone for the Management of Primary Root Caries,” In: T. Albrektsson, D. Bratthall, P. O. Glantz and J. Lindhe, Eds., Tissue Preservation in Caries Treatment, Quintessence Publishing Company Ltd., London, 2001, pp. 49-68.
[6]	A. Baysan and E. Lynch, “Effect of Ozone on the Oral Microbiota and Clinical Severity of Primary Root Caries,” American Journal of Dentistry, Vol. 17, No. 1, 2004, pp. 56-60.
[7]	J. Holmes, “Clinical Reversal of Root Caries Using Ozone, Double-Blind, Randomised, Controlled 18-Month Trial,” Gerodontology, Vol. 20, No. 2, 2003, pp. 106-114. doi:10.1111/j.1741-2358.2003.00106.x
[8]	J. R. Kanovsky and P. D. Sima, “Reactive Absorption of Ozone by Aqueous Biomolecule Solutions: Implications for the Role of Sulfhydryl Compounds as Targets for Ozone,” Archives of Biochemistry and Biophysics, Vol. 316, No. 1, 1995, pp. 52-62. doi:10.1006/abbi.1995.1009
[9]	S. Kermani, A. Ben-Jebria and J. S. Ultman, “Kinetics of Ozone Reaction with Uric Acid, Ascorbic Acid, and Glutathione at Physiologically Relevant Conditions,” Archives of Biochemistry and Biophysics, Vol. 451, No. 1, 2006, pp. 8-16. doi:10.1016/j.abb.2006.04.015
[10]	T. Thornberry and J. P. D. Abbatt, “Heterogeneous Reaction of Ozone with Liquid Unsaturated Fatty Acids: Detailed Kinetics and Gas-Phase Product Studies,” Physical Chemistry and Chemical Physics, Vol. 6, 2004, pp. 84-93. doi:10.1039/b310149e
[11]	M. F. Diaz, F. Hernandez, O. Ledea, J. A. Gavin Sazatornil and J. Moleiro, “1H NMR Study of Methyl Linoleate Ozonation,” Ozone: Science & Engineering: The Journal of the International Ozone Association, Vol. 25, No. 2, 2003, pp. 121-126. doi:10.1080/713610666
[12]	J. J. Thiele, M. G. Traber, T. G. Polefka, C. E. Cross and L. Packer, “Ozone-Exposure Depletes Vitamin E and Induces Lipid Peroxidation in Murine Stratum Corneum,” Journal of Investigative Dermatology, Vol. 108, 1997, pp. 753-757. doi:10.1111/1523-1747.ep12292144
[13]	A. Wisthaler and C. J. Weschler, “Reactions of Ozone with Human Skin Lipids: Sources of Carbonyls, Dicarbonyls, and Hydroxycarbonyls in Indoor Air,” Proceedings of the National Academy of Sciences, Vol. 107, No. 15, 2010, pp. 6568-6575. doi:10.1073/pnas.0904498106
[14]	J. Tonzetich, “Direct Gas Chromatographic Analysis of Sulphur Compounds in Mouth Air in Man,” Archives of Oral Biology, Vol. 16, No. 6, 1971, pp. 587-597. doi:10.1016/0003-9969(71)90062-8
[15]	H. Shechter, “Spectrophotometric Method for Determination of Ozone in Water,” Water Research, Vol. 7, No. 5, 1973, pp. 729-729. doi:10.1016/0043-1354(73)90089-4
[16]	M. Grootveld, E. B. Henderson, A. J. Farrell, D. R. Blake, H. G. Parkes and P. Haycock, “Oxidative Damage to Hyaluronate and Glucose in Synovial Fluid during Exercise of the Inflamed Rheumatoid Joint: Detection of Abnormal Low-Molecular-Mass Metabolities by Proton NMR Spectroscopy,” Biochemical Journal, Vol. 273, No. 2, 1991, pp. 459-467.
[17]	J. Grigor and A. J. Roberts, “Reduction in the Levels of Oral Malodor Precursors by Hydrogen Peroxide: In Vitro and in Vivo Assessments,” Journal of Clinical Dentistry, Vol. 3, No. 4, 1992, pp. 111-115.
[18]	A. E. Martell and R. J. Motekaitis. “The Determination and Use of Stability Constants,” VCH Weinham, Hoboken, 1988.
[19]	C. J. L. Silwood, E. Lynch, S. Seddon, A. Sheerin, A. W. D. Claxson and M. C. Grootveld, “1H-NMR Analysis of Microbial-Derived Organic Acids in Primary Root Carious Lesions and Saliva,” NMR in Biomedicine, Vol. 12, No. 6, 1999, pp. 345-356. doi:10.1002/(SICI)1099-1492(199910)12:6<345::AID-NBM580>3.0.CO;2-C
[20]	J. D. B. Featherstone and B. E. Rodgers, “Effect of Acetic, Lactic and Other Organic Acids on the Formation of Artificial Carious Lesions,” Caries Research, Vol. 15, 1981, pp. 377-385. doi:10.1159/000260541
[21]	J. Tonzetich and P. A. W. Carpenter, “Production of Volatile Sulphur Compounds from Cysteine, Cysteine and Methionine by Human Dental Plaque,” Archives of Oral Biology, Vol. 16, No. 6, 1971, pp. 599-607. doi:10.1016/0003-9969(71)90063-X
[22]	J. Tonzetich, P.W. Johnson and S. K. Ng, “Pathways of Cystine Metabolism in Human Saliva,” Archives of Oral Biology, Vol. 24, No. 1, 1979, pp. 35-39. doi:10.1016/0003-9969(79)90172-9
[23]	R. Battino, T. R. Rettich and T. Tominaga, “The Solubility of Oxygen and Ozone in Liquids,” Journal of Physical Chemistry Reference Data, Vol. 12, No. 2, 1983, pp. 163-178. doi:10.1063/1.555680
[24]	A. Bender, et al., “Adaptive Antioxidant Methionine Accumulation in Respiratory Chain Complexes Explains the Use of a Deviant Genetic Code in Mitochondria,” Proceedings of the National Academy of Sciences, Vol. 105, No. 43, 2008, pp. 16496-16501. doi:10.1073/pnas.0802779105
[25]	R. E Thach, K. F. Dewey, J. C. Brown and P. Doty, “Formylmethionine Codon AUG as an Initiator of Polypeptide Synthesis,” Science, Vol. 153, No. 3734, 1966, pp. 416-418. doi:10.1126/science.153.3734.416
[26]	J. R. Kanovsky and P. Sima, “Singlet Oxygen Production from the Reactions of Ozone with Biological Molecules,” Journal of Biological Chemistry, Vol. 266, No. 14, 1990, pp. 9039-9042.
[27]	R. Bonnett and G. Martinez, “Photobleaching of Sensitisers used in Photodynamic Therapy,” Tetrahedron, Vol. 57, No. 47, 2001, pp. 9513-9547. doi:10.1016/S0040-4020(01)00952-8