Circadian Influence on Saliva Biochemical Composition: A Pilot Clinical Investigation

doi:10.21203/rs.3.rs-5237929/v1

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Circadian Influence on Saliva Biochemical Composition: A Pilot Clinical Investigation

https://doi.org/10.21203/rs.3.rs-5237929/v1

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Objective. Saliva is a valuable non-invasive diagnostic tool, but the impact of circadian timing on its biochemical composition is not fully understood. We, therefore sought to investigate whether circadian fluctuations of unstimulated saliva affect biochemical parameters, including pH, total protein, glucose, and organic and inorganic ions.

Materials and Methods. Eight healthy, caries-free young adults donated whole unstimulated saliva at six time-points throughout the day, with each participant providing samples on three separate days, ensuring triplicate collections for each time-point. Before saliva collection, participants adhered to a standardized Mediterranean diet for three days and during collection day. Collected saliva samples were analyzed for pH by a pH-meter, protein by bicinchoninic acid test, and glucose and ions using a calibrated reflectometer. Statistical comparisons across the different time-points were conducted using ANOVA repeated measures test (p<0.05).

Results. The circadian rhythm did not result in statistical variations between-subjects on the measured time-points for calcium, phosphate, peroxide, salivary pH, or total proteins in unstimulated saliva (p>0.05). However, significant circadian variations were observed for lactate, nitrate, nitrite, glucose, and ammonium (p<0.05), with distinct peaks observed at specific times during the day.

Conclusions. Circadian rhythm appears to have a limited impact on the overall biochemical composition of unstimulated saliva in young, healthy adults. Significant fluctuations in specific analytes warrant further investigation.

Clinical Relevance: These findings may contribute to the standardization and refinement of research using saliva as a biomarker for oral and systemic conditions. The differences observed between volunteers highlight the individual diagnostics in the prevention of oral diseases.

Dentistry

General Biochemistry

Diurnal variations

Salivary parameters

Circadian rhythms

Ionic components

Oral health.

Saliva plays a fundamental role in preserving oral health [1]. This biological fluid originates primarily from the major salivary glands (90%), while the minor salivary glands and crevicular fluid contribute the remaining 10% [2]. Saliva consist of 99.5% water, 0.3% proteins, and 0.2% organic and inorganic substances, including proteins, enzymes, electrolytes, nitrogen products, and a variety of other components [3–5]. It supports functions like chewing, digestion, taste, and speech [6]. In healthy adults, daily unstimulated salivary volume ranges between 500–1500 mL with a flow rate 0.3–0.5 mL/min [5, 7]. Several important physiological properties of saliva, such as pH, buffering capacity, and saliva flow, can be influenced by dietary habits, lifestyles, polypharmacy, or chronic systemic diseases [8]. Salivary components can also vary throughout the day due to multiple factors, including diet, oral hygiene, salivary flow, and circadian rhythm [9].

The circadian system, often referred to as the body’s internal clock, regulates homeostatic processes in the body and responds to environmental stimuli, with light being the most significant [10–12]. The circadian rhythm consists of a central clock located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus, and peripheral clocks in various tissues [13, 14]. Light detected by photosensitive cells in the retina, is transmitted to the SCN via the hypothalamic retinal tract [12]. The SCN then coordinates circadian signals that are crucial for the sleep-wake cycle, hormonal secretion, and regulation of body temperature [13, 15]. Recent studies have reported that the salivary glands, like other human organs, contain a circadian clock [10, 16], suggesting that the peripheral circadian clock in the salivary glands may regulate the type, quantity, and content of saliva. Although saliva has been widely studied, there is a research gap regarding the association between salivary components and their circadian regulation. Therefore, the aim of this study was to determine whether circadian rhythm induces variations in pH, total proteins, and the concentration of ions related to a healthy oral environment, such as nitrate (NO₃⁻), nitrite (NO₂⁻), ammonium (NH₄⁺), lactate, peroxides, glucose, calcium (Ca²⁺), and phosphate (PO₄³⁻) in the unstimulated whole saliva of young adults. These findings may contribute to the standardization and refinement of research using saliva as a biomarker for oral and systemic conditions.

