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Reversal of imbalance between kynurenic acid and 3-hydroxykynurenine by antipsychotics in medication-naïve and medication-free schizophrenic patients
Brain, Behavior, and Immunity 25 (2011) 1576–1581
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Reversal of imbalance between kynurenic acid and 3-hydroxykynurenine by antipsychotics in medication-naïve and medication-free schizophrenic patients A.M. Myint a,b,⇑, M.J. Schwarz b, R. Verkerk c, H.H. Mueller d, J. Zach b, S. Scharpé c, H.W.M. Steinbusch a, B.E. Leonard a,b, Y.K. Kim e a
Division of Cellular Neuroscience, Department of Psychiatry and Neuropsychology, University of Maastricht, 6200 MD Maastricht, The Netherlands Laboratory of Psychoneuroimmunology, University Psychiatric Hospital, Ludwig-Maximilians University, D-80336 Munich, Germany Department of Clinical Biochemistry, Institute of Pharmaceutical Sciences, University of Antwerp, 2610 Antwerpen-Wilrijk, Belgium d Institute of Medical Informatics, Biometry and Epidemiology, Ludwig-Maximilians University, D-81377 Munich, Germany e Department of Psychiatry, Ansan Korea University Medical Centre, Korea University, 425-020 Ansan, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 7 March 2011 Received in revised form 6 May 2011 Accepted 12 May 2011 Available online 17 May 2011 Keywords: Schizophrenia Kynurenine Kynurenic acid 3-Hydroxykynurenine Antipsychotics
a b s t r a c t The association between the pro-inflammatory state of schizophrenia and increased tryptophan degradation into kynurenine has been reported. However, the relationship between metabolites from subdivisions of the kynurenine pathway, kynurenic acid and 3-hydroxykynurenine, remains unknown. The present study tested the relationship between these kynurenine metabolites in the plasma of medication-naïve (n = 35) or medication-free (n = 18) patients with schizophrenia at admission and following 6-week antipsychotic treatment compared to healthy controls (n = 48). The plasma concentrations of kynurenic acid (nmol/l) were lower (difference = 8.44 (13.22 to 3.65); p = 0.001) and of 3-hydroxykynurenine (nmol/l) were higher (difference = 11.24 (8.11–14.37); p < 0.001) in the patients compared with the healthy controls. The kynurenic acid/kynurenine (difference = 2.75 (5.115 to 0.336); p = 0.026) and kynurenic acid/3-hydroxykynurenine (difference = 1.08 (1.431 to 0.729); p < 0.001) ratios were also lower in the patients. After the 6-week treatment, the patients’ plasma kynurenic acid levels (difference = 3.85 (0.23 to 7.94); p = 0.064) showed a trend towards an increase, whereas plasma 3-hydroxykynurenine levels (difference = 22.41 (19.76–25.07); p < 0.001) decreased. As a consequence, the kynurenic acid/3-hydroxykynurenine ratio (difference = 4.41 (5.51 to 3.3); p < 0.001) increased. Higher initial plasma kynurenic acid levels on admission or increased kynurenic acid/kynurenine ratio after treatment were associated with reduction of clinical symptoms scores upon discharge although higher kynurenic acid/kynurenine on admission may induce higher positive symptoms score. In contrast, higher 3-hydroxykynurenine is associated with lower positive symptoms score. These results indicate that there is an imbalance in the kynurenine pathway in schizophrenia. The 6-week antipsychotic treatment may partially reverse the imbalance in kynurenine metabolism and that in turn induces clinical response. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction The involvement of kynurenine metabolites, especially kynurenic acid (KYNA), in the pathophysiology of schizophrenia has been widely reported (Schwarcz et al., 2001; Erhardt et al., 2001). The role of 3-hydroxykynurenine (3HK) in schizophrenia has also been reported (Yao et al., 2010; Condray et al., 2011). However, the balance between these neuroactive metabolites of the tryptophan breakdown pathway, called the kynurenine (KYN) pathway, remains unknown. ⇑ Corresponding author at: Laboratory of Psychoneuroimmunology, University Psychiatric Hospital, Ludwig-Maximilians University, Nussbaumstrasse 7, D-80336 Munich, Germany. Fax: +49 89 5160 5890. E-mail address: [email protected] (A.M. Myint). 0889-1591/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2011.05.005
Tryptophan metabolism occurs in the liver and other sites, including immune cells in the blood, lungs and astrocytes and microglia in the brain. Pro-inflammatory cytokines, such as interferon-c (Carlin et al., 1989; Yasui et al., 1986), stimulate indoleamine 2,3-dioxygenase (IDO), the key enzyme that degrades tryptophan into KYN in the lungs, placenta, blood and brain (Heyes et al., 1993; Mellor and Munn, 1999). Tryptophan breakdown to kynurenine has been shown to increase following peripheral inflammation. As kynurenine can pass through the blood– brain-barrier and 60% of the brain KYN comes from the periphery (Gal and Sherman, 1980), peripheral inflammation is likely to increase central KYN concentrations. After tryptophan is degraded to KYN in the brain, the metabolites from this KYN pathway contribute to neuroprotective and neurotoxic/neurodegenerative changes. KYN is further catabolised
