Evaluation of the Salivary-Free Amino Acids Levels (Glycine and Proline) as Caries Susceptibility Predictors in 8- to 10-Year-Old Children

Abstract

Objective

This research investigated the association between levels of salivary free amino acids (glycine and proline) and caries susceptibility in healthy children versus children with active caries, to inform future strategies for the detection and prevention of pediatric dental caries.

Material and Method

The study had a case–control design and was conducted with 80 healthy children (8–10 years old) recruited from private and governmental primary schools in the Rusafa of Baghdad city, Iraq, classified according to their level of caries activity (active caries, n = 40; inactive caries, n = 40) based on their caries experience and Snyder test results. Then, unstimulated whole saliva was collected for amino acid analysis performed by reversed-phase high-performance liquid chromatography.

Statistical Analysis

Statistical analysis was conducted using the Shapiro–Wilk test for symmetric distribution of the quantitative variable, the independent t-test was utilized for making comparisons, and Pearson's correlation was used to verify if there is a linear relationship between two quantitative factors that are normally distributed.

Results

Glycine was the most abundant amino acid in the saliva, followed by proline from 113 components identified in the saliva. Glycine levels decreased significantly (p < 0.05) with the active caries group, regardless of gender differences. On the contrary, a significant positive correlation was established between increased levels of salivary proline (p < 0.05) and the active caries group.

Conclusion

The higher salivary glycine and proline levels may serve as potential biomarkers for assessing caries susceptibility and informing preventive strategies.

Keywords

salivary-free amino acids - glycine - proline - dental caries susceptibility - caries predictors

Introduction

Dental caries is a biomediated, dynamic, multifactorial, and preventable disease. It starts with microbial changes in complex biofilms.[1] Host characteristics, such as salivary composition, dietary habits, and preventive behaviors,[2] also affect it.[3] [4] [5] Nearly 500 million youngsters have untreated caries in their primary teeth, according to a recent World Health Organization report,[6] this is considered a risk indicator. Dental caries adversely affects the child's quality of life, growth, and development. Furthermore, it could cause problems for the patient's socialization, school performance,[7] and self-esteem.[8] According to Wagle et al, healthy teeth in childhood are critical for developing healthy permanent teeth later on, as well as for maintaining a healthy diet and appearance.[9] [10] [11] [12] [13] Hence, dental caries is considered a public health issue,[14] although it is largely preventable. It can cause pulp lesions and premature tooth loss if left untreated.[15] Accordingly, it is necessary to highlight the importance of preventive strategies focused on the action of salivary components.

Human saliva, a dynamic and complex biological fluid,[16] can reflect a person's oral physiological condition because it has direct contact with and encompasses the teeth, dental plaque, and oral microbial community.[17] Salivary factors like pH, proteins, buffering capacity, and enzymes have been studied extensively as biological markers of dental caries.[18] [19] In general, saliva comprises water (98.5%), organic (1.0%), and inorganic (0.5%) components.[20] In addition to these, saliva also contains metabolites and other important compounds. It includes a significant amount of low-molecular-weight peptides and free amino acids.[21] These chemical compounds can be exchanged with plaque fluid, making them potential nutrients and energy sources for the microbial plaque and products of their metabolic pathways.[22] Thus, these molecules can serve as a reflection of bacterial activity and caries susceptibility.[23]

The building blocks of proteins[24] and the nitrogenous backbones of substances like hormones and neurotransmitters are generally referred to as amino acids. According to chemistry, an amino acid is an organic molecule that has two functional groups: one for an amino (-NH2) and one for a carboxylic acid (-COOH).[25] There is interest in salivary-free amino acid levels in children as a possible marker of dental caries.[26] [27] Understanding salivary-free amino acid levels in children is relevant for estimating their caries susceptibility

Therefore, the current study has investigated glycine and proline levels within salivary composition for associations with caries susceptibility in a cohort of Iraqi children by using reversed-phase high-performance liquid chromatography (RP-HPLC). The study's ultimate goal is to use these results to develop preventive strategies for dental caries.

