Prospective study on the association between serum amino acid profiles and depressive symptoms among the Japanese working population

Objective Accumulating evidence suggests that amino acids, particularly tryptophan and glutamate, play an important role in the pathology of depression, but prospective epidemiologic data on this issue is scarce. We examined the association between circulating amino acids and the risk of depressive symptoms in a Japanese working population. Methods Participants were 841 workers who were free from depressive symptoms and provided blood at baseline and completed 3-yr follow-up survey. 30 varieties of amino acid concentrations in serum were measured using liquid chromatography/mass spectrometry. Depressive symptoms were defined using the Center for Epidemiologic Studies Depression Scale. Logistic regression was used to calculate the odds ratios of depressive symptoms according to serum amino acids with adjustment for lifestyle factors. Results A total of 151 (18.0%) workers were newly identified as having depressive symptoms at the follow-up. Baseline tryptophan and glutamate concentrations in serum were not appreciably associated with the risk of depressive symptoms. Risk of depressive symptoms tended to increase with increasing arginine concentrations; the multivariable-adjusted odds ratio for the highest versus lowest tertile of serum arginine was 1.65 (95% confidence interval: 0.96–2.83; P for trend = 0.07). No clear association was found for other amino acids. Conclusions Results of the present study do not support a significant role of circulating amino acids in the development of depressive symptoms among Japanese.


Study procedure and participants
As part of the Japan Epidemiology Collaboration on Occupational Health Study, a nutritional epidemiological survey named the Furukawa Nutrition and Health Study was conducted during periodic health examinations of workers from two locations of a manufacturing company and its affiliates. Details of the study procedure were described elsewhere [22,23]. The survey has been performed every three years since 2012 in A factory and 2013 in B factory. In the present study, we treated the 2015-16 survey, when serum amino acids were measured, as the baseline and the 2018-19 survey as the follow-up for the assessment of depressive symptoms.
Prior to the health checkup, we asked all workers to participate in the survey and fill out two questionnaires, one specifically designed for diet and the second for overall health-related lifestyle. We asked participants to donate 7-mL venous blood for study. We obtained health checkup data, including the results of anthropometric and biochemical measurements and disease history.
The protocol for the study was approved by the ethics committee of the National Center for Global Health and Medicine, Japan. Prior to each survey, written informed consent was obtained from all participants.

Participants
Of 2350 eligible participants at the baseline survey, 2067 employees participated (response rate: 88%). Of these, 2056 participants completed the questionnaire assessing their healthrelated lifestyle at the baseline survey. We initially excluded 627 participants with depressive symptoms (Center for Epidemiologic Studies Depression [CES-D] � 16) and 67 participants with a history of the following diseases diagnosed by a physician: cancer (n = 23), cardiovascular diseases (n = 19), chronic hepatitis (n = 2), kidney diseases (e.g. nephritis) (n = 6), pancreatitis (n = 2), and mental disorders, including depression and neurotic disorder (n = 16). Some participants had two or more conditions meriting exclusion. We made these exclusions to eliminate potential reverse causality; dietary changes as a result of such diseases may have affected serum amino acid concentrations. We then excluded 111 with missing data on serum amino acids and covariates at the baseline survey. Of the remaining 1251 participants, 841 (745 men and 96 women aged 22-68 years) participated in the follow-up survey and filled out the study questionnaire (Fig 1).

Other variables
The survey questionnaire enquired about marital status, job grade, night and rotating shift work, overtime work, smoking, alcohol drinking, sleep duration, physical activity during work and housework or in commuting to work, and leisure-time physical activity. Physical activity during work and housework or in commuting and leisure time was expressed as the sum of metabolic equivalents (METs) multiplied by the duration of time (in hours) across physical activities of different levels. The psychological work environment was also assessed via the Japanese version of the Job Content Questionnaire [26], and the job strain score was calculated according to a standard procedure. Body height and weight were assessed to the nearest 0.1 cm and 0.1 kg, respectively, in a standardized procedure with participants wearing light clothes and without shoes. The body mass index was calculated as weight in kilograms divided by the square of height in meters (kg/m 2 ). Dietary habits during the preceding one-month period were assessed using a validated brief self-administered diet history questionnaire (BDHQ) [27]. The dietary intake for energy and selected nutrients, including folate, vitamin B6, vitamin B12, n-3 polyunsaturated fatty acids, magnesium, and zinc, were estimated using an ad hoc computer algorithm for the BDHQ [28], with reference to the Standard Tables of Food Composition in Japan [29,30].

