Efficacy of enzymatic mouth rinses for immobilisation of protective enzymes in the in situ pellicle

https://doi.org/10.1016/j.archoralbio.2009.10.004Get rights and content

Abstract

Aim

Mouth rinses containing enzymes are designed for patients suffering from xerostomia. The objective of the present in situ study was to investigate the efficacy of these rinses for targeted accumulation and immobilisation of protective enzymes in the acquired pellicle.

Methods

A number of six healthy subjects carried bovine enamel slabs fixed on individual upper jaw splints for pellicle formation in situ. After 1 min, they rinsed with biotène or BioXtra for 10 min, respectively. Enzyme activities of lysozyme, peroxidase and glucoseoxidase in the in situ pellicle and in the saliva were assayed before as well as 0, 20 and 40 min after the rinses. The assays for the respective enzyme activities were based on fluorogenic substrates. Separate experiments were performed for the different enzymes and mouth rinses, respectively. Statistical evaluation was carried out with the Kruskal–Wallis test.

Results

None of the investigated rinses had any significant impact on the activities of lysozyme, peroxidase and glucoseoxidase detectable in the in situ pellicle or in the saliva (Kruskal–Wallis test, p > 0.05). Despite the fact that both products should contain lactoperoxidase activity according to manufacturers’ instructions, no peroxidase activity was measurable in the pure mouth rinses.

Conclusion

With the tested enzymatic mouth rinses targeted accumulation and immobilisation of protective enzymes in the in situ pellicle did not seem possible.

Introduction

Numerous people suffer from xerostomia due to Sjögren's syndrome, irradiation or a broad range of medications reducing the secretion of the salivary glands.1, 2, 3 Accordingly, there is a strong demand for saliva substitutes mimicking the rheological and functional properties of the saliva.4, 5, 6, 7 These mouth rinses are designed to substitute the lacking saliva as well as to enrich protective biomolecules in the oral cavity.8 Due to the high clearance of the oral fluids, a tenacious immobilisation of the applied protective proteins on the tooth surfaces in the proteinaceous pellicle layer is demanded.8 Beside lactoferrin, several enzymes are typical proteins in these oral health care products.6, 7, 8 The most abundant antibacterial enzyme in the saliva is lysozyme representing a relevant part of the innate host defence mechanisms.9 Lysozyme hydrolyses the petidoglycan layer of several bacteria. Due to its polycationic nature, this protein has additional antimicrobial properties independent of the enzymatic activity based on the activation of bacterial autolysins.10, 11 Studies on patients suffering from Sjögreńs syndrome indicate that the unstimulated salivary flow rate may decrease to 3% of that observed with the healthy population.12 However, data on enzymes in the oral fluids of patients suffering from xerostomia differ considerably. Lysozyme concentrations that are 12-fold higher than in controls have been recorded.13 In contrast, in another study, salivary lysozyme concentrations in irradiated patients were only slightly reduced, but due to the low salivary flow rate, the amount of protective proteins was decreased considerably.14 Interestingly, pellicles of patients suffering from xerostomia after irradiation and of healthy subjects with physiological salivary flow rate differ only slightly with respect to the general protein composition and the ultrastructure.15

A substitute for human lysozyme is gained from hen egg white. The three-dimensional structure of human and hen egg white lysozyme is rather similar, both are composed by the same amount of amino acids, about 50% of the primary structure is similar.16, 17, 18 Accordingly, hen egg white lysozyme is a reasonable additive for oral health care products.6, 7, 8 Another relevant protective enzyme in the saliva is peroxidase, which is important for the cleavage of radicals and the elimination of oxidative stress in the oral cavity.9, 19 In addition, peroxidase catalyses the reduction of H2O2 and the oxidation of thiocyanate (SCN), yielding the antibacterial hypothiocyanate (OSCN).20 However, in contrast to other enzymes, peroxidases are inactivated irreversibly by their substrate hydrogen peroxide.19, 21 Therefore, the accumulation of this anti-oxidative enzyme is of considerable interest for patients suffering from xerostomia.7 Cheap peroxidase is gained from bovine milk or from horseradish, respectively.21 Due to its high similarity to human peroxidase, lactoperoxidase is often added to enzymatic mouth rinses.20, 22

Also enzymes hampering the carbohydrate metabolism of glycolytic bacteria such as glucoseoxidase are typical additives of oral health care products, though they are no physiological components of the saliva.6, 8 However, small amounts of glucoseoxidase might be present in the oral fluids due to secretion by fungi.23 Furthermore, glucoseoxidase is used as food preservative.23

Despite these considerations, until now, there is no evidence that enzymatic mouth rinses could enhance the oral defence capacity, if the salivary flow rate is within the normal limits as reviewed by Tenovuo.7 Therefore, the aim of the present in situ study was to investigate the efficacy of commercially available enzymatic mouth rinses for targeted immobilisation of protective enzymes in the physiological pellicle layer. It was hypothesised that an accumulation of lysozyme, peroxidase and glucoseoxidase activity in the in situ pellicle can be achieved with the tested products biotène and BioXtra.

Section snippets

Mouthrinses

The following enzymatic mouth rinses were tested in the experiments: biotène (Laclede International, Brussels, Belgium, PZN 3819841) and BioXtra (John O. Butler, Kriftel, Germany, LOT 1581-685, PZN 1553847) (Table 1).

Subjects and samples

Six healthy volunteers, members of the laboratory staff, participated in the study. According to a visual oral examination by an experienced dentist, the subjects showed no signs of gingivitis or caries. The subjects had a physiological salivary flow rate. Informed written consent

Results

Both mouth rinses were tested for the activities of the enzymes contained according to manufacturers’ declarations. Both solutions contained lysozyme activity. Glucoseoxidase activity was measurable in biotène. However, though lactoperoxidase is a component of both products, neither in biotène nor in BioXtra any peroxidase activity was measurable (Table 1).

Discussion

To the best knowledge of the authors, the present in situ study was the first investigation considering the effect of enzymatic mouth-rinses on enzyme activities in the saliva and in the pellicle layer.7, 8 The chosen set up allowed direct measurement of enzyme activities respecting oral conditions. Thereby, the rinses were adopted after short time pellicle formation to simulate rinsing immediately after oral hygiene procedures. As in many previous studies, in situ pellicle formation was

Conclusion

With the tested enzymatic mouth rinses, targeted accumulation and immobilisation of protective enzymes in the in situ pellicle is not possible.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

The study was supported by a grant from the DFG (Deutsche Forschungsgemeinschaft, # HA 5192/1-2 # HA 2718/3-3).

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