Assessment of Salivary Cortisol Concentrations as a Level of Stress Indicator among Individuals Undergoing Dental Extraction Procedure.

BACKGROUND: Anxious patients tend to exaggerate the aversive events before the actual dental therapy. The anxiety and fear are the major factors that regulate cortisol levels. AIM: Our study aims to estimate the salivary cortisol among patients subjected to dental extraction and correlate any existence between pre- and post-extraction cortisol levels and vital parameters compared to the control group.
METHODOLOGY: The study sample included sixty individuals, thirty patients indicated for a dental extraction, and thirty healthy patients between the ages of 18 and 60 years. We collected saliva samples from the control group and pre- and post-extraction samples from the study group participants and vital parameters. We used quantitative ELISA Kit, Diagnostics Biochem Canada Inc. (DBC), to estimate the salivary cortisol level. We evaluated the values using the Chi-square test, Pearson correlation test, and paired t-test using the JMP 15; JMP Pro, Version 15 (SAS) Microsoft® Windows® for × 64.
RESULTS: The mean value of cortisol concentration of saliva was significantly greater in the preextraction group (6.13 ± 0.53 μg/dl) than after extraction group (3.17 ± 0.14 μg/dl) (P < 0.001). There were no significant associations between hemodynamic parameters and salivary cortisol concentration except for the postextraction systolic blood pressure (BP). A comparison of hemodynamic parameters between the study and control group revealed substantial differences in systolic BP.
CONCLUSION: The patient's anticipation and anxiety toward dental therapy elevate the cortisol level. Dental surgeons should treat the patients more conveniently and effectively without any strains and provide a comfortable atmosphere to avoid stress-related consequences. Copyright:

INTRODUCTION

The cortisol hormone is a secretion from the adrenal gland in the zona fasciculate of the adrenal cortex.[1] The hypothalamus combined effort, pituitary, and adrenal gland (hypothalamic-pituitary-adrenal [HPA] axis) control cortisol secretion.[23] Cortisol hormone influences the processes of metabolism, immunology, and anti-inflammatory actions. The hormone maintains a circadian rhythm.[4] Dental therapy in anxious patients elevates stress levels.[1256] Stress response excites the HPA axis to secrete cortisol, which in turn brings changes in metabolism, vascular reactivity, includes heart rate (HR), blood pressure (BP), and oxygen saturation (O2) and affects the sensitivity of the nervous system.[7]

METHODOLOGY

We selected sixty participants from the outpatient department to take part in the present study. We included thirty patients indicated for extraction in the study group, and thirty sex-and age-matched healthy volunteers with no systemic complications partaken in the control group. The study inclusion criteria were healthy, nonsmoking, and nondrinking patients who are between the age of 18 and 60 years and required tooth extraction. Exclusion criteria: we excluded the patients with any local infection, swelling, or pus discharge. The patients with any systemic illness (metabolic and endocrine); who were medically compromised; patients using any medication or supplementation, especially corticosteroids and oral contraceptives; patients on chemotherapy or radiotherapy; and patients on therapy for mental illness were all excluded from the study.

Furthermore, we excluded the long-term, unemployed patients, since they would have already stressed due to work pressure or other lifestyle factors, from the research that may influence the body cortisol levels. We have obtained approval from the institute's Human Research Ethical Committee to proceed with the study. We received informed consent from the participants of both groups. The experimental group had 17 females and 13 males, and the control group had 15 in both genders.

We advised the patient to rinse the mouth with water to clear off all food debris from the oral cavity just 5 min before the salivary sample collection. We assessed the metabolic factors such as BP, HR, and oxygen saturation (O2). We performed all the procedures between 9 am and 12 noon to circumvent cortisol level alteration due to the circadian rhythm. Then, 2 ml of unstimulated saliva is collected from the patient by asking the patient to spit in a disposable container. Then, we transferred the collected salivary sample to a sterile plastic vial through a disposable syringe and labeled for identification.

We repeated the assessment of the metabolic parameters after the extraction procedure. We collected the second salivary sample of unstimulated saliva 20 min after the extraction with appropriate care to prevent blood contamination. We discarded the saliva with visually deductible blood contamination. Then, we transferred the samples without blood contamination to another sterile plastic vial and labeled.

We centrifuged the collected salivary samples immediately at 8000 rpm for 10 min. We stored the clear supernatant final samples at −20°C deep freezer.

We transported the salivary samples to the laboratory for the experimental procedure. We utilized the kit manufactured by DBC to assess the salivary cortisol levels with a lower sensitivity of 1 ng/mL.

