¹³C-tryptophan breath test detects increased catabolic turnover of tryptophan along the kynurenine pathway in patients with major depressive disorder

Abstract

Altered tryptophan–kynurenine (KYN) metabolism has been implicated in major depressive disorder (MDD). The l-[1-¹³C]tryptophan breath test (¹³C-TBT) is a noninvasive, stable-isotope tracer method in which exhaled ¹³CO₂ is attributable to tryptophan catabolism via the KYN pathway. We included 18 patients with MDD (DSM-IV) and 24 age- and sex-matched controls. ¹³C-tryptophan (150 mg) was orally administered and the ¹³CO₂/¹²CO₂ ratio in the breath was monitored for 180 min. The cumulative recovery rate during the 180-min test (CRR_0–180; %), area under the Δ¹³CO₂-time curve (AUC; %*min) and the maximal Δ¹³CO₂ (C_max; %) were significantly higher in patients with MDD than in the controls (p = 0.004, p = 0.008 and p = 0.002, respectively). Plasma tryptophan concentrations correlated negatively with C_max in both the patients and controls (p = 0.020 and p = 0.034, respectively). Our results suggest that the ¹³C-TBT could be a novel biomarker for detecting a subgroup of MDD with increased tryptophan–KYN metabolism.

Introduction

The kynurenine (KYN) pathway of l-tryptophan metabolism has gained increased attention in research into depression^{1,2,3,4,5,6,7}. Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO, tryptophan pyrrolase) are the two initial and rate-limiting enzymes that convert tryptophan to KYN^1,3,8,9. IDO is activated by proinflammatory cytokines, such as interferon (IFN)-α, IFN-γ and tumor necrosis factor (TNF)-α^6,9,10,11,12, which in addition to other inflammatory molecules such as interleukins, chemokines and prostaglandins have been associated with major depressive disorder (MDD)^{2,6,11,13,14,15}. The neuroactive KYN metabolites kynurenic acid (KA) and quinolinic acid (QUIN) are synthesized in the KYN–KA and KYN–nicotinamide adenine dinucleotide (NAD) pathways, respectively (Fig. 1). KA acts as an excitatory amino acid receptor antagonist, whereas QUIN has an excitotoxic effect. The activation of IDO promotes tryptophan catabolism in the KYN pathway and may cause reduced tryptophan availability for serotonin synthesis in the brain, resulting in imbalance or misregulation of the neuroactive metabolites. This mechanism may explain both MDD and the depressive symptoms observed in patients with inflammatory diseases and in those undergoing cytokine therapies^3,16,17.

Figure 1

Schematic view of ¹³C-tryptophan metabolism.

After utilization for protein synthesis, over 90% of dietary tryptophan is converted to KYN by one of two rate-limiting enzymes: by TDO in the liver under healthy conditions and by IDO in various human tissues (e.g., intestines, lungs, placenta, kidneys, spleen, blood and brain) under inflammatory conditions. The ¹³CO2 derived from ¹³C-tryptophan is produced by the conversion of 3-OH-KYN into 3HAA via the KYN–NAD pathway (box with solid line) as the post-KYN metabolism. In addition, although ¹³CO2 is produced to a minor degree in the tryptophan–serotonin and anthranilic acid pathways, it is not produced by the KA (box with dotted line) or xanthurenic acid pathways. Bold arrows indicate quantitatively major pathways. Minor pathways and pathways without release of ¹³CO2 are omitted. Red circles indicate the ¹³C-labeled carbon. Abbreviations: IDO, indoleamine 2,3-dioxygenase; KA, kynurenic acid; KYN, kynurenine; NAD, nicotinamide adenine dinucleotide; TDO, tryptophan 2,3-dioxygenase; 3HAA, 3-hydroxyanthranilic acid; 3-OH-KYN, 3-hydroxy-l-kynurenine.

To clarify this mechanism, a number of clinical studies have used blood and cerebrospinal fluid (CSF) samples from patients undergoing cytokine therapy. These have evidenced decreased tryptophan levels, increased KYN levels, increased KYN/tryptophan ratios (used to estimate peripheral IDO activity) and correlations between depressive symptoms and levels of tryptophan and its metabolites^{15,16,17,18,19}. However, contradictory results have also been reported^20,21.

