Many researchers working in different fields use stress measurement in saliva samples. It needs an optimal method when evaluating stress-related parameters. At the same time, saliva samples should be collected and administered simultaneously. The most commonly used saliva stress markers are cortisol and amylase.
In the current study, a biosensor was used to determine the amounts of cortisol and α-amylase in saliva. The level of compatibility and comparison of other measurement methods used with reference to the new measurement method were examined using statistical analysis methods. In a second step, it was discussed whether there is a difference in saliva cortisol and amylase levels during stress.
In 23 healthy young adults (17 female and 6.male), the mean age was 20.5± 0.95 years (range:20-24 years). The mean age in the female and male groups were 20.2± 0.43 and 21.17±1,60 (p=0.001) respectively. The mean and standard deviations of cortisol and amylase concentrations obtained in salivary samples taken in the morning, pre-stress and post-stress are shown in Table 1.
As a result of the studies conducted in young adults, who can use all three methods in case of stress, it was determined that there are differences in cortisol concentrations. It was observed that the cortisol level in the saliva taken in the morning hours was high, it decreased pre-stress, and the salivary cortisol levels increased post stress. It was observed that the salivary amylase concentration was decreased in the morning hours and increased before and after stress.
The performance of the methods used in determining the levels of cortisol and alpha-amylase were examined in 3 different times in the morning, pre-stress and post-stress in young adult individuals. In Figure 1, the findings of ELISA, biosensor and autoanalyzer methods for the young adult individuals examined in the study are presented.
While each method reveals salivary cortisol concentrations close to each other in all three times, salivary amylase concentrations were found only in the ELISA method (Table I). In ELISA and Biosensor method, post-stress salivary cortisol levels were found to be higher than pre-stress. In the autoanalyzer method, cortisol levels were found to be higher in the morning saliva samples than pre and post-stress (Table 1). Details of post-hoc analysis for comparative analysis of different methods between different study groups are given in Table 2.
When we pooled all the salivary samples of the subjects into 3 methods, 69 samples were obtained for each method. The relationship between methods tested by Spearman’s rho correlation analysis. There were significant positive correlations between ELISA and Biosensor levels (r=0.965**; p=0.0005) (Figure 2).
In the evaluation of salivary cortisol levels, the correlation measured with the cortisol biosensor is in good agreement with the measurements made by the traditional ELISA method. On the other hand, cortisol biosensor values appear to be slightly higher than the cortisol ELISA. The difference is that, firstly, the biosensor has higher sensitivity than ELISA. Secondly, the current reduction caused by impurities increases the biosensor results because the current is inversely proportional to the solution concentration.
Saliva cortisol measurement results obtained by three methods were evaluated statistically. Regression analysis was carried out to examine the relationship between them and this relationship was expressed as the correlation coefficient. The values obtained from all three measurement systems were evaluated by Spearman correlation analysis and a significant difference was found between the three methods.
The distribution of cortisol results between ELISA method and Autoanalyzer method is as shown in Figure 3. Regression analysis for these two methods; R2 = 0.012, a = 6.2378 b = 0.0811. When we put the equation of the curve in the equation y = a + bx, y = 6.238+ 0.0811x was found. It was proved that the results of ELISA method and autoanalyser method were statistically different. (r=0.109;p=0.374).
The present data showed for the mean concentration of salivary cortisol measured autoanalyzer was significantly different from that measured by ELISA.
The distribution of cortisol results between ELISA method and biosensor method is as shown in Figure 4. As a result of the regression analysis for these two methods, R2 = 0.839, a = 2.971, b = 0.748. When we put the equation of the curve in the equation y = a + bx, y = 2.971+ 0.748x was found. It was proved that ELISA method and biosensor method results were not statistically different (r=0.916; p=0.0007).
When we compared the reference method Biosensor method with other methods, it was determined that the results of the ELISA method were similar to the Biosensor, but it was determined that they were different with the other method, Chemiluminescence immunoassay.
The distribution of cortisol results between Autoanalyzer method and Biosensor method is as shown in Figure 5. Regression analysis for these two methods; R2 = 0.019, a =9.983, b = 0.153 When we put the equation of the curve in the equation y = a + bx, y = 9.983 + 0.153x was found. It was proved that the results of the autoanalyser method and biosensor method were statistically different (r=0.138; p=0.264).
Saliva α-amylase levels could not be determined in autoanalyzer and biosensor. The biosensor method developed is not suitable for determining the level of amylase. It was observed that the correct measurement range of the ELISA kit used for amylase was narrow and there was no reproducibility in the study with the saliva sample. We think that this is due to the lack of method validation for saliva samples, although the kit is compatible with all body fluids.
As a result of the studies conducted, it was seen that the biosensor method developed for cortisol is an alternative method due to low cost, fast result, specificity and high detection / information efficiency compared to ELISA.