Effect of 1% H₂O₂ on Three Salivary Stress Biomarkers, Cortisol, Alpha-Amylase, and sIgA

Abstract

Background

Due to the COVID-19 pandemic, several associations worldwide have been recommending the use of 1% hydrogen peroxide solution as a preprocedural mouth rinse before dental treatments to reduce viral load in saliva. This protocol is also employed in stress studies, especially in the context of dental treatment that uses salivary biomarkers as an indicator. However, the effect of 1% hydrogen peroxide as mouth rinse on salivary biomarkers remains unclear.

Objective

This study aims to investigate the effects of 1% hydrogen peroxide solution as a preprocedural mouth rinse on 3 salivary stress biomarkers—salivary cortisol, salivary secretory IgA, and salivary α-amylase—both on chemical influence and mechanical irrigation.

Materials and methods

Ninety healthy participants with confirmed negative Reverse Transcription Polymerase Chain Reaction results for COVID-19 at most 2 days prior to the experiment were included in this study. All participants were randomly allocated into 3 groups: experimental (1% hydrogen peroxide solution), positive control (distilled water), and negative control (no mouth rinse). Saliva samples were collected before and after mouth rinsing with the respective solutions. Salivary biomarkers were analysed using specific enzyme-linked immunosorbent assay kits.

Results

Salivary cortisol and salivary α-amylase did not significantly differ before and after rinsing, whilst salivary sIgA levels decreased in all 3 groups. Nonetheless, there were no significant differences in the changes of these biomarkers across the 3 groups.

Conclusions

This study shows that using 1% hydrogen peroxide solution as a preprocedural mouth rinse for universal precaution does not alter the levels of these 3 salivary biomarkers.

Introduction

Starting in December 2019, COVID-19 spread from Wuhan, China,^1,2 and became a pandemic and a health crisis as declared by the World Health Organization (WHO).³ Possible symptoms of COVID-19 include fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle aches, headache, loss of taste or smell, sore throat, congestion, nausea and vomiting, and diarrhoea.⁴ In August 2020, 19.9 million confirmed cases and more than 730,000 mortalities due to COVID-19 were reported.⁵ The cumulative COVID-19-infected cases drastically rose to more than 111 million worldwide in February 2021.⁵ Routes of transmission of COVID-19 are direct and nondirect contact with virus-containing secretions, such as saliva and respiratory secretions or their droplets⁶ and aerosols.⁷

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is the agent of COVID-19, which is a positive-sense single-stranded enveloped RNA virus that belongs to the betacoronavirus genus from the Coronaviridae family.^8,9 The primary receptor for SARS-CoV-2 is angiotensin-converting enzyme 2 (ACE2), which plays an important role in viral attachment and entry mechanisms. In addition to the lungs, ACE2 receptors can be found in a variety of other human tissues.¹⁰ It is abundant in oral epithelial cells,¹¹ particularly on the dorsum of the tongue.¹² Moreover, the concentration of ACE2 receptors in the minor salivary glands is higher than in the lungs.¹² These findings suggest that the oral cavity has become a high-risk route for SARS-CoV-2 infection. It has been demonstrated that SARS-CoV-2 could remain in the oral cavity for up to 2 days before infecting the lower respiratory tract.¹³ Viral load in the saliva is found within the early stage of infection,¹⁴ especially during the first week following symptom onset,¹⁵ and the viral load can be detected asymptomatically for a prolonged period.^16,17 A cough or 5 minutes of normal conversation produces around 3000 droplets, but a sneeze produces more than 40,000 droplets that can disperse several meters into the air.¹⁸ However, the route of infection via aerosols can produce more particles and splash further than sneezing, especially in aerosol-generating procedures (AGPs) of health care facilities,¹⁹ including extubation, intubation, tracheotomy, positive pressure ventilation (CPAP), bronchoscopy, and cardiopulmonary resuscitation.²⁰ Therefore, dental procedures are considered high-risk due to direct contact with high–viral load secretion and saliva and the procedures themselves generate aerosols, such as the use of ultrasonic devices, 3-in-1 air-water syringes, high-speed handpieces, air polishing, and air abrasion.²¹

