To investigate if salivary nitrate correlates to the daily psychological stress and anxiety in a group of human subjects.
The convenient sample recruitment method was employed; data from seventy three subjects were analyzed. The Perceived Stress Scale (PSS) and Hamilton Anxiety Rating Scale (HAM-A) inventories were used to determine stress and anxiety scores respectively. Salivary nitric oxide was measured through nitrate (NOx) levels using the Griess reaction method.
Although stress and anxiety were correlated. No significant correlation exists between salivary nitrate and daily psychological stress and anxiety in the study's participants.
While all previous studies focused NOx levels in acute stress models. This is the first study to investigate the correlation between salivary nitrates and daily psychological stress and anxiety. Although stress and anxiety were correlated, there is no correlation between salivary nitrates and daily psychological stress and anxiety. Further studies are required to investigate this correlation using other biological samples such as plasma.
Nitric oxide (NO) is an endogenous gas, produced by different cell types. It plays essential physiological roles, such as in the cardiovascular system and the central nervous system. Nitric oxide is synthesised through the conversion of L-arginine to L-citrulline by nitric oxide synthase (NOS).
Nitric Oxide concentration can be measured through its end metabolite nitrates (NOx). Nitrates can be measured red in central and peripheral tissues.
Stress and anxiety are among the most common psychological disorders that affect the human quality of life. According to epidemiological studies, almost one-third of people will suffer from stress and anxiety disorders.
It was reported in animal models
A cross-sectional study was conducted to meet the study's aim. The cross-sectional methodology was selected as it was the most appropriate method to explore the correlation between each anxiety and stress with nitric oxide.
The study used the convenience sample technique in recruiting the participants. The inclusion criteria were Jordanian, aged 18 years and older, and being willing to participate in the study. The participants were contacted during their visit to the one of the health care centres in Madaba city in April 2015.
To ensure homogenous data collection and reduce errors, selected senior pharmacy students from the American University of Madaba were trained for data collection by one of the authors. The study's aim, objectives, data collection procedure, and inclusion criteria were all explained. The students contacted each participant to report to their centres and asked eligible personnel for their willingness to participate in the study, explaining the study's objective and providing them with the study information sheet. The demographical data sheet and the questionnaire were distributed for the participants to be self-completed in Arabic. Ethical approval to conduct the study was obtained from the Latter-day Saint Charities in Jordan. Written informed consent and information sheet were provided, participation was completely voluntary, and participants had the right to withdraw from the study at any time.
The participant demographical sheet, Hamilton Anxiety Rating Scale, and the Perceived Stress Scale were collected from each participant. In addition, saliva was collected and a nitrate assay was performed.
The Arabic version of PSS was used to assess participants' stress levels. The scale consists of 14 items measuring stress in the last month, which was developed by.
The Arabic version of the Hamilton Anxiety Rating Scale (HAM-A) was used to measure the anxiety levels among participants. It was developed to assess the severity of anxiety symptoms.
This scale has shown adequate validity and reliability measures with a coefficient alpha of 0.84 to 0.86. In the current study, the scale shows acceptable reliability with a coefficient alpha of 0.74.
Saliva collection and storage were carried out according to standard protocols.
The accumulation of nitrate, an indicator of the production of nitric oxide (NO), was determined with a colorimetric assay with a Greiss reagent.
SPSS version 21.0 statistical software was used to analyse the data. Descriptive (including mean and standard) deviations were used to analyse the demographical data. An independent t-test and Mann-Whitney U test were used to examine the differences in anxiety, stress, and NO based on the participants' demographics. An independent t-test and Mann-Whitney U test were used to examine the differences in each NO and stress with anxiety categories.
Spearman's test was used to examine the correlation between each anxiety, stress, and NO concentration. In addition, Pearson's test was used to examine the correlation between anxiety and stress scores. A significant level was set for less than 0.05.
Out of the 84 participants asked to participate in the study, 75 participants accepted and signed the informed consent form indicating a response rate of 88.1%. However, 73 were included in the analysis as two participants did not complete most of the data package.
As shown in
Differences between anxiety, stress and salivary NOx concentration and participants' demographics were aimed to examine the homogeneity of study participants. As each anxiety and stress scores showed to be normally distributed, an Independent t-test was used to assess the differences in each anxiety and stress with each demographics. As shown in
As NOx values were not normally distributed, the Mann-Whitney U test was used to examine the differences in NOx concentration based on participants' demographics. Mann-Whitney U test showed no significance difference among all demographics.
Mann-Whitney U test was used to examine the differences in NOx concentration based on anxiety categories; less than 17, and 17 or more. The result showed no significant difference. In addition, the independent t-test was used to examine the differences in stress based on anxiety categories. The independent t-test showed significantly higher stress scores (M=35.33, SD=6.91) among participants with higher anxiety category (more than 17), compared to the lower anxiety category [M=28.21, SD=6.28; t (73)=3.54, p=0.001, two tailed].
Spearman's rho test was used to examine the correlation of salivary NOx concentration with anxiety and stress scores. No significant correlation was found (r=-0.117, n=73, p=0.323) and stress (r=-0.084, n=73, p=0.48).
Pearson's test was used to examine the correlation between anxiety and stress scores. A positive correlation was demonstrated (r=0.481, n=73, p=001).
This is the first pilot study to correlate salivary nitrates levels to stress and anxiety in humans. We report a non-significant correlation. Although the vast majority of published data on animals confirmed this correlation, however our results are contradictory. This controversy may be attributed to many factors. Firstly, the difference in the model employed i.e., this is a human-based study, while previous studies were conducted on stressed animals. Secondly, the type of stress and anxiety measured herein is chronic daily life stress and anxiety compared to an acute induced stress in previous works. Thirdly, nitrate levels were measured in saliva not in hippocampal or cortical tissue. Fourthly, the sample size used in this study is considered relatively low.
Psychological and physical stress contributes to anxiety.
A possible explanation for the different results is that the stress type, duration and intensity in animals may not be representative to humans. The PSS and HAMA-A inventories were aimed at asking participants their daily stressful conditions during the last month. However, referred animal models were stressed by immobilisation for hours. Similarly, the only study on human salivary nitrate used an acute stress method.
Another explanation for the results may be the salivary samples used. Human studies in this field are scarce; one similar study pointed out the increase in plasma nitrates in depressed patients.
Although this is the first study that employs human salivary nitrates, however it remains a pilot study. The study did not measure basal salivary nitrates in control population. Future investigation with a larger sample size can provide more confirmatory results and drive more substantial conclusions.
In conclusion, the authors report no significant correlation between stress, anxiety and salivary nitrates in this pilot investigation. The results may be attributed to a difference in methods, models and sample size. Further studies have to be performed to explore and compare salivary with hippocampal and cortical NOS and nitrate expression in models of stress and anxiety.
The authors would like to thank LDS Jordan represented by Prof. Brent Strong for their support. Also, special thanks to our senior pharmacy students for their participation. This study is funded by the American University of Madaba. The authors declare no conflict of interest.