Subjects

Healthy adult volunteers (n = 8, 21.87 ± 0.83 (mean ± SD) years old), including dental students and users of the University’s Dental Clinics, were invited to participate through social media advertisements. Detailed information about the study’s methodology, scope, and parameters was provided in an online talk. Each participant underwent a clinical examination, including a general medical history and an intra- and extraoral evaluation by a dentist calibrated in the ICDAS criteria for caries assessment [17]. Inclusion criteria were adults aged 18–35 who were systemically healthy, had adequate oral hygiene and gingival health, did not present cavitated caries lesions according to ICDAS criteria (codes 3–6), and did not have active non-cavitated ICDAS lesions as per Nyvad’s activity criteria [18]. Exclusion criteria included volunteers with conditions known to cause decreased or altered salivary flow, such as autoimmune diseases or allergic reactions, those undergoing treatment with corticosteroids, or smokers.

The study design and protocol were approved by the Scientific Ethics Committee of the University of Talca (ACT No. 14/2021), and the study was conducted in accordance with the Declaration of Helsinki and international ethical guidelines for biomedical research involving humans (CIOMS 2016). Written informed consent was obtained from volunteers who agreed to serve as saliva donors.

Protocol standardization: To minimize the influence of dietary patterns on the results, a personalized Mediterranean diet was prescribed by a nutritionist, taking into account individual requirements for macro and micronutrients, body composition, and physical activity. This dietary regimen began three days prior to sampling and was maintained throughout the entire 24-hour saliva collection cycle. On the collection day, participants were required to refrain from eating or drinking for at least one hour before each sampling and to fast for eight hours prior to the first collection of the day (7:00 AM). Food was provided to participants who had difficulty obtaining the prescribed diet. For oral hygiene, participants were instructed to brush their teeth without toothpaste on the day of sampling, at least one hour before each collection, but to refrain from brushing before the first sampling (7:00 AM). To ensure adherence, research assistants sent reminders via instant messaging and offered assistance as needed. Preliminary tests were conducted (data not shown) to optimize the collection of unstimulated total saliva, determining the appropriate collection time and the minimum volume required for each analysis. Each subject was assigned a numeric code, which was concealed from the researchers processing the saliva samples and those analyzing the data.

Study design and collection of saliva samples: Three individual collection series were carried out for each volunteer. For each series, unstimulated whole saliva samples were collected by volunteers at six time-points, evenly spaced at 4-hour intervals throughout the day: 7:00 AM, 11:00 AM, 3:00 PM, 7:00 PM, 11:00 PM, and 3:00 AM the following day. Each volunteer was asked to provide at least 25 mL of saliva per collection, excluding any foam generated. The saliva was collected in a sterile tube containing 250 µL of 0.1 M protease inhibitor cocktail (PMSF, Sigma-Aldrich, MD, USA), which had been pre-cooled on ice. Once the minimum volume was reached, saliva was mixed with the PMSF by gently stirring and immediately stored at -20°C. The study analyzed differences in pH and the concentrations of calcium (Ca²⁺), phosphate (PO₄³⁻), nitrate (NO₃⁻), nitrite (NO₂⁻), ammonium (NH₄⁺), lactate, glucose, total proteins, and peroxides throughout the 24-hour cycle to assess variations among the different time-points on the same day, as well as differences at the same time-point across the three different collection days.

Saliva sample processing and analysis

Frozen samples were thawed at 4°C and centrifuged at 3,400 rpm for 20 minutes at 4°C. From the supernatant, 1 mL was used for pH and Ca²⁺ measurement. The remaining supernatant was filtered through a 0.22 µm pore size filter (Rapid Flow, Nalgene, Rochester, NY, USA) and aliquoted into Eppendorf tubes for the determination of the concentrations of the aforementioned compounds.

pH: To verify saliva acidity, the pH of the samples was measured in triplicate using a microelectrode (5990-45 Cole-Parmer, 625 East Bunker Ct, Vernon Hills, IL, USA) coupled to a pH meter (Orion Star A211, Thermo Fisher Scientific, MA, USA).