Before starting the test, all the samples and reagents were brought to room temperature and were kept ready to use. We prepared the cortisol- Horseradish peroxidase (HRP) conjugate solution and buffer for readily use. The required number of coated wells were secured in the holder. Fifty microliters of salivary control and study samples were dispensed into the correspondingly labeled wells using a multichannel pipette. One hundred microliters of cortisol enzyme conjugate were discharged into each well. The sample plate was then incubated for 45 min on a plate shaker (approximately 200 rpm) at room temperature. We removed the sample plate and rinsed the wells three times with 300 μL of the diluted buffer. The sample plate was taped against tissue paper to make it desiccated. Then, 3,3',5,5'-Tetramethylbenzidine solution of 150 μL was dispensed into the wells. The sample container was incubated for 20 min at room temperature on a plate shaker. The experiment was then terminated by administering 50 μL of 2N HCL to each well, and immediately, the optical density was read at 450 nm with a microwell processing reader. We recorded the values. Then, we tabulated the values and statistically analyzed the data by the Chi-square test, Pearson correlation test, and paired t-test to obtain the significance and test the hypothesis. We analyzed the examined groups' data using the statistical package JMP®: JMP Pro, Version 15 Soft ware, Microsoft® Windows® for x 64; SAS Institute Inc., Cary, North Carolina, USA, 1989-2019. for × 64.

RESULTS

Comparing the experimental and control group's hemodynamic parameters revealed substantial differences in systolic BP before and after extraction [Table 1].

Table 1

Comparison of hemodynamic parameters of the study group and control group

	SBP	DBP	HR	SpO2
Before extraction and control group	0.0003*	0.981	0.999	0.999
After extraction and control group	0.0003*	0.999	0.998	0.999
Preextraction and postextraction group	0.995378	1	1	1

*Significant at probability <0.001 level. SBP: Systolic blood pressure, DBP: Diastolic blood pressure, HR: Heart rate, SpO2: Oxygen saturation

Our study shows increased preextraction salivary cortisol concentration in the study group. The preextraction mean salivary cortisol concentration was significantly higher (6.13 ± 0.53) than postextraction (3.17 ± 0.14) in the study group [Table 2 and Figure 1].

Table 2

Salivary cortisol values for the control group and experimental group

Group (n)	Mean	SD	SEM	Mean
				Upper 95%	Lower 95%
Control (30)	4.4063333	0.4344119	0.0793124	4.5685454	4.2441213
Preextraction (30)	6.126	0.532668	0.0972514	6.3249015	5.9270985
Postextraction (30)	3.1676667	0.1412428	0.0257873	3.2204076	3.1149257

SD: Standard deviation, SEM: Standard error of mean

Figure 1

Mean values of the cortisol in the study and the control groups

The present study did not display the significant correlation between salivary cortisol levels and vital parameters except the postextraction systolic BP [Table 3].

Table 3

Comparison of the hemodynamic parameters with salivary cortisol level in the study groups

	SBP	DBP	HR	SpO2
Before extraction (n=30)
Pearson correlation	0.322	0.190	0.289	-0.300
P	0.083	0.3156	0.121	0.107
After extraction (n=30)
Pearson correlation	0.363	0.213	0.147	0.174
P	0.048*	0.259	0.439	0.358

*Significant at probability<0.05 level. SBP: Systolic blood pressure, DBP: Diastolic blood pressure, HR: Heart rate, SpO2: Oxygen saturation

There was significant difference noted when comparing the experimental and control group salivary cortisol levels (P < 0.01) [Table 4]. There was also, statistical significance exists between the pre- and post-extraction levels of the study groups (P < 0.001) [Table 5]. Normal quantile plots displayed the cortisol level distribution data and the median cortisol value of the pre- and post-extraction study groups [Figures 2 and 3].

Table 4

Comparison of pre- and post-dental extraction salivary cortisol concentration (μg/dl) with the control group

Group description	Study group			Control group (n=30)			P
	Mean	SD	SEM	Mean	SD	SEM
Preextraction (n=30)	6.13	0.53	0.097	4.41	0.43	0.079	<0.001
Postextraction (n=30)	3.17	0.14	0.026				<0.01

SD: Standard deviation, SEM: Standard error of mean

Table 5

Comparison of pre- and post-extraction salivary cortisol difference estimation of paired differences

Mean	SD	SEM	95% CI for μ_difference	T-score	Correlation (r)	df	P
2.9583	0.4001	0.0730	2.8089-3.1077	40.50	0.95	29	0.00001*

*Significant at P < 0.0001 level. df: Degrees of freedom, SD: Standard deviation, SEM: Standard error of mean, CI: Confidence interval

Figure 2

Box and whisker plot and normal quantile plot for preextraction cortisol levels

Figure 3

Box and whisker plot and normal quantile plot for postextraction cortisol levels

DISCUSSION

Cortisol is primarily 90%–95% protein bound (transcortin) in serum and usually measured by free cortisol analyzes in serum. Under various clinical settings, the concentration of protein-bound cortisol level changes, and thus, the intensity of overall serum cortisol varies.[89] The unbound fraction increases at levels that surpass the transcortin saturation on specific conditions. In those situations, the serum cortisol level serves as a tool to assess the clinical condition's impact. However, it is very expensive and more time consuming and is not appropriate for routine clinical needs.[10] Saliva is a blood spiegel. Saliva is an admirable reservoir for cortisol.[6] Salivary cortisol levels correlate strongly with the biologically active “free” fraction in serum.[47] The salivary cortisol concentration is independent of the flow rate, mucous, and serous saliva content.[11] The noninvasive design with a more effortless collection technique with lower cost makes salivary sampling a beneficial assay to assess rapid changes in endogenous cortisol levels with accuracy and high patient acceptability in stress assessment studies.[712]