Statistical analyses
Differences in characteristics at the baseline survey between participants with and without depressive symptoms at the follow-up survey were assessed using the independent t-test (continuous variable) and the χ 2 -test (categorical variable). In addition, Pearson correlation coefficients across amino acid concentrations at baseline survey were calculated (n = 841). The association of baseline serum amino acids with subsequent development of depressive symptoms at the follow-up survey was evaluated by multiple logistic regression analysis (n = 841). We calculated the odds ratios (ORs) and 95% confidence intervals (CIs) of depressive symptoms for each tertile of serum amino acids at the baseline survey using the lowest tertile category as a reference. There is no consensus on optimal concentrations of circulating amino acids; we thus divided participants into the tertile of amino acid concentrations. We adjusted for age (year, continuous), sex, and site (surveyed in April 2015 or in May 2016) in the first model. The second model was further adjusted for marital status (married or other), job grade (low, middle, or high), night or rotating shift work (yes or no), overtime work (<10, 10-<30, or �30 h/month), job strain (quartile), physical activity at work and housework or while commuting to work (<3, 3-<7, 7-<20, or �20 METs-h/day), leisure-time physical activity (not engaged, >0-<3, 3-<10, or �10 METs-h/week), smoking habit (never-smoker, former smoker, current smoker consuming <20 cigarettes/day, or current smoker consuming �20 cigarettes/day), alcohol drinking habit (nondrinker, drinker consuming 1-3 days/month, weekly drinker consuming <1 go/day, 1 to <2 go/day, or �2 go/day; one go contains approximately 23 g of ethanol), sleep duration (<6, 6-6.9, or �7 h/day), body mass index (kg/m 2 , continuous), total energy intake (kcal/day, continuous), and intake of folate (μg/1000 kcal, continuous), vitamin B6 (mg/1000 kcal, continuous), vitamin B12 (μg/1000 kcal, continuous), n-3 polyunsaturated fatty acids (% energy, continuous), magnesium (mg/1000 kcal, continuous), and zinc (mg/1000 kcal, continuous). The nutritional factors we adjusted for have been linked to depressive symptoms [31][32][33][34][35][36]. We further adjusted for CES-D score. All models were adjusted for baseline values of potential confounding variables (described above). Trend associations were tested by assigning ordinal numbers (1-3) to tertile categories for each serum amino acid. To confirm the robustness of our findings, we repeated the above analysis after excluding participants whose blood sampling had not been performed after overnight fasting (n = 25). For other analyses, we considered a two-sided P <0.05 as statistically significant. All statistical analyses were performed using Stata, version 14.2 (StataCorp, College Station, TX, USA). Table 1 presents the baseline characteristics of the study populations according to their depressive symptoms at follow-up survey. Of the 841 participants free from depressive symptoms at baseline, 151 (18.0%) developed depressive symptoms (CES-D score �16) at follow-up survey. Participants with depressive symptom at the follow-up survey did not receive psychiatric treatment. Compared with participants free from depressive symptoms at the follow-up survey, those who developed depressive symptoms were younger and more likely to be in a low-ranking job position with a higher job strain and CES-D score but lower folate intake at baseline.