Nitrate, Nitrite, Ammonium, Calcium, Phosphate, Lactate, Peroxides, Glucose: These analytes were determined using a reflectometer (Reflectoquant® RQflex® 20, Merck Millipore, Darmstadt, Germany) which uses test strips that change in color intensity based on the concentration of specific substrates. Detection ranges of the test strips (Reflectoquant, Merck Millipore) for each analyte were: 3–90 mg/L for nitrate, 0.5–25 mg/L for nitrite, 20–180 mg/L for ammonium, 5–125 mg/L for calcium, 5–125 mg/L for phosphate, 3–60 mg/L for lactate, 0.2–20 mg/L for peroxides, and 1–100 mg/L for glucose. Briefly, undiluted supernatants were used to obtain concentrations within the detection thresholds of the test strips. For each measurement, 100 µL of the supernatant was added to the reactive strips, and excess liquid was removed by tipping the side of the strip. Ammonium and phosphate requested 400 µL of saliva. Each analyte was measured in triplicate across all saliva samples on all three days. The accuracy of the reflectometry was confirmed using standard solutions with known concentrations of the different ions/molecules.

Total Protein Determination: Total protein concentration was determined using the Bicinchoninic Acid (BCA) colorimetric assay (Pierce, Thermo Fisher Scientific) according to the manufacturer’s instructions. The absorbance of the saliva samples was measured at 562 nm using a NanoQuant spectrophotometer (Infinite M200 PRO, Tecan, Hombrechtikon, Switzerland). Protein concentrations were assessed in triplicate for all saliva samples on all three days and calculated using a standard curve with known Bovine Serum Albumin (BSA) (Pierce, Thermo Fisher Scientific) concentrations.

Data Analysis

The values obtained for each analyte across the three assessment days were averaged for each time point and analyzed using the ICC test. The Kolmogorov-Smirnov test (p > 0.05) was used to assess the normality of data distribution. Parametric analyses were conducted using one-way repeated measures ANOVA. Differences were considered significant at a 95% confidence level (p < 0.05). All analyses were performed using SPSS 15.0 for Windows (IBM Corporation, New York, NY, USA).

Effect of circadian rhythm on salivary compounds. Figures 1 and 2 summarize the concentrations of lactic acid, nitrate, nitrite, ammonium, glucose, calcium, and phosphate in the saliva of the 8 volunteers at each of the 6 predefined time-points. Significant circadian variations were observed for lactic acid (p = 0.041), nitrate (p < 0.001), nitrite (p = 0.022), ammonium (p < 0.001), and glucose (p < 0.001) at specific times of the day.

Lactic acid concentrations exhibited a significant decrease, reaching 6.48 ± 1.46 mg/L, from its peak at 7:00 AM (11.51 ± 2.65 mg/L) (Fig. 1a). Nitrate and nitrite levels both peaked significantly at 03:00 PM, with concentrations of 70.83 ± 11.44 mg/L and 4.2 ± 0.72 mg/L, respectively, before gradually returning to near baseline levels by 7:00 AM the following day (Fig. 1b and 1c). In contrast, ammonium showed an inverse pattern relative to nitrate and nitrite, with its lowest concentration (56.38 ± 12.17 mg/L) at 3:00 PM and the highest levels at 7:00 AM and 3:00 AM (Fig. 1d).

Glucose levels peaked at 7:00 AM and 3:00 AM, with concentrations of 10.40 ± 2.10 mg/L and 7.50 ± 0.94 mg/L, respectively, and reached their lowest levels between 03:00 PM and 11:00 PM, around 4.0 mg/L (Fig. 2a).

