Dental caries

Dental caries is a serious health issue that disproportionately affects minorities and children. The amount and duration of the acidity that carbohydrates induce on the teeth of people with and without caries differs quantitatively. Studies conducted in vitro have demonstrated that a variety of environmental conditions, including pH, the availability of glucose, and growth rate, influence the urease expression by S. salivarius. The study's working hypothesis is that a cariogenic challenge causes an increase in urease activity in dental plaque and saliva in vivo. The purpose of the study is to show that exposure to carbs improves oral urease activity. The sample in this pilot study will consist of 20 healthy children ages 4 to 12 years and the appropriate methodologies will be executed to compile the study.

Background and Significance

Dental caries is a significant health problem, which disproportionably affects children and minorities. According to the 2000 report of the Surgeon General on the Oral Health in America dental caries affects 50% of the children between the ages of 5 to 9 years on the United States (Gomez, Andres, and Karen, 500). Among 5- to 17-year-olds, dental caries is more than 5 times as common as a reported history of asthma and 7 times as common as hay fever. The development of dental caries requires that the pH of the dental plaque becomes acidic, usually as a result of the glycolytic processing of dietary carbohydrates by oral bacteria (Nascimento, Gordan, Garvan, Browngardt, and Burne, 92). Many studies have shown that a quantitative difference exists in the intensity and duration of the acidity produced by carbohydrates on the teeth of caries-free and caries-active individuals (Nascimento et al., 93). The average plaque pH of caries-active individuals shows a constant lower average pH than that of caries-inactive patients. In addition, the resting plaque fluid from caries-free individuals has a greater remineralization potential than that from caries-susceptible individuals. This difference is related to a higher concentration of ammonia in the caries-free compared to the caries-active people (Toro, 250). A major source of ammonia production in dental plaque is urea (Toro, 254). Urea is present in the saliva and in the gingival crevicular fluid at concentrations that are usually higher compared to those in plasma (Toro, 256). A number of bacteria in saliva and in the dental plaque produce ureases, which are large multi-subunit enzymes that hydrolyze urea into ammonia and carbonic acid, causing a significant rise in the pH (Toro, 255).

Urea levels equivalent to those normally found in saliva (3 to 5 mM/L) could produce a rise in plaque pH equivalent to the difference in pH between plaque and saliva which has been reported previously in fasting subjects. Dental plaque pH studies have shown that simultaneous metabolism of urea and carbohydrate raises the pH minimum in the Stephan curve (Gomez et al., 501). Furthermore, the effect of carbohydrates in lowering the pH of plaques can be largely overcome for a period of 24 hours by the application of urea in a 40-50 % concentration solution for 4 minutes (Gomez et al., 502).
Interestingly, patients with chronic renal failure in addition to having higher blood urea concentration, they also have shown increased levels of salivary urea nitrogen concentration and their plaque is significantly more alkaline. Following an exposure to carbohydrate, the absolute pH drop obtained did not attain a significant cariogenic level since the baseline plaque pH was elevated (Toro et al., 253). Similarly, some studies have shown that the addition of urea to chewing gum initiates a pronounced pH recovery in dental plaque when chewed after a sucrose rinse. Studies by Toro et al suggest that the plaque cariogenicity may be inversely related to salivary urea concentrations, not only when the latter are elevated because of disease, but even when they are in normal range.
In nature, some organisms such as Bacillus pasteuri, Morganella morganii, and some isolates of E. coli with chromosomally encoded ureases can constitutively synthesize urease. However, urease expression in most bacteria seems to be regulated at the transcriptional level by environmental factors, such as urea and nitrogen availability (Nascimento et al., 92). This is apparently also true for oral bacteria. In vitro studies have shown that the urease expression by S. salivarius is regulated by multiple environmental factors such as pH, glucose availability, and growth rate (Nascimento et al, 93). Urease expression in this organism is almost completely repressed at neutral pH and low sugar concentrations, but it can be increased up to 650-fold when the pH drops to acidic levels and glucose availability increases The expression of urease in response to pH is controlled, at least in part, at the transcriptional level. Urease expression in A. naeslundii, which is a pioneer organism in the oral cavity and a highly abundant species in dental plaque, appears to be very low when the availability of nitrogen sources is high. However, the organism can produce significant levels of urease activity when the organism is grown under nitrogen-limiting conditions, which correspond to conditions of carbohydrate-excess. Thus, it may contribute to the pH-moderating activity of oral biofilms suggesting a potential for a greater involvement of A. naeslundii in prevention of caries (Toro et al., 253).