Several lines of evidence, including our recent meta-analysis²², have demonstrated decreased tryptophan levels in MDD^{23,24,25,26,27}. However, it remains controversial whether the KYN pathway is activated in MDD. Indeed, although several studies have reported increased KYN/tryptophan ratios^23,28,29, or its association with depression severity in MDD³⁰, some negative results have also been reported^24,31. These inconsistent findings, therefore, require further investigation. Furthermore, few studies have investigated the role of post-KYN metabolism: specifically, distinguishing between the KYN–KA and the KYN–NAD pathways in MDD²⁸.

Cross-sectional quantitative data, obtained from peripheral blood or CSF samples, may provide a reasonable “snapshot”, reflecting total accumulation at the sampling time and site, but cannot measure changes in the synthesis or turnover rates of the metabolites. Indeed, these rates can alter without change in the total amount of metabolites in a dynamic steady-state equilibrium³². Additionally, metabolite turnover rates could be altered after any compensatory changes for the total amount of metabolites. Furthermore, because tryptophan, KYN and 3-hydroxykynurenine (3-OH-KYN) readily cross the blood–brain barrier (BBB)^33,34,35, fluctuations in peripheral blood levels can directly affect metabolism of KYN pathway metabolites, including the synthesis of KA, 3-hydroxyanthranilic acid (3HAA) and QUIN in the brain, although these three metabolites do not readily cross the BBB^36,37. IDO is distributed in various human tissues, including the intestine, lungs, placenta, kidneys, spleen, brain and blood⁸ and when peripherally activated, IDO can affect the concentration of QUIN in the brain and CSF³⁸. Raison et al. reported that there were significant positive correlations between peripheral and central measures of KYN and QUIN, but not tryptophan and that peripheral tryptophan decreased while CSF tryptophan remained unchanged in patients receiving IFN-α therapy¹⁶. These factors point to the merit of monitoring KYN pathway activation in the whole body.

Tracer tests are advantageous for assessing in vivo metabolism of biological compounds as a more direct approach. Using a ¹³C-phenylalanine breath test, our group reported that altered phenylalanine metabolism could be identified in patients with schizophrenia³⁹. Hankes et al. administered a ¹⁴C-tryptophan breath test to patients with scleroderma and reported that expiratory ¹⁴CO₂ was decreased and labeled QUIN in the urine was increased^40,41. Therefore, they suggested that metabolism after 3HAA may be altered in these patients. They also reported that the production of ¹⁴CO₂ from ¹⁴C-labeled d-tryptophan was different from that of ¹⁴C-labeled l-tryptophan and suggested that labeled l-tryptophan should be used for studies of tryptophan metabolism. In the present study, we used a stable isotope (i.e., non-radioactive tracer) in an l-[1-¹³C]tryptophan breath test (¹³C-TBT) to examine the whole-body kinetics and metabolism of orally administered tryptophan. In this method, the ¹³C label of l-[1-¹³C]tryptophan is released as ¹³CO₂ when 3HAA, an immediate precursor for QUIN, is produced from tryptophan via the KYN–NAD pathway both peripherally and in the brain. This method also selectively observes the KYN–NAD pathway, but not the KYN–KA pathway, because ¹³CO₂ is not produced during the synthesis of KA or xanthurenic acid. Therefore, ¹³CO₂ formation measured by the ¹³C-TBT is an index of whole-body tryptophan metabolism via the KYN pathway and excludes the pathways for KA and xanthurenic acid production.

The aim of this study was to assess KYN pathway activity in patients with MDD. We hypothesized that the ¹³C-TBT would detect altered tryptophan metabolism via the KYN pathway in patients with MDD and that the ¹³C-TBT indices would correlate negatively with plasma tryptophan concentrations.

Methods

Participants

We enrolled 18 patients with MDD (age range: 25–62 years; 10 women) and 24 healthy volunteers (age range: 21–61 years; 12 women), matched for age and sex and who were biologically unrelated. Participants were recruited through advertisements in free local information magazines, by word of mouth and via a website announcement; all were Japanese residents from the western part of Tokyo. Patients were also recruited from the National Center of Neurology and Psychiatry (NCNP) Hospital, Tokyo, Japan.