Calcium (Ca²⁺) and phosphate (PO₄³⁻) concentrations did not show statistically significant variations over the 24-hour period (p > 0.05) (Fig. 2b and 2c). The mean values for Ca²⁺ ranged between 29.29 ± 1.08 mg/L and 34.45 ± 1.79 mg/L, while PO₄³⁻ levels ranged from 83.87 ± 3.42 mg/L to 92.50 ± 3.29 mg/L. Despite the lack of statistical significance, both ions showed a similar pattern, with the highest values seen at the 7:00 AM time-point (34.45 ± 1.79 mg/L and 92.5 ± 3.29 mg/L, for Ca²⁺ and PO₄³⁻, respectively). Peroxide concentration levels were not detected by the reflectometry technique used (data not shown).

Salivary pH. The mean pH values showed slight variations throughout the day, but no statistically significant differences were detected (p > 0.05), indicating overall stability in salivary pH (Fig. 2d). However, the lowest values were observed at the 3:00 AM and 07:00 AM time-points.

Effect of circadian rhythm on total salivary proteins. As shown in Fig. 3, total salivary protein concentrations remained relatively stable over the circadian cycle, with a peak of 1718 ± 860 µg/mL at 7:00 PM, followed by a drop to 1461 ± 596 µg/mL at 3:00 AM. However, these fluctuations did not reach statistical significance (p > 0.05).

Our study revealed significant circadian fluctuations in some salivary components, including lactic acid, nitrate, nitrite, ammonium, and glucose. Lactic acid and glucose exhibited pronounced peaks in the early morning, while nitrate and nitrite levels peaked in the afternoon, particularly after lunch. Conversely, ammonium levels showed an inverse pattern, with higher concentrations observed at the beginning and the end of the 24-hour cycle. In contrast, calcium and phosphate levels, as well as total salivary proteins, remained relatively stable throughout the day, with no statistically significant variations. These findings suggest that while some salivary metabolites are influenced by circadian rhythms, others maintain consistent levels, highlighting the complex regulation of salivary composition over a 24-hour period. Previous studies have suggested that salivary components vary throughout the day due to multiple factors, including circadian rhythms. Saliva can serve as an indicator of local, systemic, and infectious disorders, making it an attractive tool for diagnosis and therapeutics [1, 4].

Lactic acid. The significant peak in salivary lactic acid levels in the morning may be attributed to various physiological and behavioral factors. Tékus et al. [19] demonstrated that lactic acid levels in saliva increase following maximum intensity exercise, highlighting the contribution of systemic metabolic activity to salivary lactate concentrations. While our study did not involve exercise, the early morning rise in lactic acid could similarly reflect heightened metabolic processes associated with waking. Additionally, the role of oral bacterial activity should not be overlooked. Bacterial metabolism, particularly before the first toothbrushing of the day and overnight, can lead to the production and concentration of lactic acid in saliva. It is well known that oral bacteria, especially cariogenic streptococci, are capable of producing significant amounts of lactic acidic, contributing to the overall lactic acid levels in the oral cavity [20].

Nitrate and nitrite. Nitrate (NO₃⁻) and nitrite (NO₂⁻) are key ions involved in various physiological processes, including vasodilation, antimicrobial activity, and the regulation of blood pressure through their role in the nitric oxide (NO) pathway. These ions are primarily derived from dietary sources, particularly nitrate-rich foods such as leafy greens and certain vegetables. The circadian fluctuations of NO₃⁻ and NO₂⁻ in saliva, as observed in our study, align with a previous study that showed that both ions significantly increased following a nitrate-rich meal and remained elevated for several hours post-consumption [21]. This dietary influence is evident in our results, where nitrate and nitrite concentrations peaked at 3:00 PM, shortly after lunch. These peaks suggest a strong diet-dependent modulation of these ions, highlighting the impact of meal timing and composition on their salivary concentrations. The postprandial rise in nitrate and nitrite may offer protective benefits, including enhanced antimicrobial activity in the oral cavity and the potential for systemic nitric oxide production, which has been linked to oral and systemic benefits [22].

