Estimation of Serum Cortisol Levels in Oral Lichen Planus Patients with Electrochemiluminescence.

OBJECTIVES: The objectives of this study were to compare the serum cortisol level in oral lichen planus (OLP) patients with normal healthy individuals and to compare the serum cortisol level in patients with erosive and nonerosive forms of OLP.
MATERIALS AND METHODS: A case-control study involving 60 patients within the age group of 20-50 years was carried out. Group A included 15 subjects with nonerosive lichen planus, without skin involvement; Group B included 15 subjects with erosive lichen planus, without skin; and Group C included 30 subjects with no apparent lesions of the oral mucosa and the skin. From the cases and controls, 7mL venous blood was taken from median cubital vein two times (between 8 and 10 AM, and 4 and 6 PM) by using a 10 mL syringe from which 5 mL was taken for estimation of serum cortisol.
RESULTS: The serum cortisol levels of the erosive OLP patients were significantly higher than those of nonerosive OLP patients and controls. There was no significant difference in the serum cortisol levels between nonerosive OLP in comparison with controls.

INTRODUCTION

Lichen planus is a chronic inflammatory mucocutaneous disease that frequently involves the oral mucosa with a prevalence of 0.04–2% of the general population.[12] About half of the patients with skin lesions have oral lesions, whereas about 25% present with oral lesions alone.[3] In 60% of cases oral lesions precede the skin lesions. Cutaneous lesions are typically present as small (2 mm) pruritic, white-to-violaceous flat-topped purple papules, which can increase in size to as much as 3 cm.

Psychological factors are strongly associated with lichen planus, in particular high stress and anxiety levels.[4] High anxiety level causes increase in serum cortisol level and has been used as an indicator in the anxiety evaluation studies.[5] In conditions such as pain and anxiety, many metabolic and endocrine changes occur, and among these a rise in the levels of serum cortisol is one of the most important physiological effects. Hence, cortisol is also known as a stress hormone and is secreted from the adrenal gland.[6] It is suggested that anxiety with its presumed effects on the immune system constitutes one of the major causative agents of oral lichen planus (OLP).

This study aimed to estimate the serum cortisol levels in patients with OLP by electrochemiluminescence (ECL) and compare these with normal healthy individual. It also aimed to compare the serum cortisol levels in patients with erosive and nonerosive forms of OLP. In ECL, the electrochemically generated intermediates undergo a highly exergonic reaction to produce an electronically excited state that emits light on relaxation to a lower-level state. This wavelength of the emitted photon of light corresponds to the energy gap between these two states. Photon detection is conducted with the help of photomultiplier tubes or silicon photodiode or gold-coated fiber-optic sensors. The importance of ECL techniques for bio-related applications has been well established.[7]

MATERIALS AND METHODS

A case–control study involving 60 patients within the age group of 20–50 years was carried out. Group A included 15 subjects with nonerosive lichen planus, without skin involvement; Group B included 15 subjects with erosive lichen planus, without skin; and Group C included 30 subjects with no apparent lesions of the oral mucosa and the skin. From the cases and controls, 7mL venous blood was taken from median cubital vein two times (between 8 and 10 AM, and 4 and 6 PM) by using a 10 mL syringe from which 5 mL was taken for estimation of serum cortisol. The collected blood was then transferred into a 5-mL clot activator and kept for 4 minutes. Then, it was transferred to wells of the centrifuge and centrifuged at a speed of 2000–3000 rpm for 10 minutes. After that, the centrifuged blood was loaded into the rack of Vitros ECiQ, orthoclinical diagnostics automated machine, along with calibrated cortisol reagent in Vitros cortisol reagent pack and incubated for 15–20 minutes. The results were generated from the automatic processor unit of Vitros ECiQ. Data were collected and analyzed using the SPSS software, version 22. Student’s t test and one-way analysis of variance (ANOVA) were applied for statistical evaluation.

RESULTS

Demographic data analysis of Group A

In this group, the age of the subjects ranged from 20 to 50 years. Majority (9/15, 60%) of these cases were within 41–50 years, followed by 31–40 years (5/15, 33.33%), and least in 20–30 years (1/15, 6.6%). Females comprised 66.66% of this group (10/15), whereas males formed the remaining 33.4% of the group (5/15).

Demographic data analysis of Group B

In this group, the age of the subjects ranged from 20 to 50 years. Majority of the patients (9/15, 60%) were in the age group of 41–50 years, followed by 31–40 years age group (5/15, 33.33%) and the least (1/15, 6.6%) belonged to 21–30 years age group. Females comprised 66.66% of this group (10/15) whereas male comprised the remaining 33.33% of the group (5/15).

Demographic data analysis of Group C

In this group, the age of the subjects ranged from 20 to 50 years. Majority of the patients (18/30, 60%) in this group were in the age group of 41–50 years, followed by 10/30 in the age group of 31–40 years (33.33%) and the least patients (2/30, 6.6%) were in the age group of 21–30 years. Female comprised 66.66% (20/30) of group and males comprised 33.33 (10/30) of the group.

Mean cortisol value analysis

The mean cortisol value in Group A (nonerosive lichen planus) in the morning was 282.4667 and in the evening was 253.3333, that in Group B (erosive lichen planus) in the morning was 563.4000 and in the evening was 497.0000, and that in Group C (control) in the morning was 255.5000 and in the evening was 235.9667. The average of morning and evening values of cortisol for Group A (nonerosive lichen planus) was 267.9000, that for Group B (erosive lichen planus) was 530.2000, and that for Group C (control) was 245.733. The mean serum cortisol levels of the erosive OLP patients (Group B) were significantly higher than those of nonerosive OLP patients (Group A) and controls (Group C).