Consensus diagnoses were made for MDD by at least two experienced psychiatrists according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV)⁴², based on the Japanese version of the Mini-International Neuropsychiatric Interview (MINI)⁴³, careful examination of medical records, observations and additional unstructured interviews.

Healthy volunteers were screened by psychiatrists using unstructured interviews and the MINI. We confirmed that they had no history of psychiatric illness or contact with psychiatric services. Participants were excluded from both the patient and control groups if they met the DSM-IV criteria for intellectual disability, had a history of substance dependence or abuse, or had a history of severe head injury. We also excluded participants if they had a current serious physical disorder, particularly an inflammatory or liver disease (where tryptophan is primarily metabolized) or if they received any immunotherapy. All participants underwent blood tests for aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein, albumin, total bilirubin, blood urea nitrogen (BUN) and creatinine levels to exclude liver and kidney dysfunction. Individuals with abnormal blood test results were excluded.

The present study was conducted in accordance with the principles of the Declaration of Helsinki. The capacity of patients to understand the nature and purpose of the study and to provide consent was confirmed by an experienced psychiatrist. After explaining the study aim and protocol, signed informed consent was obtained from each participant. The study was approved by the ethics committee of the NCNP, Japan.

Principle of the ¹³C-TBT

The kinetic values for l-[1-¹³C]tryptophan were assumed to be the same as for unlabeled tryptophan⁴⁴. The following theory underpinned the ¹³C-TBT. After protein synthesis, dietary tryptophan, an essential amino acid for humans, is primarily metabolized along the KYN pathway in the liver, which accounts for over 90% of tryptophan catabolism³⁵; in contrast, only 1%–3% of dietary tryptophan is metabolized via the serotonin pathway³⁴. Under healthy conditions, the conversion of tryptophan to KYN via N-formyl-l-kynurenine is initiated by TDO, which is activated by stress and high glucocorticoid levels^1,2,45. Thus, this is the product of elevated hypothalamic pituitary-adrenal (HPA) axis activity, which is often implicated in the development of depressive disorders^46,47. In conditions where the immune system is stimulated, IDO is induced by proinflammatory cytokines^2,3,7,48. Crucial to the test is that the ¹³CO₂ derived from orally administered ¹³C-tryptophan can be produced by the conversion of 3-OH-KYN into 3HAA via the KYN–NAD pathway, but not by the KA or xanthurenic acid pathways (Fig. 1). The ¹³CO₂ is also produced to much lesser degrees by minor pathways, such as the tryptophan–serotonin and anthranilic acid pathways. The ¹³CO₂ measured by the ¹³C-TBT was, therefore, expected to reflect whole-body tryptophan–KYN–3HAA metabolism initiated by IDO or TDO.

The maximal Δ¹³CO₂ (C_max; %), time to reach the C_max (T_max; min), area under the Δ¹³CO₂-time curve (AUC; %*min) and the ratio of the total amount of exhaled ¹³CO₂ to the administered dose of ¹³C-tryptophan (i.e., the cumulative recovery rate, CRR; %) were calculated from the Δ¹³CO₂ (%) at each sampling point. Observations were made by infrared (IR) spectrometry in which IR absorption intensities of ¹²CO₂ (2380 ± 10 cm⁻¹) and ¹³CO₂ (2280 ± 10 cm⁻¹) were measured, as described previously^39,49.

Protocol for the ¹³C-TBT

The ¹³C-TBT was performed as previously described for the ¹³C-phenylalanine breath test^39,50. Blood samples were collected after an overnight fast and stored at −20 °C until biochemical analysis (AST, ALT, total protein, albumin, total bilirubin, BUN, creatinine and tryptophan levels) by the SRL Corporation (Tokyo, Japan). The plasma tryptophan level was measured by liquid chromatography–mass spectrometry. A dose of 150 mg/body of l-[1-¹³C]tryptophan (99 atom% ¹³C; Cambridge Isotope Laboratories, Cambridge, UK) was orally administered in 100 ml of water at 1000 h. Breath samples were collected in 250-ml breath-sampling bags (Otsuka Electronics Co., Ltd., Osaka, Japan) at 10, 15, 20, 30, 45, 60, 90, 120, 150 and 180 min after ingestion of ¹³C-tryptophan. The ¹³CO₂ /¹²CO₂ ratio in exhaled air was analyzed by IR spectrometry (UBiT-IR300 and UBiT-AS10, Otsuka Pharmaceutical, Tokyo, Japan).