Materials and Methods

Sample Size Determination

Sample size calculation was performed using G*Power version 3.1.9.7, as described by Faul et al.[28] The parameters included a statistical power of 80%, α error of probability = 0.05, and an effect size (F) of 0.65. According to Cohen's conventional classification, this effect size corresponds to a medium effect.[29] Based on these inputs, the required total sample size was calculated to be 80 subjects (40 subjects for each group).

Study Design and Population

The study had a case–control design that consisted of 80 healthy children, aged 8 to 10 years, of both genders, from elementary schools in Rusafa, Baghdad, Iraq. This study received review and approval from the Research Ethics Committee of the College of Dentistry/ Mustansiriyah University, Iraq (approval Number: REC145). The research adheres to the current Human Research Guidelines, with full ethical approval granted on December 1, 2023. There were 39 females and 41 males. Each child who was tested for eligibility met all the inclusion criteria. Before enrolling patients in the study, a consent form was signed by the parents of all study participants. All recommendations to prevent cross-contamination were followed.

Inclusion Criteria

1. Children aged 8 to 10 years with different caries activity.

2. Absence of signs of gingivitis or periodontal disease.

Exclusion Criteria

1. Children are out of the age range.

2. Children with appliances in the mouth.

3. Children with any systemic diseases.

4. Children with antibiotic treatment or any other drug over the last 2 weeks.

Clinical Assessment of Dental Caries

The initial clinical assessment of dental caries among the participating children was conducted through a standardized oral examination using the dmft/DMFT index, following the diagnostic and scoring criteria recommended by the World Health Organization.[30] All observed carious lesions, whether decayed, missing due to caries, or filled, were recorded once per tooth and systematically documented in study-specific case sheets to establish an accurate baseline of caries experience. This baseline clinical evaluation served as the first step in determining caries activity.

To further strengthen the assessment and differentiate between active and inactive caries patterns, the Snyder test was subsequently employed as a validated metabolic assay for estimating acidogenic activity and classifying participants into two groups: those with active caries and those with inactive caries, in line with established methodological evidence demonstrating the test's reliability in identifying high-risk individuals.[31] This combined diagnostic approach provided a robust framework for evaluating the caries status and microbiological activity of the study population.

Saliva Collection

After informing the participants of the Navazesh protocol[32] between 9:00 and 11:00 a.m., saliva samples were taken.

Whole Stimulated Saliva Sample

All participants were instructed to chew a typical piece of gum (∼1 g) for 60 seconds to increase salivary production and release plaque into the salivary fluid.[33] Following that, the saliva was gathered using a sterile, one-use plastic cup. Samples were taken to the laboratory in a cooling box containing ice for the caries activity to be assessed by the Snyder test agar.[31]

Whole Unstimulated Saliva Sample

Whole unstimulated saliva samples, which were collected by expectoration, were kept in a disposable, sterile plastic cup for free-amino acid analysis.[33]

Sample Preparation

After collection, for 10 minutes, the samples were centrifuged at 10,000 revolutions per minute (rpm) and 4°C to remove insoluble materials, cell debris, and food residue. Until the laboratory analysis, the resulting supernatants were frozen at –80°C. Then, a 2.0-mL Eppendorf tube was filled with 800 mL of acetonitrile and 400 mL of thawed saliva, and to precipitate the proteins, the mixture was agitated violently for 1 minute. Following 15 minutes of standing at 4°C and 10,000 rpm, the mixture was centrifuged for 20 minutes. The supernatant was prepared for HPLC analysis following filtration via 0.22 μm syringe filters.[34]

