Salivary Peroxidase System Research Articles

Biofilm inhibition and antimicrobial activity of a dentifrice containing salivary substitutes

A healthy mouth harbours the continuous combined action of a salivary defence system with that of a salivary peroxidase system, containing lactoferrin, lysozyme, immunoglobulin and growth factors. This system maintains neutral pH and creates an oral environment where harmful bacteria are inhibited, thus preventing the formation of biofilms. The objective of this clinico-microbiological trial was to evaluate the anti-plaque effect of a dentifrice containing salivary substitutes, compared with a placebo-control dentifrice and to assess the effect of dentifrice on oral bacterial count. The design was a randomized controlled, double-blind, parallel study comparing a placebo-dentifrice to a dentifrice formulation containing salivary substitutes. Toothpaste slurry rinses were used over a 96-h period by 20 volunteers who refrained from all other oral hygiene procedures. Commercially available fluoride toothpaste was used as control. Plaque was scored and unstimulated salivary samples were collected at day 0 and after 4 days. A microbiological analysis was carried out for the salivary samples. Data were analyzed by using Student's t-tests. There was a statistically significant mean difference in plaque scores after using test paste (1.19 + 0.31) in comparison with those using placebo toothpaste (1.95 + 0.33). The difference between mean increase in colony forming units for the test and the placebo group was (25.2 + 8) x 10(5) and (17.5 + 6.01) x 10(5), respectively, which was statistically significant. The findings of the study support the hypothesis that toothpaste containing salivary substitutes prevents dental biofilm formation and exhibits antimicrobial property when compared with a placebo dentifrice.

Effect of iron-deficiency anaemia on saliva secretion rate and composition in the rat

Iron-deficiency anaemia in man may be associated with dry mouth symptoms, such as atrophic glossitis and ‘burning mouth’, and a low serum iron concentration or iron-deficiency anaemia with aphthous stomatitis or non-ulcerative conditions of the oral mucosa. This study was made on one group of growing rats and another group of adult rats. The iron-deprived rats in both groups gained less weight than control rats. Saliva secretion rate was significantly lower in growing, but not in adult, iron-deprived rats than in the corresponding control groups. The activity of salivary peroxidase was significantly lower in both growing and adult rats with iron-deficiency anaemia than in their control groups. The conclusion is that iron-deficiency anaemia in the rat impairs the protection provided by the salivary peroxidase system, e.g. to hydrogen peroxide, and that saliva secretion rate may be reduced.

Lysozyme enhances the inhibitory effects of the peroxidase system on glucose metabolism of Streptococcus mutans.

The combined effect of the salivary peroxidase system and lysozyme on the glucose uptake of Streptococcus mutans NCTC 10449 was investigated. The bacteria were grown to late-exponential phase, washed, re-suspended in buffer at pH6, and incubated with (1) 50 micrograms/mL lysozyme from human milk for 60 min; (2) 7-15 mumol/L hypothiocyanous acid/hypothiocyanite for 10 min; and (3) lysozyme for 60 min prior to addition of and incubation with hypothiocyanous acid/hypothiocyanite for 10 min. Glucose uptake was initiated by adding the bacterial suspensions to 10 mL of pre-warmed 50 mumol/L glucose containing 0.98 mumol/L D-(U-14C-)-glucose, and the mixture was incubated in a shaking water-bath at 37 degrees C. Samples were withdrawn at various time intervals, rapidly filtered through 0.45-microns membranes, washed with ice-chilled buffer, and the incorporated radioactivity determined. Lysozyme stimulated S. mutans glucose uptake slightly, but significantly inhibited S. rattus glucose metabolism. A 20-30% inhibition of radiolabeled glucose incorporation was observed with hypothiocyanous acid/hypothiocyanite alone. Incubation of the bacteria with lysozyme prior to addition of hypothiocyanous acid/hypothiocyanite containing peroxidase resulted in a total inhibition of the glucose uptake. In contrast, lysozyme in combination with hypothiocyanous acid/hypothiocyanite without peroxidase gave only a 30-50% inhibition. The addition of 5 mmol/L dithiothreitol after incubation with lysozyme and hypothiocyanous acid/hypothiocyanite eliminated the inhibition of the bacterial glucose uptake. The viability of S. mutans was not affected by treatment with any of the components used. Our results indicate that physiological concentrations of lysozyme and the salivary peroxidase system components have a synergistic effect which results in a significant inhibition of glucose metabolism by S. mutans.