Clinical status and antidepressant medication

Current depression severity in patients was assessed using a 21-item version of the Hamilton Depression Rating Scale (HAMD-21) administered by a single experienced psychiatrist. Antidepressant doses were converted to imipramine-equivalent (IMIeq) doses according to published guidelines^51,52.

Statistical analysis

The Statistical Package for the Social Sciences (SPSS) version 11.0 (SPSS Japan, Tokyo, Japan) was used to conduct all statistical tests. Averages were expressed as mean ± standard deviation (SD). Statistical differences in the demographic data between groups were compared using the χ² test and t-test for categorical variables and continuous variables, respectively. We then performed stepwise multiple regression analyses with two models, using the ¹³C-TBT indices (C_max, AUC and CRR_0–180) as the dependent variable: in model 1, diagnosis, sex, age and body weight were entered as possible predictors; and in model 2, diagnosis, sex, age, body weight and plasma tryptophan concentration were entered as possible predictors.

A 2 × 10 (group × sampling point) repeated measures analysis of covariance (ANCOVA) was conducted to examine group differences in Δ¹³CO₂ or CRR over time, controlling for sex, age and body weight. Greenhouse–Geisser corrections were used for lack of sphericity. We used ANCOVA (controlling for sex, age and body weight) and t-tests to compare ¹³C-TBT indices between the two groups. The T_max, a discrete variable, was compared between the two groups using the Mann–Whitney U test. The correlations of each ¹³C-TBT index (AUC, CRR_0–180 and C_max) with the liver parameters and the plasma tryptophan concentrations were examined by Spearman’s and Pearson’s correlation analyses, respectively. Cramer’s V, Cohen’s d, η² and partial η² were calculated as measures of effect size for χ² test, t-tests, ANOVAs and ANCOVAs, respectively. In the patients, the correlations of each ¹³C-TBT index (AUC, CRR_0–180 and C_max) with IMIeq and HAMD-21 total score were examined using Spearman’s correlation analysis. A stepwise multiple regression was employed to assess independent predictors of C_max, AUC and CRR_0–180, using sex, IMIeq, HAMD-21 total score and plasma tryptophan concentration as candidate predictors. Statistical significance was two-tailed and set at p < 0.05.

Results

Demographic and clinical characteristics

The demographic and clinical characteristics of the patients and controls are presented in Table 1. There was no significant difference in sex, age, body weight, body mass index, education length, liver or kidney blood test results, or plasma tryptophan concentrations between the two groups.

Table 1 Demographic and clinical characteristics of patients with major depressive disorder and healthy controls.

Predictors for ¹³C-TBT indices

Stepwise regression analyses were performed (Table 2). In model 1, diagnosis and sex were identified as significant predictors for C_max, AUC and CRR_0–180. In model 2, diagnosis and sex remained significant predictors for C_max, AUC and CRR_0–180, but the plasma tryptophan concentration was not a significant predictor in the model.

Table 2 Stepwise multiple regression for l-[1-¹³C]tryptophan breath test indices as dependent variables.

Increased C_max, AUC and CRR in MDD

Figure 2 shows the time course of mean values of Δ¹³CO₂ (Fig. 2a) and CRR (Fig. 2b) in patients and controls. The repeated measures ANCOVA, controlling for sex, age and body weight, indicated significant differences between the two groups in both Δ¹³CO₂ and CRR over time. The repeated measures ANCOVA with Greenhouse–Geisser adjustment revealed significant interactions between the diagnosis and sampling point for both the Δ¹³CO₂ and the CRR, indicating different patterns of change over time between the two groups. The Δ¹³CO₂ and CRR values were higher in the patients than in the controls at all sampling points during the 180-min test (Fig. 2 and Table S1).

Figure 2

(a) Time course for the Δ¹³CO2 in expired air after the ¹³C-tryptophan breath test was applied to patients with major depressive disorder (MDD) and to healthy controls. (b) Time course for the cumulative recovery rate (CRR; %) in expired air for patients with MDD and for healthy controls. Abbreviations: MDD, major depressive disorder group; CON, control group; Time, time after ingesting the solution of ¹³C-tryptophan (150 mg).