Prevention of virus transmission during daily life is the primary strategy to break the chain of COVID-19 transmission. The WHO has guided community members on new lifestyle recommendations such as physical distancing, mask-wearing, and hand cleaning.²² One of the najor preventive dental protocols that has been widely accepted is the reduction of viral load in the saliva. Dental associations and centers for disease control and prevention in several countries worldwide, including the United States,^23,24 Australia,²⁵ and Germany,²⁶ have recommended the use of mouth rinse as a preprocedural step before dental procedures. Several studies have suggested that mouth rinses have the potential to reduce viral load in saliva after rinsing for at least 30 seconds.^9,27, 28, 29 At the same time, some studies have claimed that mouth rinses do not show a noticeable effect on viral load reduction.^26,30,31 Those studies have explained that mouth rinses may inhibit viral infection activity, but Reverse Transcription Polymerase Chain Reaction can detect residual RNA, indicating that viral load remains present. Povidone-iodine (PVP-I) and hydrogen peroxide (H₂O₂) are highlighted amongst the candidate active ingredients or compounds that have been screened and shown a virucidal effect on SARS-CoV-2.^9,32 In this study, H₂O₂ was selcted for its effect on three salivary biomarkers.

In diluted form, H₂O₂ is a colourless chemical compound and an odourless water-soluble liquid. It shows an antimicrobial effect and has proved to be effective against numerous human viruses, including coronavirus and influenza viruses.³³ H₂O₂ targets the viral lipid envelope, particularly that of SARS-CoV-2.³⁴ It releases oxygen-free radicals and disrupts the lipid membrane,³⁵ and the oxidation properties of H₂O₂ cause viral particles to degrade.³⁶ H₂O₂ is commonly used to reduce plaque and improve recovery after oral surgery.³⁷ A low concentration of H₂O₂ is sufficient to demonstrate antiviral activity; a study found that 0.5% concentration revealed moderate antiviral activity without severe cell death,⁹ and 1% H₂O₂ may effectively reduce viral load in the oral cavity.³⁸ H₂O₂ has been confirmed to be safe when used at 3% concentration for 6 months continuously³⁹ or 6% concentration^37,40, 41, 42; hence, 1% H₂O₂ can be harmlessly utilised as a preprocedural mouth rinse prior to dental surgical procedures.

Preventive measures should be taken during dental procedures that involve contact with the oral cavity, which is a high-risk area for infection, and during other processes involving close contact with this area, such as collecting saliva samples to detect biomarkers. Because the collection of saliva samples is an essential process in research and studies, particularly in the dental field, precise standard protocols must be followed to prevent viral transmission. Measuring stress levels during dental operations using salivary biomarkers as indicators is an experiment that entails saliva collection and performing dental procedures. The main salivary biomarkers for stress detection are salivary cortisol, salivary α-amylase, and salivary secretory IgA (s-IgA), which can be observed at various intervals during periods of stress.

Salivary cortisol measurement has been extensively studied as an indicator of stress.^43,44 Salivary cortisol concentration is directly proportional to the concentration of unbound cortisol in serum.⁴⁵ Cortisol level increases in response to physical or emotional stress through the hypothalamic-pituitary-adrenal axis. Approximately 5% of cortisol in the bloodstream is free unbound cortisol and is transported to the salivary glands via acinar cells, where it becomes salivary cortisol.⁴⁶ Salivary cortisol levels have been validated as a viable measure of active, free cortisol and a potential biomarker of overall stress.⁴⁷ Salivary cortisol is a hormonal stress response, whereas s-IgA is an immune stress response and can be utilised as a marker of overall state of the immune system.⁴⁸ Several investigations have reported that s-IgA levels increase in acute stress situations49, 50, 51, 52, 53 but decrease in long-term stress.⁵⁴ s-IgA is modulated by a distinct pattern of autonomic activation.⁵⁵ Finally, α-amylase is considered a novel stress biomarker regulated by the sympathetic adrenomedullary system (SAM).56, 57, 58 In conclusion various studies have confirmed that changes in the levels of the foregoing biomarkers occur due to stressful situations.^47,57,59,60