The fear and discomfort play a crucial role in day-to-day dental practice but are often underestimated.[7] The literature research pointed out that highly anxious patients tend to overestimate the intensity of the expected pain and aversive dental events even if they have never experienced such a specific previous experience.[1314] Many studies conveyed that apprehension before the local anesthesia administrations and consequent uneasiness toward dental extractions confirm a wide variety of stress-related physical and psychological difficulties that lead to more stress the actual procedure. Thus, dental treatment anticipation acts as a vital stimulus for cortisol released by the adrenal cortex and subsequently increases serum cortisol level.[115] The changes in the cortisol level indicate the amount of stress. The substantial increase in preextraction salivary cortisol concentration specifies the patient's strain toward dental therapeutics.[16] An increase in cortisol, especially in patients with preexisting systemic diseases, raises blood glucose levels, delays regaining of regular metabolic status, and impairs defense mechanisms and routine wound healing.[17] It subsequently will increase BP and HR and decrease oxygen saturation, resulting in further complications. Analysis of the study results showed that stress before dental extraction is much stronger than other dental therapeutic procedures.

Agani et al. had displayed a significant rise in preextraction systolic and diastolic BP, while there was no significance in pulse rate.[18] Following Agani et al., the present study also revealed significant differences in systolic BP before and after extraction between experimental and control groups.[18] Few authors reported a considerable increase in the experimental group preoperative pulse rate than the control group.[1920]

Alfayad and Al-Hadithy stated that the experimental group patients showed significantly higher serum cortisol levels before minor oral surgery due to phobia and anxiety toward dental surgical work.[20] Hempenstall et al. reported a marked decrease in cortisol level after dental therapy, precisely similar to our findings.[17] Steer and Fromm reported that their patients had developed postoperative cortisol insufficiency.[21]

On the contrary, Banks and Franksson and Gemzell have described that preoperative nervousness is not a stimulus to cortisol secretion.[2223] Few authors had concluded that significant cortisol upsurges after the extraction procedure than the preoperative period.[167222425] Miller et al. had reported that the stress related with dental extractions persists and prolongs in the postoperative period.[5]

Miller et al. had established higher cortisol concentration in patients during dental extraction than other dental procedures.[5] Fewer studies specified that the response of adrenal stress associated with prolonged and painful dental procedures was higher than short and painless dental treatments.[1820] They also presented that the more anxious patient had shown even more stress than less anxious patients.[1826]

Some of the studies had analyzed the relationship between salivary cortisol and hemodynamics. Gadicherla et al. stated that no significant relation was detected between the hemodynamic parameters and salivary cortisol concentration, except for postextraction diastolic BP.[7] Gregg et al. stated that although there was the rise of stress-related salivary cortisol, only a weak correlation exists with hemodynamic changes.[27] The present study established a moderate correlation between salivary cortisol levels and postextraction systolic blood pressure. At the same time, there was no significance in other hemodynamic parameters and cortisol levels.

Dental treatment in patients with hypertension requires special attention because any stressful procedure can induce cardiovascular disease and trigger acute complications such as heart attack and stroke.[28] The decreased oxygen saturation level may lead to complications and prevails in emergencies such as respiratory collapse, syncope, and cyanosis. These medical emergencies sometimes necessitate the dentist to render essential life support measures to the patient.[29] The dentist needs to recognize such patients to avoid future consequences and maintain their vital signs within the standard limit during dental therapy.[30] The dentists can comfort the patient by following ways such as reassuring and communicating sufficient information about the dental procedures, reducing the wait time before therapy, and using distraction methods such as videos, music, and chewing of sugarless gum or substitutes. The sufficient relaxation time for subsequent dental appointments and a pleasant environment may also reduce the patient's anxiety experience.[3132]

CONCLUSION

Dental extraction and administration of local anesthesia will induce stress on patients undergoing dental extraction. Dental surgeons should minimize the fear of pain and anxiety during the procedure by providing a stress-free and comfortable atmosphere and atraumatic treatment procedure. The chairside salivary cortisol kit will quickly assess the patient's stress level during dental extraction. It is a more comfortable method to measure steroids in a small saliva volume without involving serum samples. It alerts the dental surgeons about the patient's stress level, which aids in a better understanding of patients' psychology, thus preventing complications during any dental procedure. It also gains patients' trust for the dentist to repeated visits. The dentists might implement strategies to identify and modify the patient's behavior to minimize the stress commonly generated by dental interventions.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.