Analyses of salivary components in leukemia patients receiving chemotherapy

We analyzed several salivary components in stimulated whole saliva from patients with acute leukemia who were undergoing chemotherapy. Saliva samples were collected at the time of diagnosis and longitudinally during the treatment period. Data analyses showed that patients with leukemia had significantly higher peroxidase and amylase activity and elevated concentrations of total protein at the time of diagnosis. After induction chemotherapy these parameters returned to normal values and remained constant during the observation period. At the time of diagnosis no significant differences in thiocyanate (SCN −) concentrations were found in saliva samples from control subjects and patients with leukemia. Treatment with cytotoxic agents resulted in granulocytopenia and a concomitant decrease in the SCN − concentration in saliva. The function of the salivary peroxidase system is impaired by the decrease in SCN − concentration, which may be a contributing factor to some of the oral complications that occur in patients undergoing chemotherapy.

The salivary peroxidase system: thermodynamic, kinetic and antibacterial properties.

Enzymes are specific catalysts which optimize the rates of reactions vital to living organisms. If the oxidation of thiocyanate (which occurs, in vivo, in human saliva and salivary glands) is a vital reaction for the maintenance of oral health, then the enzyme (salivary peroxidase), which catalyzes this reaction, should function optimally under in vivo conditions. Studies have shown that salivary peroxidase maintains the SCN- oxidation reaction in an apparent state of dynamic equilibrium, that the kinetic properties of the enzyme provide optimum rates of reaction under in vivo conditions, and that the antibacterial properties of the products of the reaction are optimum under those conditions where unlimited bacterial metabolism provides the greatest threat to host tissues. The evidence indicates that the salivary peroxidase enzyme has evolved in such a way as to maximize its protective value in the oral cavity.

Effects of variations in pH and hypothiocyanite concentrations on S. mutans glucose metabolism.

Hypothiocyanous acid (HOSCN) and hypothiocyanite (OSCN-) were generated by the antibody-independent salivary peroxidase (SP) system. The metabolism of Streptococcus mutans NCTC 10449 was examined by uniformly labeled glucose incorporation studies. We found that the SP-system causes a pH-dependent inhibition of 14C-labeled glucose uptake, and that the effects of HOSCN/OSCN- are bacteriostatic. The results also showed that, at low pH, bacteria required more time to recover fully from HOSCN/OSCN- inhibition. When control experiments were performed in the absence of HOSCN/OSCN-, but the pH was varied, we found a positive correlation between pH and the rate of 14C-glucose incorporation. The results also showed that pH did not affect the maximum incorporation of 14C-glucose, demonstrating that S. mutans can adapt to pH changes in the environment. Based on the data obtained, we postulate that the antibody-independent SP system plays an important role in the regulation of the metabolism of oral streptococci.

Relationship of the human salivary peroxidase system to oral health.

The human salivary peroxidase system (SPS) contributes in several ways to the maintenance of good oral health. The SPS is one of the non-immunoglobulin defense factors which regulate the quantity and species distribution of oral micro-organisms. The SPS also prevents toxic accumulations of hydrogen peroxide (H2O2) and it inactivates many carcinogenic and mutagenic compounds. The salivary glands secrete a peroxidase enzyme (salivary peroxidase) as well as the thiocyanate ion (SCN-, derived from diet). The enzyme catalyzes the oxidation of SCN- by hydrogen peroxide (H2O2). The H2O2 is excreted by oral bacteria and by host cells in amounts which vary with the state of cellular metabolism, the diet and other factors. Oxidized forms of SCN- temporarily inhibit the growth, respiration and metabolism of most species of oral bacteria. The major oxidized form generated in the mouth is the hypothiocyanite ion (OSCN-) which must reach a minimum threshold concentration before bacterial inhibition occurs. This threshold concentration varies from species to species. The concentration of OSCN- in the mouth rises and falls with the availability of H2O2. This natural rise and fall, together with bacterial variation in sensitivity to OSCN- inhibition, suggests a role for the SPS in the regulation of the oral microflora. As a result of the rapid consumption of H2O2 by the SPS, host cells are protected from a toxic build up of this potent oxidizing agent. The major product of the reaction, OSCN-, does not harm human cells. Many carcinogenic and mutagenic compounds may serve as substrates for the SPS and be oxidized to less harmful compounds.

