REVIEW ARTICLE

Activity and effects of urease and arginine deiminase in saliva and oral human biofilm

Évelyn Reyes Beltrán¹; Javier Martín Casielles¹; Ismael Yevenes López²; Miguel Neira Jara²; Patricia Palma Fluxá³; Valeria Gordán Veiga⁴; Gustavo Moncada Cortés¹

¹ Operative Dentistry, School of Dentistry, Universidad de Chile
² Chemistry, School of Dentistry, Universidad de Chile
³ Microbiology, School of Dentistry, Universidad de Chile
⁴ Operative Dentistry Department, College of Dentistry, University of Florida, Gainesville, FL, USA

ABSTRACT

The purpose of this review is to present the emerging findings on the potential anti-cariogenic properties of the bioactive molecules of oral metabolism related with ammonia production. The literature analysis supports a new preventive dimension in the knowledge of dental caries disease by studying the published evidence provided by in vitro and clinical studies, in which the ammonia produced from urea and arginine in the oral environment represents an important endogenous inhibitory factor in the development of dental caries lesions. This fact would support the hypothesis that ammonia production by urease and the arginine deiminase system could potentially inhibit the development of dental caries by neutralizing acids and stabilizing the oral microbiota, thus enhancing the conditions for oral health maintenance. This review presents studies on the oral enzyme activity which may constitute a promising field in the definition of new lines of research in cariology, particularly in vivo and in situ, aimed at establishing the effectiveness and clinical application of these compounds in the prevention of dental caries.

Key words: ammonium, urease, arginine deiminase, saliva, biofilm, dental caries.

INTRODUCTION

Out of all the diseases that affect the oral cavity, dental caries is the one with the greatest prevalence among the world's population, thus generating an epidemiological problem in all the countries to a greater or lesser extent, and affecting nearly 100% of the population in most of them.¹

Dental caries represents the greatest oral health problem even in highly industrialized countries, where it affects between 60 and 90% of the school population and most of the adult population. It is also the prevalent bucco-dental disease in Latin America and Asia, being less common and severe in African countries.² In the United States it is still an important public health problem, as it is the most frequent chronic disease among the population, affecting more than 96% of adults, and 99.5% of individuals aged 65 or older have experienced caries in this country.^{3, 4}

In spite of the development of preventive dentistry, dental caries is still a big oral health problem; that is why researchers constantly explore new preventive and treatment strategies, as well as evaluation of cariogenic risk in different conditions.⁵

It is commonly accepted nowadays that oral bacteria colonizing teeth form a community called biofilm that exists in dynamic balance with the organism defenses and is generally compatible with the integrity of dental tissues.^6-8 Transition from oral health to oral disease, such as dental caries, is characterized by structural and metabolic changes in the oral biofilm ^7,9 creating a kind of acidification that favors proliferation of acidogenic and aciduric microbiota harboring Streptococcus mutans and Lactobacillus spp, to name just a few, which produce a rapid fermentation of carbon hydrates, thus generating pH decrease up to a point in which significant demineralization of teeth occurs. Continuous plaque acidification promotes the establishment of acidogenic microbiota. This microbiota has been associated with increasing vulnerability to dental caries.^{8, 10-14}

Extended acidification of dental plaque, as a result of the metabolic activity of cariogenic microbiota, leads to teeth demineralization.

Once sugar is added, pH of the dental plaque quickly decreases, reaching minimum values that gradually return to their initial level.¹⁵ Development of dental caries is a prolonged process that involves demineralization and remineralization cycles. When the demineralization phase predominates, carious lesion develops.

THE BIOFILM ALKALI

Some risk factors for dental caries have been identified and studied,^16-18 as well as the transition from a caries-free individual to a state of cariogenic activity—a process that could be related to a reduced potential of generating alkali in the plaque, along with an acidogenic environment increase—. Although many studies have focused on the acidogenic nature of dental caries, the way of increasing the alkalization phase and pH homeostasis has not been fully explored.