Statement of Hypothesis and Specific Aims

The working hypothesis in this study is that urease activity in dental plaque and in saliva in vivo increases during a cariogenic challenge, thus providing additional protection against the development of dental caries.
The specific aim of this pilot study is to demonstrate that urease activity in the mouth increases after exposure to carbohydrates. To achieve this aim we will compare the levels of urease activity in the plaque and in the saliva of children 4 to 12 years of age, during fasting and after rinsing with a 10% sucrose solution.

Research Design and Methodologies

Subject recruitment

The sample in this pilot study will consist of 20 healthy children ages 4 to 12 years. This study focuses on children mainly because dental caries affects 50% of the children between the ages of 5 to 9 years in the United States suggesting that early caries risk assessment and prevention should begin early in childhood. In addition, they present a unique intra-oral bacterial environment that may have particular effect on caries susceptibility.

Clinical procedures

Subjects will be required to refrain from eating and drinking anything but water for a minimum of 6 hours prior to sample collection. They will also have to refrain from oral hygiene procedures for at least 6 hours prior to sample collection to achieve sufficient plaque accumulated for collection. All samples will be collected between 8 am and noon.
Initially, supragingival plaque will be collected from all available smooth dental surfaces on the upper and lower right quadrants using a periodontal scaler and pooled. Plaque will be placed in pre-weighed micro-centrifuge tubes. Saliva samples will be collected using a mucous trap attached to the dental suction. A minimum of 2 ml of saliva will be collected. Following the first sample collection subjects will be asked to take 5 ml of a sterile 10% sucrose solution and swirl it around for 1 minute then spit it out. After 1 minute, a temporary drop in plaque pH will occur due to bacterial metabolism of the carbohydrate. The pH returns to baseline levels normally within 30 minutes following the exhaustion of the carbohydrate source (Toro et al). Thus, a second plaque and saliva sample will be collected by the same method 30 minutes following the oral rinse from the quadrants on the opposite side of the mouth. Plaque and saliva samples will be placed immediately on ice and will be transferred to the laboratory, where they will be weighed again. The difference in the weight of the tubes between pre- and post-sampling will correspond to the wet weight of the respective plaque sample. Plaque and saliva samples will be washed once in sterile 10 mM sodium phosphate buffer pH 7.0 in cold, in order to remove any background urea. Plaque samples will be re-suspended in 300 ul the same buffer. Saliva samples will be re-suspended in 500 μl of the same buffer (Toro et al, 254).

Measurement of urease activity

25 μl of plaque or 50 μl saliva suspensions will be incubated at 37oC in a mixture containing 50 mM potassium phosphate buffer pH 7.0 and 50 mM urea for 1.5 hours. After the incubation, the reaction mixture will be centrifuged at 12,000 rpm for 3 min and the supernatant will be assayed for ammonia content using the Nessler's ammonia color reagent with ammonium sulfate as the standard. Urease activity will be normalized to protein content as determined by the method of Bradford. The units to be used to measure urease activity will be defined as nmoles of urea hydrolyzed per min per mg of protein (Gomez et al, 498).

Statistical analysis

Descriptive statistics for urease activity in the dental plaque and saliva including mean, standard deviation, median, and range (min.-max.) will be computed in order to have an epidemiological profile of the sample. A paired t-test will be used to compare pre- and post- rinse urease levels.

Potential outcomes

The observations presented above suggest that oral bacteria may have a greater capacity to produce urease during conditions of a cariogenic challenge, when the carbohydrates are present in excess and the plaque pH becomes acidic. If it could be demonstrated that this occurs in vivo, it would indicate that urease activity in plaque and in saliva might be an important endogenous caries preventing mechanism. Upon developing further data to support our hypothesis, the determination of urease activity in each individual may be used as a diagnostic tool for early caries risk assessment and prevention early in childhood.

Works Cited

Gomez, Andres, and Karen E. Nelson. "The Oral Microbiome of Children: Development, Disease, and Implications Beyond Oral Health." Microbial Ecology. 73.2 (2017): 492-503. Print.
Nascimento, M M, V.V Gordan, C W. Garvan, C M. Browngardt, and R A. Burne. "Correlations of Oral Bacterial Arginine and Urea Catabolism with Caries Experience." Oral Microbiology and Immunology. 24.2 (2009): 89-95. Print.
Toro, E, MM Nascimento, E Suarez-Perez, RA Burne, A Elias-Boneta, and E Morou-Bermudez. "The Effect of Sucrose on Plaque and Saliva Urease Levels in Vivo." Archives of Oral Biology. 55.3 (2010): 249-54. Print.