The protective effect of peroxidase and thiocyanate against hydrogen peroxide toxicity assessed by the uptake of [ 3H]-thymidine by human gingival fibroblasts cultured in vitro

The hypothiocyanite ion (OSCN −) is the principal oxidation product of the salivary peroxidase-thiocyanate (SCN −)-hydrogen peroxide antimicrobial system. Supplementation of human saliva in vitro and in vivo with low amounts (< 1.0 mM) of hydrogen peroxide increase the concentration of salivary OSCN − ( in vivo up to 0.3 mM). Elevated concentrations of OSCN − are strongly antimicrobial and may therefore be protective against dental caries. However, as OSCN − is a highly-reactive oxidizing agent, its possible toxic effect on human cells was studied using gingival flbroblasts as target cells. Concentrations of OSCN − (up to 300 μM) had no effect on [ 3H]-thymidine incorporation into the cells. However, fibroblasts were sensitive to peroxide so that 100 μ M of H 2O 2 caused over 80 per cent reduction in [ 3H]-thymidine incorporation. The toxicity of H 2O 2 could be entirely prevented by adding lacto-peroxidase and SCN − to the cell culture before the addition of peroxide. Thus, conversion of toxic H 2O 2 to non-toxic OSCN − in fibroblast culture by lactoperoxidase and SCN − suggests a dual role for the salivary peroxidase system: protection of human cells from H 2O 2 toxicity and antimicrobial action against oral pathogens. Furthermore, the elevated concentrations of OSCN − which produce inhibition of bacterial metabolism did not damage human cells.

A mouthrinse which optimizes in vivo generation of hypothiocyanite.

We report here the properties of a mouthrinse which enhances one of the natural defense factors in human saliva, the salivary peroxidase system. Concentrations of the antimicrobial agent, the hypothiocyanite (OSCN-) ion, can be increased in vivo to bacteriostatic levels by use of a mouthrinse which is 4 mM (0.014%) in hydrogen peroxide and 1 mM (0.0097%) in potassium thiocyanate at pH 5.5. The volume of the rinse, the H2O2 concentrations, and the pH were shown to be determinants of the concentration of OSCN- generated by the rinse.

Detection of the hypothiocyanite (OSCN −) ion in human parotid saliva and the effect of pH on OSCN − generation in the salivary peroxidase antimicrobial system

Human whole saliva contains the hypothiocyanite ion (OSCN −) which is the principal antimicrobial product of the salivary peroxidase system. The peroxidase system requires a source of peroxide in order to produce OSCN − and in the human mouth this source has been assumed to be primarily the peroxidogenic oral bacteria. However, we report here studies which show that samples of stimulated human parotid saliva collected directly from Stenson's duct have concentrations of OSCN − which are similar to those found in human whole saliva. Thus, the peroxidogenic bacteria are not an absolute requirement for the generation of significant levels of OSCN − in the human mouth. Supplementation of human whole saliva with components [thiocyanite (SCN −), hydrogen peroxide (H 2O 2)]of the peroxidase system produces a 10-fold or greater increase in OSCN − concentration. However, the magnitude of this increase is critically dependent upon pH and upon the relative and absolute concentrations of SCN − and H 2O 2. The pH dependence of OSCN − generation is similar for human whole saliva and for the lactoperoxidase/SCN −/H 2O 2 system. The optimum is in the range 6.5–7.0. Samples of parotid saliva adjusted to pH 6.5 and supplemented with appropriate amounts of SCN − and H 2O 2 show increases in OSCN − concentrations which are similar to those observed with whole saliva. The results show that there is a significant source of H 2O 2 within the parotid gland, that the OSCN − generating potential of parotid saliva is similar to that of whole saliva and that the enhancement of OSCN − levels in saliva by addition of SCN − and H 2O 2 is critically dependent upon pH and upon the relative and absolute concentrations of H 2O 2 and SCN −.