Using t-tests, the C_max, AUC and CRR_0–180 were all significantly higher in the patients than in the controls and these differences remained significant after ANCOVA controlling for sex, age and body weight (Table 3). The T_max was reduced in the patients compared to the controls at the trend level.

Table 3 l-[1-¹³C]tryptophan breath test results in patients with major depressive disorder and healthy controls.

Association of ¹³C-TBT indices with tryptophan concentrations and clinical variables in patients

Both C_max (Pearson’s correlation coefficient [r] = −0.54, p = 0.020) and CRR_0–180 (r = −0.48, p = 0.045) showed significantly negative correlations with plasma tryptophan concentrations in the patient group. In the controls, a significantly negative correlation was also observed for the plasma tryptophan concentrations with the C_max of the ¹³C-TBT (r = −0.43, p = 0.034). Further, stepwise multiple regression analysis demonstrated that plasma tryptophan concentrations were significant predictors of C_max, while sex predicted the AUC and CRR_0–180 (Table S2). In addition, both the AUC [Spearman’s rank correlation coefficient (ρ) = −0.53, p = 0.023] and CRR_0–180 (ρ = −0.50, p = 0.033) were significantly and negatively correlated with the IMIeq dose. There were no significant correlations between ¹³C-TBT indices and depression severity (HAMD-21 score; data not shown). There were also no significant correlations between the ¹³C-TBT indices and the liver tests in the participants (Table S3).

Discussion

We examined, for the first time, whole-body tryptophan metabolism using a ¹³C-TBT in patients with MDD and found that levels of exhaled ¹³CO₂ were significantly increased in patients compared with healthy controls. Although plasma tryptophan concentrations were not significantly different between the two groups, tryptophan showed significant and negative correlations with the C_max in both the patient and control groups. In our ¹³C-TBT, exhaled ¹³CO₂ is mainly attributable to the conversion of tryptophan via KYN into 3HAA. Therefore, our results support the study hypotheses and corroborate previous findings of KYN-pathway activation in MDD (i.e., the serotonin–KYN hypothesis)^48,53.

The ¹³CO₂ measured by our ¹³C-TBT can be produced by either the KYN pathway or the serotonin pathway (Fig. 1). Although just 1%–3% of dietary tryptophan is metabolized via the serotonin pathway³⁴, expiration of ¹⁴CO₂ (produced by the conversion of ¹⁴C-labeled 5-hydroxytryptophan to serotonin) can be measurably decreased in depression⁵⁴. Therefore, in our patients with MDD, the observed increase in expired ¹³CO₂ could be attributed to increased metabolism in the KYN pathway rather than the serotonin pathway. Specifically, the observed increase in ¹³CO₂ could relate to increased conversion of 3-OH-KYN to 3HAA (Fig. 1), suggesting an increased production of KYN and 3-OH-KYN (precursors of 3HAA), as well as QUIN (the immediate catabolite of 3HAA). Importantly, 3-OH-KYN and 3HAA have neurotoxic effects that are mediated by oxidative stress^2,55. In addition, QUIN not only has neurotoxic effects mediated by the activation of NMDA-type glutamate receptors^2,56 but also induces lipid peroxidation and oxidative stress⁵⁷. Therefore, our results are consistent with the neurodegeneration hypothesis of MDD⁴⁸. Although tryptophan, KYN and 3-OH-KYN are able to cross the BBB^33,34,35, KA, 3HAA and QUIN cannot do so readily^36,37. It has also been reported that there are significant positive correlations between peripheral and central measures of the KYN and QUIN, but not tryptophan¹⁶. Further studies are warranted to elucidate the relationship between concentrations of peripheral and central KYN pathway metabolites and indices of ¹³C-TBT. These neurotoxic metabolites in the KYN pathway are also implicated in several neurodegenerative disorders^11,12,58. It may, therefore, be interesting to examine the possible clinical use of the ¹³C-TBT in these disorders.