HPLC Condition

Chromatography conditions were according to the SYKAM - German HPLC method. Shortly, the robotic autosampler was programmed to automatically derivatize the hydrolyzed samples with ortho-phthaldialdehyde. After derivatization, a portion equivalent to 100 μL of each sample was injected on a Zorbax Eclipse-AAA column at 5 μm, 150 × 4.6 mm (Agilent), at 40°C, with detection at λ = 338 nm. Mobile phase A consisted of 40 mM NaH2PO4, adjusted to pH 7.8 with NaOH. On the other hand, mobile phase B consisted of acetonitrile, methanol, and water (45/45/10 v/v/v). Utilizing a gradient program, the separation was achieved at a flow rate of 1 mL/min. For 0 to 5 minutes, A was 10%/B was 90%, and for 6 to 15 minutes, A was 30%/B was 70%. Amino acid concentrations were calculated using the peak areas relative to the standard area.[35]

Statistical Analysis

Several statistical measures, including frequency (represented as a percentage), mean, and standard deviation (SD), were used to present the study's findings. The Shapiro–Wilk test was used to assess the normal distribution of the quantitative variable. The independent t-test was employed to determine statistical differences between two groups, and Pearson's correlation was used to verify the existence of a linear relationship between two normally distributed quantitative factors. Statistical comparisons were conducted using SPSS software (“Statistical Package for Social Science” version -26, Chicago, Illinois, United States). A p-value of < 0.05 was considered a statistically significant value.

Result

Demographic Characteristics

Participants in this study ranged in age from 8 to 10 years. Children with active caries (n = 40; 23 male and 17 female) made up the patient group, while children with inactive caries (n = 40; 18 male and 22 female) made up the control group, as shown in [Table 1] and [Fig. 1].

Table 1 Demographic distribution of study participants

Caries activity status group	Gender				Total
	Male		Female
	N	%	N	%	N	total%
Inactive caries	23	28.75	17	21.25	40	50%
Active caries	18	22.5	22	27.5	40	50%

Fig. 1 Distribution of caries activity status percentages by gender.

[Table 2] demonstrates the distribution of two variables, selected free amino acids (glycine and proline) concentration in saliva among individuals with inactive caries and those with active caries. The Shapiro–Wilk test was utilized to determine the normality of research data distribution, which revealed the data were normally distributed (p > 0.05).

Table 2 Normality test by the Shapiro–Wilk test for all studied variables

Variables	Groups
	Control				Active caries
	Statistic	N	p-Value	Significance	Statistic	N	p-Value	Significance
Glycine	0.955	40	0.125	Nonsignificant	0.935	40	0.055	Nonsignificant
Proline	0.958	40	0.128	Nonsignificant	0.970	40	0.133	Nonsignificant

Note: p < α: 0.05 stated to be significant.

The descriptive statistics for the salivary glycine and proline in the study groups are displayed in [Table 3]. The mean ± SD in the inactive caries group was 12.507 ± 0.390 for salivary glycine and 18.249 ± 0.438 for salivary proline; in contrast, the results in the active caries group were 3.442 ± 0.197 and 30.800 ± 1.417 for salivary glycine and proline, respectively.

Table 3 Descriptive and statistical tests of salivary glycine and proline among study groups

Salivary amino acid		Control group, N = 40	Active caries group, N = 40	t-Test	p-Value
Glycine	Minimum	11.880	3.110	131.105	0.000[a]
	Maximum	13.110	3.690
	Mean	12.507	3.442
	± SD	0.390	0.197
Proline	Minimum	17.450	28.870	53.512	0.000[a]
	Maximum	18.910	32.880
	Mean	18.249	30.800
	± SD	0.438	1.417

Abbreviation: SD, standard deviation.

^a Highly significant; p < α: 0.05 stated to be significant.

Increased levels of salivary glycine showed a highly significant correlation with inactive caries (p = 0.000), suggesting that individuals with high salivary glycine levels have a lower risk of dental caries. On the other hand, the increased levels of salivary proline demonstrated a highly significant association with the active caries group (p = 0.000), indicating that individuals with elevated proline levels in saliva had a higher risk of active caries, as shown in [Table 3].