Among the less aciduric microorganisms—and the more closely related to dental health—are Streptococcus sanguinis (S sanguinis) Streptococcus gordonii (S. gordonii),^19-22 which contribute to alkalization of the plaque by generating ammonium from arginine deiminase and urease. This ammonium production by oral bacteria positively influences the balance between the tooth's new mineralization and its demineralization and may contribute to prevent the development of cariogenic microbiota.^23-27

Levels of S. mutans observed in subjects with active dental caries were significantly higher in comparison to subjects without it. Besides, lower levels of Streptococcus sanguinis, Streptococcus gordonii, Actinomyces naeslundii, and Streptococcus salivarius were observed in subjects with active caries compared to caries-free subjects, although this difference was not statistically significant.⁵

UREASE ENZYMATIC ACTIVITY AND CARIOUS LESIONS

One of the studies reviewed showed a positive relation between the number of Streptococcus salivarius and levels of urease activity was observed, as well as a negative relation between the number of Streptococcus mutans and levels of arginine deiminase.²⁸

Similarly, the findings of an in vitro study, in which a Streptococcus mutans was modified so that it would produce urease, demonstrated increase of urease activity, while obtaining substantial reduction of environment acidification.²⁹

Two significant sources of alkali in the plaque are arginine and urea. Urea is hydrolyzed by the urease enzyme, which is part of some oral bacteria. Interestingly, the ammonium generated from urealysis may be able to lead to a pH increase in the plaque, despite a diet rich in carbohydrates.^{25, 30-32} Ammonium may also be produced from arginine by the enzyme arginine deiminase. There is ample in vitro evidence to support the hypothesis that the ammonium produced from urea and arginine, by means of ammonium production via urealysis, and by the arginine deiminase system, could be an important endogenous factor for inhibition of cariogenic microbiota and development of dental caries, by neutralization of acids and stabilization of oral microbiota, which is related to oral health.^{5, 16, 23, 33} It has been concluded that small differences in the concentration of urea and in the quantity of the urease enzyme may significantly inhibit the progression of dental caries.34

The hydrolysis generated by the urease bacterial enzyme produces ammonium and CO₂ and it is one of the best ways for the production of alkali in the oral cavity.35 Urea reaches the oral cavity by means of salivary secretions and the crevicular fluids, and its concentration oscillates between 1 and 10 mm in healthy individuals.^36-38

There are several reports in this direction, either as in vitro studies^{15, 24, 29, 31, 33, 39-44} in animals²⁹ or as indirect clinical observations.^{18, 23, 40, 45, 46} The literature also supports, although in an indirect way, the fact that caries risk is directly associated to the loss of the potential to generate alkali.^{18, 25, 31} Therefore, the alkalinogenic potential of a biofilm could be established as a possible strategy in the control of dental caries disease.

It has been demonstrated that there exists a more alkaline plaque in subjects with resistant caries, in comparison to subjects with susceptible caries.⁴¹

Also, the production of alkali in plaque and saliva of individuals with diverse cariogenic status has been studied: caries-free subjects present higher levels of activity of arginine deiminase compared to subjects with active caries, in both saliva and plaque. Likewise, caries-free subjects presented urease levels three times higher than subjects with active caries in plaque samples.²⁸

A positive relation between higher levels of alkali production and caries resistance has been demonstrated; as considerably high levels of urease activity and arginine deiminase were observed in individuals without caries records compared to individuals with active caries.⁵ Some available data indirectly support the idea that alkali production affects the plaque pH and its homeostasis. Part of this result, according to the researchers, is related to the greater concentration of ammonia generated in the plaque of these caries-resistant subjects.⁴⁰

Another interesting observation is the one related with nephropatic patients. Some clinical studies have showed a high concentration of salivary urea in patients with chronic renal failure,^{47, 48} who presented a significant low incidence of dental caries^{36, 49, 50} in spite of consuming a diet based on carbon hydrates. ⁴⁵

The reason for the low activity in the production of alkali in individuals with active caries may be related to the lack of urea as substratum, which generates a lower number of urease-producing organisms and less activity of such enzyme. Urea is usually found at high concentrations in dental plaque, which is rapidly hydrolyzed by the relatively high levels of urease in dental plaque and saliva.⁵¹ Nevertheless, it has been observed that individuals with low levels of urease have a reduced capacity of compensating glycolytic acidification, even if they are provided with a great amount of urea.⁵¹

It may well be that individuals with lower levels of urease activity have a reduced number of ureaseproducing bacteria. By supporting this idea, a recent study showed a considerable positive association between the number of Streptococcus salivarius and the urease levels.