We observed significant increases in ¹³C-TBT indices in the patient group, but found no significant difference in the plasma tryptophan concentrations between patients and controls. This result is inconsistent with previous data, including the results of our own meta-analysis^{22,23,24,25,26,27}. The inconsistency may have occurred due to the small sample size and the modest severity of depression in our patients (mean HAMD-21 score of 12.6), as well as the modest effect size. Indeed, our meta-regression analysis found a negative correlation between plasma tryptophan concentrations and depression severity²². Moreover, some previous studies have reported no significant difference in plasma tryptophan levels between patients with MDD and healthy controls^59,60,61. Our results may suggest that ¹³C-TBT is more sensitive than plasma tryptophan levels at detecting increased metabolism in the KYN pathway.

As shown in Table 3, differences existed between the 2 groups in most of the pharmacokinetic parameters, including the AUC, CRR_0–180 and C_max, but not the T_max. All these parameters are affected by gastric emptying, digestive absorption, metabolism rates and respiratory status⁶². The AUC reflects the global amount of exhaled ¹³CO₂ during the test session. However, although the CRR_0-180 also reflects the global process, it may be a better index because it takes into account the body surface area. The CRR may therefore be the best parameter for biomarker research in depression, particularly given the high statistical significance. Finally, while T_max is particularly subject to the influence of digestion, C_max could be more relevant because it more closely reflects the metabolic rate.

There are several limitations to this study. First, the sample size was small and the patients with MDD had only modestly severe disease. In addition, depression severity was not measured for the control sample. Despite conflicting results³¹, there is normal variability in mild depression symptoms in the healthy population together with evidence of a relationship between depressive symptoms and the KYN/tryptophan ratio³⁰. Such methodological limitation might have minimized, rather than exaggerated the difference between the MDD and control groups in ¹³C-TBT indices. Further studies are needed to address these issues and should include larger samples, patients with more severe forms of MDD and assessment for depressive symptoms among healthy controls. A second limitation is that we included 6 patients who had co-morbid diagnoses of dysthymia or an anxiety disorder. There is some evidence suggesting that there is a different KYN metabolism pathway between anxiety and depression⁶³. To examine the possible influence of the comorbidity on the current results, we re-analyzed the data, excluding the 6 patients with MDD comorbidities. However, the results were essentially unchanged and the significant differences in ¹³C-TBT indices remained between the 2 groups (data not shown). In addition, some studies have shown positive associations of suicidality with KYN and QUIN^29,64,65,66. Almost all of the patients in our study (17 of 18) had no history of having attempted suicide. However, because the MDD group in our study might be the heterogeneous group with different biological mechanisms, further studies will be needed with a sample of MDD patients without comorbidity or suicidal behavior. Third, only 10 patients were drug-free, with a further 8 being antidepressant-treated. The AUC and CRR_0–180 correlated negatively with the IMIeq dose. Therefore, because some antidepressants have anti-inflammatory effects⁶⁷, inhibit TDO⁶⁸, or can affect KYN/tryptophan³¹, it is possible that antidepressant treatment minimized the differences in the indices of the ¹³C-TBT and concentrations of plasma tryptophan between the patients and controls. Fourth, although we measured plasma tryptophan, we did not measure any KYN pathway metabolites. Unfortunately, no conclusive relationship can be determined between the measures in this study and the ¹³C-TBT indices, without knowing the data for other KYN pathway metabolites. In addition, we did not obtain data on inflammatory markers such as C-reactive protein, proinflammatory cytokines, or cortisol levels, which may affect depressive symptoms through the KYN pathway³¹. Thus, additional studies are required to examine the relationship between these data and the ¹³C-TBT indices. Lastly, we did not assess the effects of vitamin B6 deficiency, which can interfere with the breakdown of KYN⁶⁹. Pyridoxal phosphate, the active form of vitamin B6, is needed for the conversions of KYN to anthranilic acid and KA and for that of 3-OH-KYN to 3HAA and xanthurenic acid.

In conclusion, the ¹³C-TBT, a noninvasive tracer test, provided evidence of increased tryptophan metabolism along the KYN–NAD pathway in patients with MDD. Consistent with this, ¹³C-TBT indices correlated negatively with plasma tryptophan concentrations. Therefore, whole-body tryptophan metabolism detected by our ¹³C-TBT could be a novel and sensitive biomarker for the diagnosis and subtyping of MDD, with the potential to enhance the development of new drugs for the treatment of MDD. Moreover, when ¹³C-TBT is combined with the ¹³C-phenylalanine breath test³⁹, discrimination between MDD and schizophrenia may be possible.