Results
The correlations among amino acid concentrations at baseline of the cohort are presented in Fig 2. Three BCAAs (isoleucine, leucine, and valine) were highly correlated with each other (r = 0.80-0.83), as well as glutamine and glutamate (r = −0.77). Other amino acids were not highly correlated with each other (r <0.64).
As shown in Table 2, there were no significant associations between tryptophan and glutamate concentrations and subsequent risk of depressive symptoms after adjusting for multiple confounders (model 2). The null association was robust when the analyses were performed after additional adjustment for baseline CES-D score (model 3) as well as after the exclusion of participants in whom blood sampling had not been performed after overnight fasting (model 4). Likewise, no significant associations were found for other amino acid concentrations (model 4) (Tables 3-6). Regarding serum arginine concentrations, a marginally significant trend was found for the risk of depressive symptoms after adjusting for multiple confounders (model 2) (P for trend = 0.09). The association were virtually unchanged after additional adjustment of baseline CES-D score (model 3); the multivariable-adjusted odds ratio for the highest versus lowest tertile of serum arginine was 1.65 (95% CI: 0.96-2.83; P for trend = 0.07). We confirmed similar findings after excluding participants whose blood sampling had not been performed after overnight fasting (model 4) (P for trend = 0.054).

Discussion
In this prospective study of Japanese employees, we found no significant association between tryptophan or glutamate concentrations and subsequent risk of depressive symptoms after adjustment for a wide range of dietary and lifestyle factors. For serum arginine, a marginally significant trend was found for risk of depressive symptoms. To our knowledge, this is the first prospective study to investigate the association between serum amino acid profiles and depressive status.
Tryptophan, an essential amino acid, is a precursor of serotonin synthesis, which is thought to be involved in the pathophysiology of depression [37]. Consistent with this pathophysiological evidence, a meta-analysis indicated that patients with depressive disorder had significantly lower plasma tryptophan concentrations than healthy controls [10]. Contrary to our expectation, serum tryptophan concentrations were not associated with subsequent risk of depressive symptoms in the present cohort. This null finding is consistent with those of two cross-sectional studies among the community-dwelling elderly in Japan [21] and in Mexico [17]. Results of these population-based studies including ours do not support the hypothesis that higher circulating concentrations of tryptophan are associated with a prevalence or risk of depressive symptoms. Mechanistic evidence suggests that glutamate system plays a major role in the pathogenesis of depressive disorders [38], which has been supported by a meta-analysis that showed significantly higher blood glutamate concentrations in patients with major depressive disorder than in controls [11]. In the present study, however, we found no measurable relationship between serum glutamate concentrations and subsequent risk of depressive symptoms. Our findings agreed with those of a cross-sectional study of the free-living elderly in Japan [21], which found no association, but contradict with the results of another cross-sectional study of community-dwelling elderly women in Mexico, which showed that depressed individuals had an increased plasma glutamate concentrations compared with control individuals [17]. Given these data, we speculate that alternation of circulating glutamate concentrations in depressive symptoms might be less sensitive for Asian populations. Further investigations will be necessary to support or refute this assumption.
We found a marginally significant trend of an increased risk of depressive symptoms with increasing serum arginine concentrations. To date, a cross-sectional study of communitydwelling elderly women in Mexico found that depressive patients had a significantly higher concentrations of arginine than healthy controls [17], whereas another cross-sectional study of free-living elderly in Japan found no measurable association between them [21]. Similarly, clinical studies found inconsistent results. There were no significant differences in the plasma   concentrations of arginine between depressive patients and healthy control participants [14,18,39] or even significantly lowered serum concentrations of arginine in patients with depression compared with controls [40]. Pathophysiological evidence suggests that higher concentrations of nitric oxide (NO), which is synthesized from arginine, are neurotoxic by mediating neuroinflammatory actions [41]. In addition, experimental evidence from an animal study revealed that NO synthase inhibitors enhanced the extracellular level of serotonin and dopamine in the rat ventral hippocampus, whereas the NO precursor arginine had the opposite effect [42]. Another study also added biological plausibility to the neurotoxic properties of arginine by showing that dietary arginine depletion reduced depressive-like response in male but not female mice [43]. Further researches are needed to determine whether higher concentrations of circulating arginine concentrations are associated with an increased risk of depressive symptoms.
