Comparison of Age and Gender Differences of Tryptophan Metabolites in Patients of Major Monopolar and Bipolar Depression

Background: No research has been done for the determination of plasma levels of tryptophan metabolites in patients of monopolar and bipolar depression. Methods: Ultra high-speed liquid chromatography/mass spectrometry has been used for the simultaneous determination of plasma levels of tryptophan metabolites in depressive patients. Results: No significant age and gender differences were shown in monopolar depressive patients and some differences were shown in bipolar patients. The administration of drugs such as antidepressants, antipsychotics, mood stabilizers do not seem to have affected the results. Conclusion: In patients of major monopolar depression age and gender differences of plasma levels of tryptophan metabolites disappear although significant differences are observed in healthy volunteers. Some differences of age and gender differences were shown between monopolar and bipolar depressive patients.


Introduction
.8% of the global burden of diseases is due to psychiatric disorder [1]. Depres-How to cite this paper: Tomioka, H., Masuda, J., Takada, A. and Iwanami, A. sion is considered to be the leading cause of this disability. The number of people suffering from depression substantially increased since 1990, probably due to increase in aging people [2].
Antidepressants are widely used for the treatment of major depression. However, there has been a long lasting debate and concern about their efficacy and effectiveness. Generally, clinicians have a wide choice of individual drugs and they need evidence to make the best choice for patients.
Using a meta-analysis of data of the effectiveness of antidepressants, it has recently been shown that all antidepressants are more effective than placebo in adults with major depressive disorders [3].
Serotonin, one of important metabolites of TRP has been implicated to play roles of patho-etiology of depression, there debates about roles of serotonin.
Some people doubt roles of 5-HT in etiology of depression because 5-HT concentration was not decreased in the brains of depressive patients [4].
We have recently succeeded in the simultaneous measurements of all the TRP metabolites in plasma [5] [6] [7] [8]. Using this technology we measured all TRP metabolites in plasma of depressive patients.
We analyzed plasma levels of patients of major monopolar depression and preliminarily reported that plasma levels of 5-HT, but not 5-HIAA, were lower in depressive patients [6].
However, since there are significant gender and age differences in plasma levels of TRP metabolites in healthy volunteers, we decided to analyze differences of TRP metabolites in patients of major monopolar depression and bipolar depression.

1) Patients
Outpatients of depression were recruited in this study. Patients were diagnosed using the criteria of Diagnostic and Statistical Manual of Mental Disorders, the 5th edition (DSM-5, American Psychiatric Association 2013) [9]. Fasting blood samples were taken early in the morning. Their severity of depression was checked by CGI-S [10], SDS [11], and HDR-S [12]. The history of prescriptions of drugs such as antidepressants, anxyolitics, mood stabilizers and other drugs were asked.
Sample numbers are 55 (male; 15, and female 40, and average age; 45.4 ± 11.9). The number of MDD is 36 and BD is 19. Further characteristics of patients are described below. Outpatients who showed symptoms of depression were recruited in the studies at the Department of Psychiatry of Showa University Northern Yokohama Hospital. Patients were required to have had no lifetime history of psychosis, personality disorder, or drug or alcohol misuse. We also excluded patients who had organic problems of the brains. This study was carried out from 2016 to 2019. All patients gave their consent in a written form after receiving comprehensive information on the study protocol.

2) Measurements of TRP metabolites.
Plasma factors were measured after plasma was separated from blood (3000 rpm/min. at 4˚C). Ethylenediaminetetraacetic acid (EDTA) was used as an anticoagulant.
The simultaneous measurements of TRP metabolites in plasma were performed by using an ultrahigh-speed liquid chromatography/spectrometry. Detailed methodology was described elsewhere [5] [6] [7] [8] [13]. TRP metabolic pathways are shown in Figure 1. Figure 1 shows how each metabolite of TRP is located in the map.

Statistics
Statistical significance of the differences between groups was calculated according to one-way ANOVA. When ANOVA indicated a significant difference (P < 0.05), the mean values of the treatment were compared using Tukey's least significant difference test at P < 0.05. The statistical comparison between groups is analyzed by the two-way ANOVA. Mann-Whitney U test was also used for evaluating statistical significance of the use of drugs.

1)
Tryptophan metabolites of patients with MDD. Table 1 shows that by using one way ANOVA tests in patients of MDD only KYN levels of young men were higher than young women and old men. There are no significant difference in other metabolites among 4 groups. Table 2 shows that KYN level of old men was higher that of other 3 groups and 5-HIAA level was higher that other 3 groups. The level of KYNA of old men was higher that that of young women. Table 3 shows following results.
The results of two-way ANOVA were as follows: The ratios of products per substrate (TRP) indicate the rates of the production of the products. Table 4 shows that there was no significant difference in the rates of the production of the products from TRP in patients of MDD and BD.  Table 4 shows that 5-HIAA/TRP ration was higher in BD patients than MMD patients. Table 5 shows that the presence of old men resulted in increase in 5-HIAA/TRP ratio.
2-2; Influences of drug administrations on substrate product ratios.  Table 6 shows that using U test, KYN/TRP ratio was higher in patients who took SSRI/SNRI.

