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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, Queen’s University Belfast, Belfast, N. Ireland, 2 St. Bart’s and the Royal London SMD, University of London.

Introduction 

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).

Materials and Methods

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 L-

methionine), 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 thatit 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 manyof the chemical modifications which arise on equilibration of 

intact human saliva or GCF samples with therapeutic

preparations.

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 giventheir 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.

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 aperiod 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 (HClO 4) 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 pHneutralisation with sodium hydroxide (NaOH) solution. Each

sample was again centrifuged as described above and the

supernatant collected for 1H NMR experiments.

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 spectraacquired 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, andpyruvate), 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 itscorresponding sulphoxide. Furthermore, evidence for the O3-

mediated oxidation of 3-D-hydroxybutyrate was also obtained.

1H 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 bypresaturation with gated decoupling during the delay betweenpulses. 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.

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 norequirement for knowledge of sample composition prior to analysis. Furthermore,

chemical shift values, coupling patterns and coupling constants of resonancespresent 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 theoxidative 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 (K a = 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.

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

Met-S-CH3

Met--CH2

Ace

Pyr 

Bu

a)

Figure1. (a)expanded 0.50-4.50ppm regionof the600 MHz1HNMR spectrum of apost-neutralisedHClO 4 extract of aplaque

specimen. A typical spectrum is shown. (b) expanded5.50-9.00ppm regionof the samespecimen. (c) and(d) expanded

0.50-4.50and 5.50-9.00ppm regions, respectively, of thesame specimentreated withO3.

8.5 8.0 7.5 7.0 6.5 6.0

b)

Figure1. (cont.)

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0

~Ace

Met-SO-CH3

Met-S-CH3

c)

Figure1. (cont.)

8.5 8.0 7.5 7.0 6.5 6.0

Form

d)

Figure1. (cont.)

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.

Conclusion

Results

Discussion