Here, we discuss our null findings of circulating amino acid concentrations and the risk of depressive symptoms in the context of brain amino concentrations. In most brain regions, the uptake of circulating amino acids is suggested to be limited by the blood-brain barrier (BBB) and thus amino acids concentrations in brain are maintained fairly independently of those of circulating concentrations [44,45]. Meanwhile, it is speculated that peripheral amino acids alteration might, to some extent, reflect those changes observed in the brain [46]. Studies that measured both peripheral and brain amino acid concentrations showed inconsistent data. For instance, the correlation between central and peripheral tryptophan concentrations was moderate in some (r = 0.47) [47] but poor in other studies (r = 0.14-0.15) [48,49]. Likewise, the correlation between central and peripheral glutamate concentrations was moderate in some (r = 0.67) [50] but poor in other studies (r = 0.02) [51]. To date, depression studies that measured brain amino concentrations have also shown mixed results. A study found significantly lower brain tryptophan concentrations in depressive patients compared with control [52], while another study showed no significant difference between those groups [53]. Similarly, some studies observed significantly higher brain glutamate concentrations in depressed participants compared with controls [54,55], whereas other studies showed no difference [56,57] or even decreased of glutamate concentrations in patients with depression compared with controls [58]. Given these inconsistency regarding the correlation of amino acid concentrations between blood and brain and the relationship of central amino acid concentrations with depression, further studies are required to clarify the significance of circulating forms of amino acids in the development of depression. The strengths of this study include its prospective design, use of validated measurements for nutrient intake (i.e., the BDHQ) as well as depressive status (i.e., CES-D score), and adjustment for various dietary and lifestyle factors. In addition, as the study survey was carried out in a company during a periodic health check-up and had a high participation rate, the possibility of bias linked to selective survey participation is low. We also acknowledge limitations of our study. First, 32.7% did not take part in the follow-up survey, which may lead to selection bias. Second, serum amino acids might be less stable than those in plasma, but serum samples provide more sensitive results for amino acid detection [11,59]. Third, since we made , night or rotating shift work (yes or no), overtime work (<10, 10-<30, or �30 h/month), job strain (quartile), physical activity at work and housework or while commuting to work (<3, 3-<7, 7-<20, or �20 METs-h/day), leisure-time physical activity (not engaged, >0-<3, 3-<10, or �10 METs-h/week), smoking habit (never-smoker, former smoker, current smoker consuming <20 cigarettes/day, or current smoker consuming �20 cigarettes/day), alcohol drinking habit (nondrinker, drinker consuming 1-3 days/month, weekly drinker consuming <1 go/day, 1 to <2 go/day, or �2 go/day; one go contains approximately 23 g of ethanol), sleep duration (<6, 6-6.9, or �7 h/day), body mass index (kg/m 2 , continuous), total energy intake (kcal/day, continuous), and intake of folate (μg/1000 kcal, continuous), vitamin B6 (mg/1000 kcal, continuous), vitamin B12 (μg /1000 kcal, continuous), n-3 polyunsaturated fatty acids (% energy, continuous), magnesium (mg/1000 kcal, continuous), and zinc (mg/1000 kcal, continuous). d Model 2 + CES-D score at baseline (continuous) were further adjusted for. e Further excluded 25 participants whose blood sampling was not performed after over-night fasting.
https://doi.org/10.1371/journal.pone.0256337.t006 numerous tests, chance may explain some of the significant associations. The interpretation of the present findings thus requires caution. Fourth, we used a validated questionnaire (i.e. the CES-D scale) to assess depressive symptoms rather than structured diagnostic interviews. Thus, the findings may have differed if the outcome was defined by clinically diagnosed depression. Fifth, although we considered various potential confounders, we cannot rule out the possibility of bias due to unmeasured or residual confounding factors. Finally, our study was carried out among employees of a Japanese manufacturing company, hence caution is required when generalizing the present findings.

Conclusion
We observed no prospective association between serum amino acid concentrations and depressive symptoms among Japanese workers. Few prospective studies have investigated this association, and further prospective studies among populations with different ethnicity, gender, age, and physiological and psychological conditions are warranted to assess these associations.