[Figs. 2] and [3] present the HPLC chromatograms of the standard amino acids glycine (retention time: 3.8 minutes) and proline (retention time: 4.8 minutes), respectively.

Fig. 2 High-performance liquid chromatography (HPLC) chromatogram, retentive time for standard glycine in minutes.

Fig. 3 High-performance liquid chromatography (HPLC) chromatogram, retentive time for standard proline in minutes.

In contrast, [Figs. 4] and [5] illustrate the HPLC chromatograms of amino acid concentrations obtained from a patient sample and a control sample, respectively.

Fig. 4 High-performance liquid chromatography (HPLC) chromatogram of glycine and proline concentration in the active caries group.

Fig. 5 High-performance liquid chromatography (HPLC) chromatogram of glycine and proline concentration in the control group.

[Table 4] and [Fig. 6] indicates that salivary proline and glycine exhibit a statistically significant inverse correlation in the active caries group, whereas no statistically significant association is observed in the control group.

Table 4 Correlation of salivary-free amino acids (glycine and proline) according to caries activity groups

Group		Proline
		r	p-Value
Control	Glycine	−0.050	0.761
Active caries		−0.688	0.000

Note: r, Pearson's correlation coefficient.

p < α: 0.05 stated to be significant.

Discussion

According to the previous investigations' findings, the higher caries risk potential can be affected by two factors: genetics and environment.[36] [37] [38] Among these environmental factors are microorganisms, nutrition, dental hygiene, and host factors.[39] Free amino acids contribute significantly to controlling the function and metabolic processes of cells,[40] in addition to supporting the immune response[24] and having anti-inflammatory properties.[41]

The fermentation of carbohydrate by dental plaque bacteria is the main process that results in caries development, which produces destructive organic acids such as lactate that lower the pH and cause demineralization of tooth enamel.[42] [43] Saliva plays a key role in maintaining the acid-base balance in the oral cavity. It contains buffering components such as bicarbonate, proteins, and amino acids that help neutralize acids produced by bacterial metabolism.[44] In this study, salivary glycine levels were significantly elevated in the control group compared with those in the active caries group (p < 0.05). Previous studies have reported glycine as a marker of caries-free status in saliva.[27] [45] The presence of amino acids in saliva may modify the chemical environment in the oral cavity, where some can be converted by oral bacteria into ammonia and amines, which help neutralize acids produced by caries-causing bacteria,[46] thus reducing enamel degradation. A glycine-supplemented diet reduces caries development and tooth fat concentration in humans and rats, indicating the cariostatic impact of glycine.[45]

The increased levels of salivary glycine of the inactive caries group (control group) in this investigation could be due to its antimicrobial properties, which is a known factor. For several bacteria, elevated levels of glycine stimulate degradation or profound structural changes in the outer layer.[47] Studies have confirmed glycine's impact on various bacterial species, including both Gram-positive and Gram-negative, anaerobic, and aerobic bacteria, and have reported inhibition of bacterial growth by glycine.[48] Proposed mechanisms of antimicrobial properties of glycine, in addition to disrupting cell wall remodeling, can inhibit biofilm formation[49] and enhancement of immune system functions such as reactive oxygen species production and phagocytosis.[50] [51] The defense applied by the existence of salivary glycine in participants with inactive caries is an innovation, and it requires further inquiry.

The elevated levels of salivary proline in children with active caries could be due to the degradation of salivary proteins and peptides by oral bacteria.[45] Consequently, it has previously been proposed that individuals with and without dental decay exhibit distinct proteolytic deterioration of proline-rich salivary proteins.[52] Proline-rich proteins (PRPs) are major components of saliva and form a significant part of the acquired enamel pellicle, which coats the tooth surface and performs protective functions.[53] When biofilm activity is high (as in active caries), proteolysis of the salivary membrane or saliva may increase, releasing free proline.[54]