Ammonium. Ammonium acts as a buffer, helping to neutralize acids produced by bacterial metabolism, thereby protecting against enamel demineralization and caries formation [23]. It is primarily produced through the deamination of amino acids by oral bacteria and the hydrolysis of urea by urease, an enzyme present in both dental plaque and saliva [24]. Our study observed an inverse circadian pattern, with ammonium levels peaking in the early morning and in the middle of the night, which may suggest that in the presence of low oral activity and the absence of food, ammonia levels are maintained, just reflecting natural metabolic cycles and the influence of fasting periods during sleep. Given the relationship between ammonia and caries, further research should expand these findings.

Salivary glucose. The circadian variation profile of glucose in saliva is particularly intriguing, with significant peaks at 7:00 AM and 3:00 AM the following day. This pattern partially aligns with the findings of Morris et al. [25] and Qian & Scheer [26], who reported that glucose tolerance and metabolism exhibit clear circadian rhythms, with improved glucose handling in the morning hours compared to the evening. The unexpected rise in salivary glucose at 3:00 AM may indicate the onset of preparatory metabolic processes gearing up for the upcoming wake period, a phenomenon also suggested by circadian biology studies highlighting early morning increases in glucose production and reduced insulin sensitivity [27]. This early morning surge could be attributed to hormonal fluctuations, such as increased cortisol secretion, known to elevate glucose levels in anticipation of energy demands upon waking. Since participants woke for saliva collection, a cortisol response could have been triggered, which could also explains glucose elevation.

Calcium and phosphate. Our analysis of the circadian rhythm's impact on the average concentrations of Ca²⁺ and PO₄³⁻ in saliva did not reveal statistically significant differences, suggesting that these ions are maintained consistently in the oral cavity throughout the day [28]. However, slight intra-individual fluctuations were observed, with a tendency for both analytes to increase during the morning and evening and decrease during the day. These variations align with reports of intra-individual differences in salivary inorganic composition [29–31]. A potential explanation for the slight variations in Ca²⁺ concentrations could involve the protein components of saliva. Most secreted Ca²⁺ is bound to proteins such as statherin, with a smaller proportion bound to acidic proline-rich proteins (PRPs) or cystatins [32]. These proteins play specific roles in maintaining oral homeostasis, potentially causing variations in the availability of these electrolytes according to individual physiological needs [33]. Our findings are consistent with Dawes et al. [29], who observed similar patterns, although Ferguson et al. [34] reported significant circadian effects on Ca²⁺ concentration in saliva obtained from the submandibular gland. The discrepancy may be due to differences in saliva collection methods and the type of saliva analyzed [35]. In our study, we opted for collecting whole saliva to have an overview of the actual fluid that will take contact with the hard and soft tissues of the mouth. PO₄³⁻ plays a crucial role not only in the oral cavity but also in systemic processes such as skeletal development, bone mineralization, and energy transfer [36]. Here, PO₄³⁻ concentrations showed a slight increase in the early morning hours, with a decrease during the afternoon and evening. This trend may be linked to salivary flow rates, which tend to decrease during sleep and increase during waking hours [37]. Contrary to our findings, previous studies have reported higher PO₄³⁻ levels during the day [29, 38], possibly due to differences in collection techniques and analytical methods, as well. Calcium and phosphate concentrations can be altered after sucrose rinses, suggesting a potential link between ion variability and sugars-associated cariogenic challenges [39]. The absence of significant circadian fluctuations in calcium and phosphate concentrations in our study could be attributed to the caries-free status of the volunteers.

Salivary pH. The pH and buffering capacity of saliva can alter the bioavailability of calcium and phosphate. In our study, pH levels remained relatively stable, fluctuating between 7.0 and 7.4 over the course of the day. This stability can be attributed to the robust buffering capacity of saliva, which is regulated by three primary systems: bicarbonate, phosphate, and proteins [3]. These buffering systems play a critical role in minimizing pH fluctuations within the oral cavity. The bicarbonate buffer system, which is predominant in stimulated saliva, is most active within the pH range of 5.0 to 7.0. In contrast, the phosphate buffer system, which is more prevalent in unstimulated saliva, operates effectively at a pH range of 7.0 to 7.26 [40]. The protein buffer system, however, is primarily active at acidic pH levels (pH 4.0 to 5.0). Given the pH range observed in our samples, it is likely that the phosphate system was the principal buffering mechanism, complemented by the bicarbonate system.

