What if an ordinary urine sample could help reveal mental stress before it develops into depression or anxiety? This study tests that idea by combining six urinary neurotransmitters into a stress index. Almost ten percent of the population develop depression or anxiety disorder during their lifetime.

Because people exposed to high stress are more likely to develop mental disorders, the goal is to detect and remove mental stress before depression or anxiety disorder develops. The study aimed to develop an objective screening test that quantifies mental stress in workers, so they can recognize and remove it before the disorder develops.

Diagnosing depression and anxiety disorders still relies on subjective assessments by doctors, making pre-disease states difficult to identify. That creates a need for markers that can detect stress before a disorder develops.

Cortisol is a reliable biomarker for measuring stress levels, but its measurement requires strict control of sampling timing because of circadian fluctuations. The study turns instead to neurotransmitters and their urinary measurements, because urine and saliva offer greater convenience and ease of collection than serum or plasma.

Traditional self-report questionnaires such as the Beck Depression Inventory, Patient Health Questionnaire-9, and CES-D were designed to confirm depression or anxiety disorders, so they are limited for identifying underlying mental stress in healthy individuals. The Brief Job Stress Questionnaire, or BJSQ, assesses mental stress along with employees’ work environments and personal lives, making it more sensitive to mild mental stress and underlying stress among healthy individuals.

The study acknowledges that urinary neurotransmitter levels may not always reflect neurotransmitter levels in the brain. Even so, urinary neurotransmitters are proposed as potential biomarkers for monitoring mental health conditions and assessing the degree of mental stress.

The screening test targets six neurotransmitters in urine: dopamine, serotonin, five-HIAA, GABA, HVA, and VMA, using urine samples from healthy volunteers. Figure one lays out both the study workflow and the biochemical targets.

Panel A follows one hundred healthy volunteers—forty-nine male and fifty-one female—from overnight fasting through serum, saliva, and urine collection, questionnaire responses, a medical interview, and laboratory testing. Panel B maps the urinary neurotransmitters and related metabolites, including dopamine, norepinephrine, GABA, serotonin, homovanillic acid, vanillylmandelic acid, and five-hydroxyindoleacetic acid, giving the analytical measurements a biological pathway context.

Participants started fasting from nine p.m., and sampling and self-report questionnaires were conducted the next morning. Serum, urine, and saliva were obtained, but only urine was used in this study. All participants answered the BJSQ and CES-D.

The BJSQ comprises fifty-seven items, while the CES-D comprises twenty items, with positive-item scores reversed before summing. Participants were interviewed by medical doctors about smoking, alcohol drinking habits, and familial health histories. Participants judged unhealthy by the doctor, including cases of serious renal failure, hepatic failure, or diabetes, were excluded.

The participants then underwent laboratory tests covering thirty-six items related to hepatic, renal, and metabolic functions. Each analyte was quantified using the ratio of its peak area to the internal standard area, with deuterium-labeled compounds of the target analytes used as internal standards.

The concentrations of the six analytes were divided by creatinine concentration to correct a dilution factor, and creatinine was analyzed simultaneously with the six neurotransmitters by liquid chromatography mass spectrometry. To generate the stress index, all analyte concentrations were transformed to logarithms.

The values were then normalized by zero-mean-centering and unit-variance-scaling. The stress index was obtained as a linear combination of these values with weight factors, and the weights were optimized using the Excel-solver program to minimize the p-values of Student’s t-tests between two groups.

All regression coefficients of the six standard curves exceeded zero point ninety-nine, and relative errors from predicted to observed concentrations were less than fifteen percent across the stated concentration ranges, except for the later qualification concerning HVA.

Within-run and between-run accuracies and precisions using authentic standards were generally less than fifteen percent, while HVA slightly exceeded the tolerance in one within-run measurement. Standards and urine extracts were both stable at four degrees Celsius for forty-eight hours after preparation on the liquid-chromatography autosampler.

The method’s accuracy, precision, and stability were considered acceptable, but HVA requires careful interpretation because of larger errors. Figure two compares total scores from the job-stress questionnaire, BJSQ, with the depression-focused CES-D.