In summary, the COVID-19 pandemic has necessitated changes in daily life, including health care practices. Dental health care providers must take precautions to minimise the risk of transmission by using mouthwash as a preprocedural step to reduce the viral load in saliva. The most recent guidelines states that preprocedural mouthwash by rinsing with 1% H₂O₂ must be done prior to any procedures. Several studies have reported that using H₂O₂as a preprocedural mouthwash can reduce the viral load in saliva, suggesting that preprocedural mouthwash could also impact salivary biomarker measurement and potentially lead to changes in research involving salivary biomarker and biosensor development using saliva as an analyte following the guidelines. To our knowledge, there has been no study on the effects of preprocedural mouthwash on salivary biomarkers. Therefore, this study was conducted to provide insights into the utilisation of salivary biomarkers for research in the post–COVID-19 era and aims to investigate the effects of 1% H₂O₂ solution on 3 salivary biomarkers: salivary cortisol, salivary α-amylase, and salivary s-IgA, with regard to physical irritation and chemical influence.

Materials and methods

Participants

In this study, 90 healthy participants comprising 40 males and 50 females with negative RT-PCR results for COVID-19 within 2 days before the experiment were enrolled. The mean age of the participants was 32 years, with a standard deviation of 8.8 years. Prior to their inclusion in the study and saliva collection, all participants were informed about the objective and procedures of the study and provided their informed consent through a consent form. All procedures involving human participants in this study had been approved by the Institutional Review Board of Faculty of the Dentistry/Faculty of Pharmacy, Mahidol University, with COA No. MU-DT/PY-IRB 2019/019.1004 amendment in April 2021.

Sample size estimation

To estimate the required sample size, the G*Power program⁶¹ was employed. A significance level (α) of 0.05 was selected, which reflects the accepted threshold for Type I error. Additionally, power of the test (1-β) of 0.80 was chosen, signifying the desired probability of correctly rejecting a null hypothesis. Upon the comparison between before and after, 2-tailed paired t test must be conducted, so it was set in the sample size estimation. Sample size calculation was independently performed for each of the salivary biomarkers under investigation, and the larger of these sample sizes was adopted as the estimated sample per group. The effect size, variability, and the estimated sample size of each salivary biomarker were as follows: salivary cortisol⁶² (effect size: 0.65, variability: 0.28, estimated sample size: 4), salivary s-IgA⁶³ (effect size: 6.00, variability: 7.85, estimated sample size: 16), and salivary α-amylase⁶⁴ (effect size: 10.60, variability: 3.32, estimated sample size: 4). As the consequence of this estimation process, it was initially determined that 16 participants per group would be sufficient. Nevertheless, in accordance with the principles of the central limit theorem, it is generally accepted that sample sizes equal to or greater than 30 are often considered sufficient for assumption of the theorem, regardless of the population's distribution. Consequently, for this study 30 participants per group were desired, leading to a total of 90 participants being enrolled in the investigation. Statistical robustness and adherence to academic standards were ensured.

Allocation concealment

Prior to undergoing the saliva collection process, participants were randomly assigned to 1 of 3 groups based on numeric representations. Simple randomisation was applied to each ordered numeric representation in a group allocation procedure using Microsoft Excel. Even distribution of 3 groups was assigned in the numeric representation. The list of group allocation was revealed by relevant officials in the consent process and then provided representative letter “A,” “B,” and “C” together with participant numerical representation before underwent into the experimental room. The experimental room for saliva collection was a negative-pressure room isolated from the dental unit. Each participant had to give the representative letter to the researcher in the experimental room to receive a container that contained the solution corresponding to the representative letter: “A” for 1% H₂O₂, “B” for distilled water, and “C” for no container. After the saliva collection process, the container used to collect the saliva was labelled with participant numeric representations and then brought to the laboratory assessor without identification. With this concealment, participant allocation was identified from numeric representations as “A” for the experimental group, “B” for the positive control group, and “C” for the negative control group in the statistical analysis process.