Total Salivary Proteins. Although extensive research has explored the protein components present in saliva, the relationship between these proteins and circadian fluctuations remains underexplored. Daily fluctuations in protein concentration have been reported, suggesting that salivary protein levels can vary depending on the method of collection and the time of day [30]. In a more recent metabolomics study, substantial daily fluctuations in amino acids ranged from 20–200% [41]. These findings suggest that while specific components of saliva may exhibit circadian variation, the overall impact on total protein levels might be less pronounced. In our study, total salivary protein concentrations displayed minor fluctuations throughout the day, with a peak in the evening and a slight reduction during the night. However, these variations did not reach statistical significance (p > 0.05), suggesting that proteins play a constant role in oral homeostasis, including lubrication, antimicrobial activity, and buffering capacity. While the slight fluctuations observed could be attributed to circadian influences, they do not follow a clear or consistent pattern. Therefore, it is important to recognize that these variations might simply reflect natural, non-significant daily oscillations rather than a defined circadian rhythm.

Study limitations and implications. One of the primary limitations is the pilot nature of this study. A limited number of participants reduces the generalizability of the findings and increases the risk of statistical anomalies. Various factors, such as hormonal fluctuations, stress, or sleep disturbances, could have influenced the volunteers on these specific days, potentially skewing the results. To obtain more reliable and generalizable results, future studies should consider more participants under consistent conditions. Furthermore, the study analyzed saliva samples collected over just three independent days. While this approach provides a snapshot of circadian variations, it may not be sufficient to establish reproducible trends over time. Another limitation concerns the standardization of the diet provided to the volunteers. Although a standard diet was provided to minimize its influence on the salivary variables analyzed, it was standardized based on general population data, assuming light physical activity and average nutritional requirements. However, individual caloric needs can vary significantly, and the lack of personalized nutritional assessments may have compromised the effectiveness of this standardization. As dietary intake can significantly impact salivary composition, a more individualized dietary approach would likely enhance the accuracy and relevance of the findings. Additionally, the viscous nature of saliva, due to mucopolysaccharides and mucoproteins, posed challenges for analytical measurements, potentially decreasing the accuracy of the measurements [28]. To mitigate this, saliva samples were centrifuged and filtered to remove components that could interfere with the accuracy of the determinations. Despite these precautions, the inherent variability and complexity of saliva as a biological fluid may still have introduced measurement inconsistencies.

Understanding how circadian rhythms affect salivary composition could improve the use of saliva as a non-invasive diagnostic tool, especially for conditions with circadian patterns. Its easy collection and biochemical diversity make saliva ideal for daily health monitoring. While our study was conducted on healthy individuals, people with certain diseases might show greater circadian variations in saliva, which could offer valuable insights for diagnosis and treatment.

Circadian rhythm appears to have a limited impact on the overall biochemical composition of unstimulated saliva in young, healthy adults, but a potential role for selective substances that warrant further investigation.

Declaration of Conflicting Interests

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethics approval

The study design and protocol were approved by the Scientific Ethics Committee of the University of Talca (ACT Nº 14/2021), and the study was performed in accordance with the postulates of the Declaration of Helsinki and international ethical guidelines for biomedical research in humans CIOMS 2016.

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Funding and Acknowledgments

This study was funded by a Chilean Government Grant ANID/Fondecyt 1210188 to RAG. The funder had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript. This manuscript was submitted in partial fulfilment of the requirements for the graduation from the Dental School, Faculty of Dentistry by M. Antonieta Albornoz-Bravo, Albert Sobarzo-Vergara, J. Francisco Reyes-Parraguez and M. Elisa Valenzuela-Aravena at the University of Talca. The authors thank their work and insights. The authors also thank to Javiera Villalobos, Denisse Echeverría and Felipe Muñoz for their collaboration in the design and data analyses.

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The authors declare no competing interests.

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Circadian Influence on Saliva Biochemical Composition: A Pilot Clinical Investigation

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