In panel A, the points follow a fitted curve, with dashed ninety-five percent confidence bounds, indicating how the two score sets relate while also showing individual variation. Panel B shows that CES-D responses cluster strongly at the low-stress category, while BJSQ responses are distributed more evenly across the four categories.

This matters because it supports checking self-reported BJSQ responses against a related measure, while recognizing that the questionnaires capture different kinds of stress. Participants were assigned to high- and low-stress groups for every questionnaire item: the high-stress group chose three or four, while the low-stress group chose one or two.

Student’s t-tests compared the two groups for six neurotransmitters across all fifty-seven BJSQ items, and markers with p-values below zero point zero one were listed in Table 1. Urinary dopamine responded to dissatisfaction, gloominess, physical fatigue, and pain.

Table one reports significant responses of six urinary transmitters, comparing high and low total BJSQ-score groups among one hundred participants. For dopamine, for example, the average difference is zero point one seven three for “This job does not suit me well,” while HVA shows differences of two point six for a poor working environment and two point four for poor sleep.

The table also provides group sizes, ninety-five percent confidence intervals, and p-values, linking transmitter concentrations to specific stress-related responses. The high-stress group included fifty participants, and the low-stress group included fifty volunteers.

Laboratory tests of thirty-six items showed no significant differences between the groups, meaning there were no biases between the groups except for mental stress. Urinary dopamine decreased in the high-stress group to zero point six three seven micrograms per milliliter, compared with zero point eight zero one in the low-stress group, with a p-value of zero point zero zero four two.

HVA, the end-point metabolite of dopamine, also decreased in the high-stress group, from fourteen point zero micrograms per milliliter in the low-stress group to eleven point six, with a p-value of zero point zero two zero. Table three compares average urinary neurotransmitter concentrations for fifty high-stress and fifty low-stress participants, using means, standard deviations, ninety-five percent confidence intervals, average differences, and p-values.

This matters because the authors are evaluating urine-based neurotransmitters as non-invasive biomarkers of mental stress. Figure three compares six urinary neurotransmitters between high- and low-stress groups, each containing fifty participants; Student’s t-test markers identify reported differences for DA and HVA.

Panel B then shows the combined stress index in separate training and test sets, with p-values of zero point zero one two and zero point zero one six. The index combines log-transformed, standardized analyte values using optimized weights, providing a compact measure of the pattern across all six neurotransmitters.

The stress index used one hundred participants divided randomly into a training set of sixty-six and a test set of thirty-four, with equal numbers of high- and low-stress people in both sets. The weights for dopamine and HVA were zero point seventy-six and zero point thirty-six, much higher than the weights for the other neurotransmitters.

In the training set, the index values were minus zero point twenty-one for high stress and zero point twenty-one for low stress, with a p-value of zero point zero one two. In the test set, the values changed from minus zero point thirteen for high stress to zero point twenty-nine for low stress, with a p-value of zero point zero one six.

The study found that the concentration of each urinary neurotransmitter varied depending on different stress factors. Dopamine responded to dissatisfied moods or physical fatigue, GABA related to appetite, HVA responded to working environments, sleep disturbances, and isolation, VMA was associated with uncontrollable situations, and five-HT was linked to anxiety.

Both dopamine and HVA responded to total stress severities, indicating that the stress index generated from the six urinary biomarkers could be a promising indicator for overall stress severities. Because the study included only healthy volunteers and not patients with depression, these urinary neurotransmitters show promise as biomarkers for detecting mental stress in healthy individuals.

A limitation is that the influence of sampling timing on urinary neurotransmitter levels still needs to be assessed. Previous research showed diurnal variations in urinary HVA and VMA levels. The study developed simultaneous analysis of urinary neurotransmitters using mass spectrometry.

It found that urinary neurotransmitter concentrations changed with various stress factors, and that a stress index from six urinary biomarkers was a promising indicator of total stress severities. The findings may provide clues for developing screening tests for mental stress or depression, but further research and validation are required to establish the utility and accuracy of urinary neurotransmitter analysis as a diagnostic tool for mental stress.

The study found that urinary dopamine and HVA, especially within a six-marker stress index, could distinguish high- and low-stress healthy volunteers. But timing effects and further validation still matter before clinical use.