Elsevier

Archives of Oral Biology

Volume 58, Issue 10, October 2013, Pages 1349-1356
Archives of Oral Biology

Interactions between hyaluronic acid, lysozyme, and the glucose oxidase-mediated lactoperoxidase system in enzymatic and candidacidal activities

https://doi.org/10.1016/j.archoralbio.2013.06.015Get rights and content

Abstract

Objective

To investigate interactions between hyaluronic acid (HA), lysozyme, and the glucose oxidase-mediated lactoperoxidase (GO-LPO) system in enzymatic and candidacidal activities.

Design

The influences of HA (0.5, 1.0, and 2.0 mg/mL) and lysozyme (30 μg/mL hen egg white lysozyme) on the enzymatic activity of GO-LPO system (25 μg/mL bovine LPO, 1 mM KSCN, 10 units/mL GO, and 30 μg/mL glucose) were determined by measuring oxidized o-dianisidine production. The influence of the GO-LPO system on lysozyme activity was determined by measuring the turbidity of a Micrococcus lysodeikticus suspension. The effects of interactions between HA, lysozyme, the GO-LPO system on candidacidal activity were examined by pre-incubating various combinations of components. Candidacidal activity was determined by comparing the numbers of colony forming units using Candida albicans ATCC strains 10231, 18804, and 11006.

Results

HA inhibited the enzymatic activity of the GO-LPO system in a dose-dependent manner. HA inhibited the candidacidal activities of the GO-LPO system. However, the inhibitory activity of HA was not significantly different according to concentration of HA. The GO-LPO system enhanced the enzymatic activity of lysozyme, though lysozyme did not affect the enzymatic activity of the GO-LPO system. The candidacidal activities of the GO-LPO system and lysozyme were not additive.

Conclusions

HA inhibited the enzymatic and candidacidal activity of the GO-LPO system. The GO-LPO system enhanced the enzymatic activity of lysozyme, but the candidacidal activities were not additive.

Introduction

Hyaluronic acid (HA) is present in human saliva and may contribute to the lubricating and healing properties of saliva, thereby assisting in protecting the oral mucosa.1, 2 HA also has anti-Candida activity.3 Due to its viscoelastic properties and non-immunogeneity,4 HA can be a candidate substance for effective saliva substitutes for patients with dry mouth as a solution containing HA has rheological properties which might mimic the viscosity of human saliva at shear rates for routine oral functions.5 A relationship between decreased levels of salivary HA and dry mouth symptoms has also been reported,6 suggesting that HA is important in protecting and lubricating the oral mucosa. In addition, the wound repair and potential anti-Candida activities of HA3, 7 can provide additional benefits to patients with dry mouth, who are susceptible to oral mucosal injuries and candidiasis.8

The most widely used antimicrobial host proteins in oral health care products are lysozyme and the peroxidase system.9 One lysozyme supplement in these products is hen egg white lysozyme (HEWL). The bactericidal activity of lysozyme is muramidase-dependent and uses cation-dependent or structure-related mechanisms.10, 11, 12 Its antifungal activity is also well known.13, 14, 15 The peroxidase system supplements are the glucose oxidase-mediated lactoperoxidase (GO-LPO) system, which comprises bovine lactoperoxidase (bLPO), glucose oxidase (GO), and SCN. The GO-LPO system interacts with peroxidases (salivary peroxidase and myeloperoxidase), SCN, H2O2, and glucose in whole saliva. GO degrades glucose and produces H2O2, which serves as a substrate for bLPO and peroxidase in saliva to form antimicrobial OSCN from SCN.16 Furthermore, by using glucose, GO can hamper the carbohydrate metabolism of glycolytic cariogenic bacteria.16, 17, 18 In addition, the GO-LPO system has antifungal activities.19

Many antimicrobial proteins in saliva interact with each other in vitro. These interactions can result in additive, synergistic, or inhibitory effects on mutans streptococci, lactobacilli, or fungi.20, 21, 22 For example, interactions have been reported between sIgA and peroxidase,23 lactoferrin and peroxidase,24, 25 lactoferrin and lysozyme,26 lysozyme and histatins,27 and lysozyme and the peroxidase system.15, 28

Although these observations are in vitro, such concerted effects likely exist in mixed saliva or oral health care products, where all the components are present simultaneously. Therefore, HA molecules may also interact with antimicrobial molecules in human saliva or saliva substitutes. HA and peroxidase have been suggested to form complex molecules,29 and HA affects the enzymatic and candidacidal activities of lysozyme and the peroxidase system.5, 30 However, there is no information as to how HA affects the enzymatic and anticandidal activities of the GO-LPO system or how lysozyme and the GO-LPO system affect each other. In this study, we investigated the influences of HA on the enzymatic and anticandidal activities of the GO-LPO system and the interactions between lysozyme and the GO-LPO system.

Section snippets

HA, lysozyme, and glucose oxidase-mediated lactoperoxidase system

HA (1630 kDa, Sigma–Aldrich Chemical Co., St Louis, MO, USA) was used at three concentrations: 0.5, 1.0, and 2.0 mg/mL. HEWL (final concentration of 30 μg/mL) (Sigma–Aldrich) served as the lysozyme source. The GO-LPO system included bLPO (final concentration of 25 μg/mL), potassium thiocyanate (KSCN, final concentration of 1 mM), GO (final concentration of 10 units/mL), and glucose (final concentration of 30 μg/mL) (Sigma–Aldrich). All components were solubilized with simulated salivary buffer (SSB,

Influence of HA and lysozyme on the enzymatic activity of the GO-LPO system

HA inhibited the enzymatic activity of the GO-LPO system in an HA or glucose concentration-dependent manner (Fig. 1). HEWL did not affect the enzymatic activity of the GO-LPO system. The mean (±SD) differences in OD between with HEWL and without HEWL was −0.86 ± 3.60% at 20 μg/mL glucose, 0.63 ± 2.60% at 40 μg/mL glucose, and −0.18 ± 2.17% at 60 μg/mL glucose.

Influence of the GO-LPO system on the enzymatic activity of lysozyme

GO, KSCN, and glucose did not affect the enzymatic activity of HEWL. bLPO alone and mixtures containing bLPO significantly enhanced the enzymatic

Discussion

Developing effective saliva substitutes requires understanding and mimicking both the rheological and biological properties of human saliva.32, 33 Therefore, a practical method to develop effective salivary substitutes for xerostomic patients is to identify or develop substances with viscoelastic patterns similar to human whole saliva and supplement them with antimicrobials that restore the diminished biochemical properties. The best salivary substitute in a viscoelastic aspect is one with

Funding

This work was supported by the National Research Foundation of Korea Grant through the Oromaxillofacial Dysfunction Research Center for the Elderly (No. 2012-000911) at Seoul National University in Korea.

Competing interests

None declared.

Ethical approval

Not required.

Acknowledgements

This work was supported by the National Research Foundation of Korea Grant through the Oromaxillofacial Dysfunction Research Center for the Elderly (No. 2012-000911) at Seoul National University in Korea.

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