Saliva collection

All participants were randomly assigned to 3 groups as mentioned: experimental group (G1) with an average age of 32.5 years (SD = 8.7 years), positive control group (G2) with an average age of 32.5 years (SD = 8.9 years), and negative control group (G3) with an average age of 31 years (SD = 9.6 years). All 3 groups underwent the same saliva collection procedure but differed in the mouth rinse solution used. Initially, participants rinsed with 15 mL of water for 30 seconds before saliva collection using a sterile container and the saliva collection aid (Salimetrics) to collect at least 3 mL of unstimulated saliva without timing. Participants were then given a 5-minute rest, after which they rinsed their mouth for 30 seconds with 20 mL of the assigned solution for their group: 1% H₂O₂ solution for G1 to investigate its effect on salivary biomarkers, distilled water for G2 to compare mechanical irrigation effects on salivary biomarkers, and no solution for G3 as the negative control. Distilled water was chosen instead of normal saline solution to ensure that any change in the activity of α-amylase was not due to taste stimulation. Following the rinsing procedure, saliva was collected again using the same method as the initial collection. Figure 1 depicts the saliva collection procedure.

Fig. 1.

Experimental procedure.

The collected samples were promptly transferred to an insulated container with ice, labelled with the participant's code, and transported to the laboratory within 30 minutes. Upon arrival, all samples underwent centrifugation at 3000 rpm for 10 minutes at 4 °C. The supernatant was then transferred to a microtube 1000 µL with 100 µL of protease inhibitor solution (Roche) and stored at −80 °C for further analysis.

Salivary biomarker detection

Biomarkers for stress detection in this study are salivary cortisol, salivary α-amylase, and salivary s-IgA, which were analysed as follows:

Salivary cortisol concentration was determined from the prepared samples using Cortisol Enzyme Immunoassay Kit (Salimetrics^®, Lot 1904502), which is a competitive immunoassay kit specifically designed for saliva cortisol measurement.

Salivary s-IgA concentration was determined from the prepared samples using sIgA Indirect Enzyme Immunoassay Kit (Salimetric^®, Lot 1901529), which is an indirect competitive immunoassay kit.

Salivary α-amylase activity was investigated from the prepared samples using α-Amylase Kinetic Enzyme Assay Kit (Salimetrics®, Lot 1904503), which measures absorbance at 405 nm between 2 time points.

Statistical analysis

Paired t test was conducted separately on salivary cortisol concentration, salivary α-amylase activity, and salivary s-IgA concentration to compare between preintervention and postintervention time points for each group. A P value < .05 was considered significant. Subsequently, 1-way analysis of variance (ANOVA) was performed to compare differences in the change of these levels and activities between the groups. A P value < .05 was also considered significant. Finally, post hoc analysis was conducted to evaluate different pairs if the ANOVA indicated significant differences.

Results

Salivary cortisol concentrations

The paired t test revealed no significant differences in salivary cortisol concentration between preintervention and postintervention (Figure 2). The concentration of salivary cortisol was reported as mean ± SD in μg/dL. For the experimental group (G1), the preintervention period showed a concentration of 0.1031 ± 0.0327 μg/dL and the postintervention period displayed 0.1057 ± 0.0419 μg/dL, with a mean difference of 0.0026 ± 0.0396 μg/dL (P = .7207). Similarly, for the positive control group (G2), the preintervention period exhibited 0.2263 ± 0.2159 μg/dL whilst the postintervention revealed 0.2180 ± 0.2049 μg/dL, resulting in a mean difference of −0.0083 ± 0.0659 μg/dL (P = .4957). For the negative control group (G3), concentrations were 0.1903 ± 0.1406 μg/dL in the preintervention period and 0.1848 ± 0.1576 μg/dL in the postintervention period, with a mean difference of −0.0055 ± 0.0923 (P = .7457).

Fig. 2.