Streptococcus salivarius uses urea as a source of nitrogen for growing, on a urease-dependent path.³⁴ It has been confirmed that both the oral biofilm and the Streptococcus salivarius have a capacity to regulate the urease expression in response to pH increase, thus generating a significant impact on oral biofilm, pH, homeostasis, and microbial ecology.⁵² In order to underline the importance of this enzymatic activity, it has been suggested that urealysis in the plaque may be almost completely explained by the percentage of urealytic bacteria in the plaque's microbiota.⁴³

In the same direction, the results of an in vitro study showed that levels of urease activity produced by a mutant (or recombinant) Streptococcus mutans may be related to small increases of urease activity, considerably decreasing environment acidification.²⁹ It has been clinically proven that urease activity in the plaque is associated to caries-free subjects with high levels of enzymatic activity.⁴⁶ This agrees with previous studies in which the mean urealysis quantity in the dental plaque was 2.5 times higher than in saliva. In that study, the quantity of urealysis in the plaque was almost completely explained by the total percentage of urealytic bacteria in the plaque's microbiota.⁴³

Another important variable influencing urease activity is oral environment. The environment's pH affects both the Streptococcus salivarius capacity and the urease metabolism; pH levels under 4 may inactivate the urease of Streptococcus salivarius ^{53, 54} In terms of arginine deiminase, an elevated level of salivary arginine has been associated with caries resistance,18 and current clinical studies have started to study the capacity of the oral biofilm to produce alkali from arginine, as well as its relation to caries. The results of a recent study show significant differences between the levels of arginine deiminase activity among individuals with different caries status, thus confirming that levels of arginine deiminase activity in non-stimulated saliva were high in caries-free subjects.⁵

Additionally, this study showed a considerable positive relation between the number of Streptococcus sanguinis and the levels of arginine deiminase activity.

It is important to consider that the metabolic activity of both, arginine deiminase and urease, is highly sensitive, and the results of the trial may vary within the same bacteria species or among different bacterial strains of the same species. Another important aspect to underline is that bacteria are extremely dependent of their environment for production of both, acid and alkali.⁵

Newer studies seek to identify bacterial strains and microbial associations capable of contributing to the total arginolysis and urealysis in the oral cavity. Similarly, they pretend to establish the nature and function of the factors that control the activity that produces alkali in oral microbiology.⁵

Nevertheless, further advances are needed in this direction in order to totally understand the role of each of the involved parts in their biological intimacy; it is also critical to establish research protocols for measuring this alkalinogenic activity. Such protocols should be reproducible in the long term in order to define a line of action for the measurement of this activity and to provide cariology with a base that allows following the studies towards a better understanding of microbiology, biochemistry, and the impact of the production of alkalis in saliva and human oral biofilm on health and disease.

Knowing the activity of these enzymes may provide valuable information to explain, for example, the possible effects and biochemical mechanisms of action of therapeutic elements that contain urea such as dental whitening products or toothpaste containing arginine, which are used to reduce teeth sensitivity. The inhibitory effect of such products on the occurrence of new carious lesions and their progression rate has been reported, as well as their superior performance compared to the single use of fluorides, thus constituting a simple and economic tool to substantially reduce one of the prevalent diseases in children.^{55, 56}

CONCLUSION

Alkalization of the oral environment, self-generated by means of a given microbiota and specific substrates, may be a great contribution to the prevention of dental caries. Current evidence would indicate that ammonia production by urease and the arginine deiminase system could potentially inhibit the development of tooth decay by neutralizing acids and stabilizing the oral microbiota, thus providing conditions that favor oral health. Besides, the role of certain species in this process has been studied, showing a positive relation between this enzymatic activity and species such as Streptococcus sanguinis and Streptococcus salivarius.

CORRESPONDING AUTHOR

Gustavo Moncada C.
Avenida Kennedy 7120 Of. 301
Vitacura, RM, Santiago, Chile
Correo electrónico: gmoncada@adsl.tie.cl
Teléfono: 562-656-7051
Fax: 562-655-9082

REFERENCES