Discussion
TRP is an essential amino acid needed not only for the protein synthesis but its metabolites have important functions in many physiological and pathological processes [14]. Humans do not synthesize TRP, which must be taken from diet. The required daily dose is considered to be 3.5 mg per kg of weight [15]. Less than 1% of ingested TRP is used for protein synthesis and the majority of TRP is metabolized along three known pathways, Indole-3-acetic acid (IAA), 5-HT and KYN pathways. The majority of TRP is enters from the gut to portal circulation and metabolized in the liver. The remaining TRP, together with liver degradation products, transported to the brain, heart and skeletal muscle.
H. Tomioka et al. For many people TRP is best known for a substance to generate 5-HT and melatonin [17]. Our results will be shown and discussed later. Since 5-HT is very important in the regulation of mood, anxiety and cognition, people tend to underestimate roles of KYN as a metabolite of TRP. In mammals, the majority of free TRP is degraded through KYN pathway and many metabolites are generated in inflammation, immune responses and neurotransmission [18]. Metabolites of KYN pathway have been linked to several psychiatric and mental health disorders such as depression and schizophrenia [19]. Sixty percent of KYN are transported from the periphery through blood brain barrier (BBB) and the rest is produced locally. Stress increases many TRP metabolites [20] but these metabolites have antagonizing actions. QA is an N-methyl-p-aspartate receptor agonist and KNA is an NMDAR and α7 nicotinic acetylcholine receptor (α7nAChR) an-tagonist. The balance of the levels of these agonist and antagonist is shown to be dysregulated in depression and schizophrenia [21] Although the concentration of 5-HT has been considered to be low in depressive patients [22], 5-HT concentration in the brains of suicide victims is not low [4]. Therefore, it is not known if 5-HT concentration is decreased in the brains of depressive patients.
By using an ultra high-speed LC/MS, we wanted to know changes in plasma levels of TRP metabolites, especially 5-HT and 5-HIAA (6). Although plasma serotonin levels and 5-HT/TRP ratio were low in depressive patients, the levels of 5-HIAA/TRP were not lower in depressive patients. This result indicates that 5-HT is degraded to 5-HIAA in depressive patients almost to the same extent to healthy old and young women.
We measured the levels of KYN in healthy old men and young women, and depressive patients. TRP seems to be degraded to KYN pathways to the same extent in these three groups.
These results suggest that in depressive patients 5-HT was quickly degraded to 5-HIAA, and this seems to be a reason of low 5-HT levels in depressive patients.
Recently we found that there are significant gender and age differences in plasma levels of TRP metabolites [23]. Plasma levels of 5-HT, 5-HIAA, KYN, AA are higher in young women and old men that young men and old women, but plasma levels of 3-HKYN and XA are higher in young men and old women than young women and old men.
These results mean that there are significant age and gender difference in plasma levels of TRP metabolites.
We then measured plasma levels of TRP metabolites in MMD and BD and found that such significant gender and age difference disappear in MMD patients. Although some changes exist in BD patients, they are not so significant as shown in healthy people.
It is not probable that such changes are due to use of drugs for depression. Probably in patients of depression severe hormonal change may influence TRP metabolism.
As to a relationship between serotonin pathway and KYN pathway, Lapin IP suggested that in depression tryptophan 2,3-dixygenase in the liver shunted metabolism of serotonin away from 5-HT production to KYN production, resulting in serotonin deficiency [24]. KYN, QN and 3-HKN were shown to be anxiogenic and KNYA were anxiolytic [25]. From these results, he tried to explain the effects of anti-depressive drugs.
Our results do not support this hypothesis. Metabolites of KYN pathways were not high in depressive patients.
There have not been enough studies as to 5-HT levels in the brain of patients of bipolar depression. Serotonin levels in cerebrospinal fluids of patients of bipolar depression were shown to be high [26] or normal [27]. So, it seems to be very important to discriminate monopolar and bipolar depression to study roles of serotonin in the pathogenesis of disease.
Our results show that plasma 5-HT levels were low and metabolites of KYN pathway were not changed in patients of monopolar depression.
Comparison of TRP metabolites in healthy people and MMD or BD patients will be reported soon.
Finally, relationship of TRP metabolites levels between brain and plasma should be discussed.
It is well known that 5-HT does not pass the blood brain barrier [28]. It has been suggested that the integrity of the endothelial cell lining of the cerebrovascular bed constitutes a morphological blood-brain barrier mechanism to neurotransmitter monoamines. Circulating monoamines are prevented from entering the brain primarily at the luminal membrane of the endothelial ling.
So, we wonder whether the measurements of TRP levels in the blood may represent their changes in the brain.
We have reported changes in TRP metabolites levels in the bran and plasma after the application of electric shocks in rats [29] [30] [31] [32]. Most of TRP metabolites including 5-HT and 5-HIAA in the brain and plasma increased immediately after electric shock and returned to normal 30 min. later. These results suggest that plasma levels of TRP metabolites may represent those in the brain, of which the reasons we could not speculate. However, we can get some metabolic informations in the brain by analyzing plasma levels of TRP metabolites such as 5-HT, 5-HIAA, or KYN.

Ethics
This work has been approved by Institutional Review Board of Showa University Northern Yokohama Hospital (No. 1508-11) and has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments.

Financial Support
The present study was financially supported by grants in-aid from the Ito Memorial Foundation and NPO "International products on Food and Health".