Jitter plot of salivary cortisol concentration of all groups (blue dot shows preintervention time point, orange dot shows postintervention time point). Paired t test analysis determines the difference between 2 time points within each group. No significant difference was observed.

Salivary s-IgA concentrations

The paired t test revealed significant differences in salivary s-IgA concentration between preintervention and postintervention in all 3 groups (Figure 3). The concentration of salivary s-IgA was reported as mean ± SD in μg/mL. For G1, the preintervention period showed 239.9039 ± 146.1829 μg/mL and within the postintervention period displayed 203.2429 ± 117.2587 μg/mL. The mean difference of G1 was −36.6610 ± 73.4033 μg/mL (P = .0105*), which showed statistical significance. Likewise, for G2, preintervention concentration was 155.5997 ± 54.7395 μg/mL and post-intervention concentration was 128.0476 ± 48.4566 μg/mL, resulting in a mean difference of −27.5521 ± 30.4623 μg/mL (P < .0001*), signifying significance. For G3, preintervention concentration was 375.8842 ± 160.3208 μg/mL and postintervention concentration was 333.0587 ± 178.8998 μg/mL. The mean difference for G3 was −42.8255 ± 110.0151 μg/mL (P = .0416*) indicating statistically significant difference.

Fig. 3.

Jitter plot of salivary secretory IgA concentration of all groups (blue dot shows preintervention time point, orange dot shows postintervention time point). Paired t test analysis determines the difference between 2 time points within each group. *indicates significant difference.

Salivary α-amylase activity

The paired t test revealed no significant differences in salivary α-amylase activity between preintervention and postintervention for any of the 3 groups (Figure 4). The activity of salivary α-amylase was demonstrated as mean ± SD in U/mL. For G1, preintervention activity was 288.4610 ± 160.8617 U/mL and postintervention activity was 287.0656 ± 156.6171 U/mL, with a mean difference of −1.3954 ± 43.4409 U/mL (P = .8616). Similarly, for G2, preintervention activity was 213.3421 ± 127.3443 U/mL and postintervention was 196.2752 ± 100.1038 U/mL, resulting in a mean difference of −17.0669 ± 54.8173 U/mL (P = .0988). For G3, preintervention activity was 278.6360 ± 128.8281 U/mL and postintervention activity was 290.3347 ± 130.2117 U/mL, with a mean difference of 11.6987 ± 89.9493 U/mL (P = .4819).

Fig. 4.

Jitter plot of salivary α-amylase activity of all groups (blue dot shows preintervention time point, orange dot shows postintervention time point). Paired t test analysis determines the difference between 2 time points within each group. No significant difference was observed.

Comparison of the mean differences amongst the 3 groups

One-way ANOVA revealed no significant difference in the change of these levels or activity amongst the 3 groups (Figure 5). Mean differences in all groups were utilised for statistical analysis. Specifically, for salivary cortisol, there was no significant difference observed amongst the 3 groups (G1 = 0.0026, G2 = −0.0083, G3 = −0.0055, F = 0.2004, P = .8188). Likewise, salivary s-IgA concentrations showed no significant difference amongst the groups (G1 = −36.6610, G2 = −27.5521, G3 = −42.8255, F = 0.2885, P = .7501). Similarly, salivary α-amylase activity did not exhibit a significant difference amongst the 3 groups (G1 = −1.3954, G2 = −17.0669, G3 = 11.6987, F = 1.4379, P = .2430).

Fig. 5.

Comparisons of the changes between preintervention and postintervention of each salivary stress biomarker by one-way analysis of variance. The bar charts show the average of the changes with standard error. No significant difference was observed.

(Left: salivary cortisol; middle: salivary sIgA; right: salivary α-amylase).

Discussion

The effects of 1% H₂O₂ mouth rinse on 3 salivary stress biomarkers (cortisol, s-IgA, and α-amylase) have been investigated in this study. Pre and post -intervention salivary samples were collected from 3 groups of participants—experimental (1% H₂O₂ solution), positive control (distilled water), and negative control- were compared. The positive control was used to assess mechanical irritation, whilst the negative control was used to establish a baseline. The experimental group was compared to both controls to examine the chemical and mechanical effects.