Christgen and Becker recommend that inhibiting the metabolism and transport of proline could be an effective therapeutic approach against specific pathogens, as proline provides a carbon, nitrogen, or energy source for bacterial and protozoan pathogens. Some pathogens rely on proline as an essential respiratory substrate, while others use it to protect against stress.[55] [56] [57] Recent evidence suggests that oral bacterial biofilm can utilize free proline as an alternative energy source in the absence of sugars. In a study using nuclear magnetic resonance to monitor metabolism in a human saliva biofilm cultured on hydroxyapatite, the biofilm was found to metabolize proline into metabolites such as 5-aminopentanoate, butyrate, and propionate, with clear consumption of free proline from the medium. Under sugar-rich incubation conditions (e.g., the presence of glucose or sucrose), the same study shows that the proline metabolism pathway is almost inhibited, indicating that bacteria preferentially consume sugars when available.[54]

Therefore, an elevated level of free proline in the saliva of children with active caries does not necessarily mean that proline itself directly contributes to tooth decay. Rather, it may reflect a metabolic dynamic, a balance between the rate of degradation of PRPs or salivary proteins, and the rate of metabolism of this proline by bacteria.

In a broader view, this interpretation supports the idea that free proline in saliva can be used as an indirect biomarker of intense oral biofilm activity and high proteolysis in the oral environment, rather than necessarily as a direct pathogen itself. Nevertheless, these results have not been in agreement with other studies.[27] [58] [59] The data of the current study demonstrate higher levels of proline in participants with active carious lesions, consistent with earlier reports by Fonteles et al and with the data reported by Pereira et al.[45] [60]

Limitations

Although this study provides important preliminary indications of biochemical changes in saliva, several methodological limitations must be considered when interpreting the results. First, the sample size was small, which may limit the generalizability of the findings to broader populations. Second, the study did not include any oral microbiological analyses of the microbiome, which limits the ability to correlate the observed changes in saliva components with bacterial activity or microbial structure. Third, no comprehensive proteomic analyses were performed, limiting the understanding of the sources of changes in amino acid levels or related compounds and preventing the determination of whether these changes result from salivary protein degradation or specific metabolic pathways. Despite the statistically significant differences, it is advisable to confirm these findings in future studies designed with larger samples and incorporating advanced microbiome and proteomic analyses to further understand the potential mechanisms of the biochemical changes in saliva and to elucidate their sources more precisely.

Suggestions for Future Studies

• - Longitudinal, standardized studies that correlate the extent of PRP degradation and the concentrations of free glycine and proline with actual caries incidence across diverse populations.

• - Functional in situ biofilm investigations assessing the effects of exogenous proline or glycine supplementation on lactic acid versus ammonia production, alongside quantifying subsequent shifts in demineralization–remineralization dynamics.

• - Include microbiological analysis of saliva to explore the relationship between salivary component and oral bacteria and their impact on the results.

Conclusion

In conclusion, the current study used RP-HPLC to examine saliva samples from children with varying levels of caries activity. There were notable variations in the amino acid levels of the active and control groups in this research. Free glycine and free proline in human saliva appear to play distinct yet complementary roles within the oral environment. Glycine may exert a potential protective effect through ammonia generation and localized alkalinization, whereas proline serves as a metabolic substrate for the oral biofilm. Recent metabolomics and in vitro biofilm studies support this conceptual framework; however, well-designed longitudinal investigations are still required to establish clear causal relationships.

Fig. 6 Correlation between salivary proline and glycine of the active caries group.

Conflict of Interest

None declared.

Acknowledgment

The authors would like to thank Mustansiriyah University (http://www.uomustansiriyah.edu.iq), Baghdad, Iraq, for its support in the present work.

Ethical Approval

This study received review and approval from the Research Ethics Committee of the College of Dentistry/Mustansiriaha University, Baghdad, Iraq (Approval Number: REC145). The research adheres to the current Human Research Guidelines, with full ethical approval granted on December 1, 2023. All methods were performed in accordance with relevant guidelines and regulations.

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Publication History

Article published online:
04 February 2026