An error bar is drawn to compare the average hormone level for each group. The bar represents the variability of the data. In Graph 1, the data point rounded is the mean of the cortisol level of each group and the length of the bar represents the confidence interval (95%). These graphs also help us to compare two values visually. If they are significantly different they will not overlap. From the error bar drawn below, it can be observed that the mean average cortisol levels are different for the three groups. The erosive group is having the highest cortisol level followed by nonerosive group. The control group has the lowest mean hormone level. But for nonerosive and control groups, there does not seem much difference in the mean hormone levels.

Graph 1

Error bar for average hormone levels

Paired t test is conducted to find if the difference in the mean hormone levels in morning and evening is equal or not. From the descriptive statistics [Table 1], it can be observed that during both morning and evening, the mean and standard deviation of the hormone level are the highest for erosive group whereas they are the lowest for the control group.

Table 1

Table showing results of paired t test

Paired Differences
Std. Deviation of the Difference
95% Confidence Interval
Group	Timing	Mean	Deviation	Lower	Upper	t	df	p-value
Non Erosive	Morning-Evening	29.13333	29.08330	13.02754	45.23913	3.880	14	.002
Erosive	Morning-Evening	66.40000	24.18028	53.00941	79.79059	10.635	14	.000
Control	Morning-Evening	19.53333	11.71128	15.16027	23.90640	9.136	29	.000

From the ANOVA table [Table 2], it can be observed that the P value is less than 0.05, so the mean hormone level in the morning (F(2, 57) = 476.045, P value <0.05) for the three groups is significantly different. Similarly, for the evening and the average of morning and evening, it is significantly different.

Table 2

Table showing one-way analysis of variance

Timing	Groups	Sum of Squares	df	Mean Square	F	p-value
Morning	Between Groups	1012435.350	2	506217.675	476.045	.000
	Within Groups	60612.833	57	1063.383
	Total	1073048.183	59
Evening	Between Groups	735950.433	2	367975.217	263.684	.000
	Within Groups	79544.300	57	1395.514
	Total	815494.733	59
Average	Between Groups	868599.679	2	434299.840	386.331	.000
	Within Groups	64077.367	57	1124.164
	Total	932677.046	59

The means plot in Graph 2 shows that the average of the morning and evening mean serum cortisol values varies between different groups (nonerosive, erosive, and control). From the means plot in Graph 2, it can be observed that the mean average hormone levels are different for three groups. The erosive group is having the highest hormone level followed by nonerosive group. The control group has the lowest mean hormone level.

Graph 2

Mean plot for average of morning and evening cortisol level

DISCUSSION

In this study, an attempt was made to analyze the serum cortisol levels in patients with erosive and nonerosive OLP. Also, an attempt was made to compare the serum cortisol levels between patients with OLP and healthy patients. The mean serum cortisol analysis between patients with erosive OLP in comparison to those with nonerosive OLP (Group A) and controls (Group C) revealed a significant difference.

Ivanovski et al.[8] measured serum cortisol levels by using radioimmunoassay. In their study, the serum cortisol levels in the controls and nonerosive lichen planus patients were 248.7 ± 10.7 and 250.3 ± 11.1 nmol/L, respectively whereas in the erosive group, the serum cortisol level was 536.0 ± 55.6 nmol/L. The results of their study matches our study. Although a different method of estimation of serum cortisol was used in our study compared to the one used by Ivanovski et al., the outcome was similar. We used ECL for cortisol estimation but Ivanovski et al. measured serum cortisol levels by using radioimmunoassay. Shetty et al.[9] conducted a case–control study in 2010 comprising 15 cases each of nonerosive oral lichen planus (NEOLP) and erosive oral lichen planus (EOLP) and 10 controls. The serum cortisol levels of all 40 patients were estimated by using ECL method. We also used ECL method for serum cortisol estimation. Hospital Anxiety and Depression Score questionnaire were administered to evaluate the psychiatric status of all the patients and found that the mean serum cortisol level of the OLP group showed a very highly significant difference from the controls. The difference in the mean cortisol level between NEOLP group and control was not significant, whereas it was highly significant between the EOLP group and controls. A similar result is observed in our study also. There was difference in the anxiety score between the NEOLP and EOLP groups, but the depression scores between the two groups were not very significant. These findings suggest that psychiatric factors play a very important role in the pathogenesis of OLP and serum cortisol could be a possible indicator for the erosive nature of the lesion. One of the research group conducted a study by determining the salivary cortisol[10] levels in patients under stressful conditions, whereas the other conducted a study to determine the average plasma cortisol and 24 hour urine cortisol in patients with OLP. They stated that their study results do not support with the hypothesis that environmental factors such as stress can have an influence on the neuroendocrine system through cortisol levels. This may be due to the difference in study methods.

Although the cortisol levels tested in our study represent only one possible mechanism for psycho–neuro–immune interactions, these data suggest that cortisol and psychological status may play a role that may influence the pathogenesis of OLP, especially in the erosive form. Taken together, these may act as possible avenues by which the psychological status of an individual may affect the immune system homeostasis during onset and progression of lichen planus. Results indicate that the psychological health of the patient affects the progression of erosive lichen planus.

CONCLUSION

The following conclusions can be drawn from the study. OLP was more commonly observed in the middle-aged group. The serum cortisol levels of patients with erosive lichen planus were significantly higher in comparison to those of the controls and patients with nonerosive lichen planus. This study suggests that serum cortisol may be a predictor for the erosive nature of OLP and stress may be an aggravating factor of OLP. The overall results suggest that changes in serum cortisol levels correlate with those in the stress levels and the clinical presentation of OLP, and we need a close collaboration between physicians, dentists, and counselors/psychologists to optimize handling of these patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.