Results revealed that 1% H₂O₂ mouth rinse has no chemical or mechanical effect on salivary cortisol, s-IgA, or α-amylase activity. Concentrations of salivary cortisol and α-amylase were consistent pre- and postintervention, and although s-IgA differed, there was no statistically significant difference amongst the 3 groups.

The decrease in salivary s-IgA concentrations across all groups might be due to acute stress during the saliva collection procedure. The collection was performed in an experimental room, which was different from the normal environment of each participant and could induce stress. Previous research has suggested that acute stress caused by environmental changes can result in an increase in s-IgA synthesis as an adaptive response by the body.⁶⁵ One study reported that the increase in s-IgA concentration occurred within the first 15 minutes of acute stress and was reduced to baseline levels, thereafter; although salivary cortisol levels increased and were mainatained due to continued stress.⁵⁴ In our study, acute stress might occur during the first saliva collection during adaptation to a new environment, whilst the levels of salivary cortisol may not increase in the absence of stress. Therefore, salivary s-IgA concentration dropped from the preintervention time point to the post intervention time point.

Statistical analysis indicated no significant differences in salivary cortisol concentration amongst the groups, suggesting that the use of 1% hydrogen peroxide solution does not affect this biomarker. Salivary cortisol concentration typically increases in response to stress, usually 30 minutes after the onset of stress.⁵⁴ However, there was no difference in salivary cortisol concentration between pre-and postintervention, which is inconsistent with an increase in salivary s-IgA caused by acute stress. One possible reason is that the participants may have adapted to the environment, leading to a decrease in stress level over time.

When exposed to hydrogen peroxide, salivary s-IgA and salivary α-amylase induces increased proteolytic activity. However, no such effects were observed in this study. This may be due to the poor sensitivity of the test we used and or the duration of exposure to the H₂O₂ solution.

Moreover, based on our findings, it can be concluded that mouth rinsing with 1% H₂O₂ solution has no significant effect on salivary biomarkers, including both chemical influence and mechanical irrigation. Therefore, it can be safely used as a preprocedural step to reduce the SARS-CoV-2 viral load, which complies with the recommendations of several organisations. This finding is particularly important for stress research.

However, it is not entirely clear why 1% H₂O₂ mouth rinse had no effect on salivary cortisol in the study. It is possible that the concentration or duration of exposure to the H₂O₂ solution was insufficient to cause a measurable effect on cortisol levels. Additionally, individual variations in cortisol response to stress may have also played a role. Further research is required to completely understand the relationship between H₂O₂ mouth rinse and salivary cortisol levels.

The significance of this study primarily benefits researchers, who are focussing on the relationship between salivary biomarkers and other related clinical signs and symptoms and biosensor development using saliva as an analyte for point-of-care monitoring sensors. Salivary biomarkers such as salivary cortisol, salivary s-IgA, and salivary α-amylase are not typically evaluated in clinical dental practice, making our study more research-oriented. Moreover, this research is valuable for biosensor development, including those designed for use in dental practice. Future development of accurate biosensors could have the potential for the prediction of either disease progression or prognosis, investigation of connections between salivary biomarkers and relevant clinical signs and symptoms.

The limitation of this study is that only 3 salivary stress biomarkers, namely salivary cortisol, salivary s-IgA, and salivary α-amylase, were examined. Whilst the lack of effects on these biomarkers may suggest no impact, further investigations for other biomarkers are necessary to draw definitive conclusions.

Conclusion

The study aimed to investigate the effects of preprocedural mouth rinse with 1% H₂O₂ solution on 3 salivary stress biomarkers: salivary cortisol, salivary s-IgA, and salivary α-amylase. The findings of the study revealed that mouth rinsing with 1% H₂O₂ solution had no effect on these 3 salivary stress biomarkers. Therefore, using 1% H₂O₂ as a preprocedural step to reduce SARS-CoV-2 viral load is compatible with research involving the development of biosensors using saliva as an analyte.

Conflict of interest

None disclosed.