THERAPEUTIC OXIDATION OF HUMAN PLAQUE BIOMOLECULES BY A NOVEL ANTI-BACTERIAL OZONE DELIVERY SYSTEM

M. GROOTVELD1,2*, E. LYNCH1, B. MILLS2, C. SMITH1, A. BAYSAN1,2 and C. J. SILWOOD2

1School

of Clinical Dentistry, Queens University Belfast, Belfast, N. Ireland, 2 St. Barts and the Royal London SMD, University of London.
b)
Ace

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Introduction
The nature, rate and extent of salivary reductant consumption [e.g., that of volatile sulphur compounds (VSCs) responsible for halitosis (predominantly methyl mercaptan and hydrogen sulphide), their amino acid precursors (L-cysteine and Lmethionine), thiocyanate, urate and pyruvate] by ozone (O3) reflects the oxidising capacity of this microbicidal agent, a parameter of much relevance to its therapeutic and aesthetic actions. High resolution proton (1H) nuclear magnetic resonance (NMR) spectroscopic analysis of human saliva, gingival crevicular fluid (GCF) or appropriate chemical model systems serves as a very useful technique for the in vitro evaluation of the O3-mediated oxidation of such biomolecules, and we have recently found that it also provides much valuable regarding the molecular mechanisms associated with the potential therapeutic actions of this reactive oxygen species (ROS). Indeed, the multicomponent analytical ability of this technique allows researchers to rapidly and simultaneously monitor many of the chemical modifications which arise on equilibration of intact human saliva or GCF samples with therapeutic preparations.

Materials and Methods

Patients
All participants (n=16 for 1H NMR analysis) were patients of the Dental Hospital attending for routine oral health care (mean s.e. age 56.2 3.1 yr. ; range 50-69 yr.). Each subject had given their informed consent for both dental examinations to be undertaken and for samples of plaque to be taken for bacteriological investigation. Patients with xerostomia based on obvious clinical criteria, those who had undertaken courses of systemic antibiotic therapy or had used anti-bacterial toothpastes or mouthwashes during the previous four weeks, and any who had undergone periodontal surgery within the previous six months, were excluded from the study. Four of the patients were smokers. Only patients who had refrained from oral activities (i.e., eating, drinking, tooth-brushing, oral rinsing, smoking, etc.) for at least 2 hr. prior to sample collection were selected.
Pyr Met-S-CH3 Met- -CH2 Bu

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Figure 1. (a) expanded 0.50-4.50 ppm region of the 600 MHz H NMR spectrum of a post-neutralised HClO4 extract of a plaque specimen. A typical spectrum is shown. (b) expanded 5.50-9.00 ppm region of the same specimen. (c) and (d) expanded 0.50-4.50 and 5.50-9.00 ppm regions, respectively, of the same specimen treated with O3.

Figure 1. (cont.)

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Form

Sample preparation
Plaque specimens (weight range 1-4 mg) were weighed on a microbalance and then divided into 2 equivalent portions. The first was treated with O3 generated from the above device for a period of 20 s; the second group of portions served as controls. For 1H NMR analysis each control and O3-treated specimen was treated with 1.50 ml of 60% (v/v) perchloric acid (HClO4) solution to the sterile container, thoroughly rotamixed and then centrifuged (5,000 g) at 4C for a period of 15 min. The clear supernatant was removed and left to stand for 1.00 hr. before pH neutralisation with sodium hydroxide (NaOH) solution. Each sample was again centrifuged as described above and the supernatant collected for 1H NMR experiments.
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Met-SO-CH3

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Figure 1. (cont.)

Figure 1. (cont.)

Discussion
Consumption of methionine by O3 is of great importance to oral hygiene and clinical periodontology since both CH3SH and H2S are generated from this amino acid via metabolic pathways operational in gram-negative micro-organisms. Hence, our data indicate that O3 has the capacity to clinically alleviate oral malodour via the direct oxidative inactivation of VSCs and their amino acid precursors. High resolution, high field 1H NMR spectroscopy is a technique which offers many advantages over alternative time-consuming, labour-intensive analytical methods since (1) it permits the rapid, non-invasive and simultaneous examination of a very wide range of components present in human plaque and (2) it has little or no requirement for knowledge of sample composition prior to analysis. Furthermore, chemical shift values, coupling patterns and coupling constants of resonances present in 1H NMR spectra of such multicomponent systems provides much valuable information regarding the molecular nature of both endogenous and exogenous chemical species therein. As demonstrated here, the technique is of much value concerning multicomponent assessments of the interactions of O3 with human plaque biomolecules, and the oxidative decarboxylation of pyruvate by this oxidant evaluated in this study serves as an important fundamental example of this which may be of some relevance to its mechanisms of action. Indeed, pyruvic acid is a very powerful proton donor (Ka = 3.20 mM) being much stronger in this capacity than lactic acid (Ka = 0.14 mM), and hence may play an important role in promoting tooth demineralisation processes.

Aim
In this investigation we have employed high resolution 1H NMR spectroscopy for the purpose of conducting a multicomponent evaluation of the oxidative consumption of plaque biomolecules by O3. The ozone-delivery system employed in this study was the HealOzone Unit (CurOzone USA).
1H

Results
The expanded 0.50 - 4.50 and 5.50 - 9.00 ppm regions of a typical 600 MHz 1H NMR spectrum of a post-neutralised HClO4 extract of plaque collected from a patient are shown in Fig 1. The spectra acquired contain many resonances assignable to a wide variety of low-molecular-mass metabolites, and illustrate the multicomponent analytical ability of the technique employed. Indeed, these spectra contain well-resolved, sharp signals assignable to bacterial- or yeast-derived organic acid anions (including formate, acetate, propionate, n-butyrate, lactate, and pyruvate), amino acids (predominantly alanine and glycine) and carbohydrates such as glucose. Results acquired revealed that treatment with O3 gave rise to the oxidative decarboxylation of the electron-donor pyruvate (generating acetate and CO2 as products), and the oxidation of the volatile sulphur compound (VSC) precursor methionine to its corresponding sulphoxide. Furthermore, evidence for the O3mediated oxidation of 3-D-hydroxybutyrate was also obtained.

NMR spectroscopy

The above post-neutralised HClO4 extracts (0.60 ml) were placed in 5-mm diameter NMR tubes and 0.07 ml of 2H2O was added to provide a field frequency lock. 1H NMR spectra were acquired on a Bruker AMX-600 [University of London Intercollegiate Research Services (ULIRS), Queen Mary and Westfield College Facility, University of London, U.K.] spectrometer operating at 600.13 MHz (14.1 T) and ambient probe temperature (22 1C). 256 free induction decays (FIDs) were acquired for spectra of biopsies with initial weights > 2 mg, each of magnitude 32,768 datapoints, 4.0 s pulses (corresponding to a flip angle of 45), a 1.8 s spin relaxation delay between pulses and an acquisition time of 3.9 s. For analyte solutions derived from biopsies of low mass (i.e., 2 mg), ca. 2,500 FIDs were acquired. The spectral width was 8,403 Hz, corresponding to 14 ppm. The intense H2O/HOD signal was suppressed by presaturation with gated decoupling during the delay between pulses. Sensitivity enhancement was achieved via the employment of 0.3 Hz line broadening, followed by zero-filling of FIDs to 65,536 datapoints, giving a final digital resolution of 0.13 Hz per point.

Conclusion
In conclusion, the removal of plaque pyruvate by O3 may suppress the development and progression of tooth demineralisation. In view of these considerations, O3 may offer caries-preventative and management actions and experiments to investigate this further are currently in progress.