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into 3-hydroxykynurenine (3HK) and the NMDA receptor agonist quinolinic acid (QUINA) (Chiarugi et al., 2001; Bender and McCreanor, 1985) (Fig. 1) HK causes neuronal apoptosis (Okuda et al., 1998), while QUINA causes excitotoxic neurodegenerative changes (Schwarcz et al., 1983). However, KYN can also be catabolised into kynurenic acid (KYNA) (Fig. 1), which is an NMDA receptor antagonist (Perkins and Stone, 1982) and is therefore protective against the excitotoxic action of QUINA (Kim and Choi, 1987; Stone and Darlington, 2002). In addition to enhanced IDO activity, proinflammatory cytokines also enhance the activity of kynurenine3-monooxygenase (KMO), the enzyme that converts KYN into 3HK, which in turn enhances the excitotoxic degenerative arm of the pathway. The involvement of immune changes in schizophrenia with respect to the imbalance between T-helper type-1 (Th1) cytokines, most of which are pro-inflammatory, and Th2 cytokines, most of which are anti-inflammatory, has been reported, although the findings are controversial (Avgustin et al., 2005; Kim et al., 2004; Pae et al., 2006; Schwarz et al., 2001b; Arolt et al., 2000; Rothermundt et al., 1998; Sperner-Unterweger et al., 1999; Schwarz et al., 2001a). The involvement of inflammation in schizophrenia was also supported by the increase in the inflammatory fingerprint at the transcriptome level in schizophrenic patients (Drexhage et al., 2010). Moreover, the pro-inflammatory state and enhanced tryptophan breakdown in schizophrenia has also been reported (Kim et al., 2009). Therefore, it is possible that the high IDO and KMO activities induced by the pro-inflammatory state in schizophrenia may result in the high formation of toxic 3HK and QUINA syntheses, while KYNA formation would be decreased. However, the KYNA can also induce abnormal behaviour by interfering with glutamatergic transmission if it is present in excess. Micro-molar brain levels of KYNA have been reported to be associated with disruption of auditory sensory gating in rats, and this association was due to down-regulation of the permissive action of 5-hydroxy indole (5HI) at a-7 nicotinic acetylcholine receptors (a-7nChR) caused by high KYNA concentrations (Shepard et al., 2003). KYNA has also been shown to induce psychotic symptoms through antagonism at a-7nChR (Schwieler and Erhardt, 2003). In addition, KYNA concentrations have been found to be increased in Brodmann area 9 (Schwarcz et al., 2001) and the anterior cingulate area (Miller et al., 2006) of post-mortem schizophrenic brains. Moreover, increased KYNA levels were de-
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tected in the CSF of medication naïve and medicated male schizophrenic patients, but not in medication-free male schizophrenic patients (Nilsson et al., 2005). The role of KYNA as an endogenous NMDA-R antagonist supports the hypothesis of NMDA hypofunctioning in schizophrenia (Olney et al., 1999; Olney and Farber, 1995; Muller and Schwarz, 2006). However, Ravikumar et al. (2000) have reported increases in both QUINA and KYNA in the plasma of small groups of patients with multiple sclerosis, Parkinson’s disease, generalised epilepsy, schizophrenia, gliomas and syndrome X compared with healthy controls. Although the sample sizes were small, this study suggested that both neuroprotective and neurotoxic/neurodegenerative arms of the kynurenine pathway can be increased in those diseases. The above findings raise questions of whether (i) the balance between the two arms of KYN metabolism is disturbed in schizophrenic patients, (ii) there is any effect of KYN metabolites on clinical parameters and (iii) there is any effect of antipsychotic treatment on kynurenine metabolites in the periphery. These questions have been addressed in the present study. 2. Methods 2.1. Subjects Amongst the patients admitted to Korea University Medical Centre from 2003 to 2005, 53 medication-naïve or medication-free patients who met the DSM-IV criteria (APA, 1994) for schizophrenia and completed 6-week antipsychotic therapy as inpatients were recruited. Diagnoses were made using the DSM-IV Structured Clinical Interview (First, 1998). Patients with a history of any concomitant psychiatric illness, such as substance or alcohol abuse, infection or a known autoimmune disease, were excluded. Patients had normal values in routine blood and urine tests, electrocardiograms and electroencephalograms. All patients had acute psychotic symptoms at the time of recruitment. All patients were either medication-naive (n = 35) or medication-free for at least 4 months (n = 18). The psychopathological status of the patients was assessed by a trained psychiatrist using the Positive and Negative Syndrome Scale (PANSS) (Kay et al., 1987) and the Korean version of the Calgary Depression Scale for Schizophrenia (K-CDSS) (Kim et al., 2005). Each patient’s symptoms were assessed again 6 weeks later by the same physician. During the 6-week treatment, 20 patients were medicated with risperidone (4–12 mg), 19 patients were given amisulpride (200– 900 mg), 10 patients were administered olanzapine (10–30 mg) and 4 patients were medicated with aripiprazole (15–30 mg). A total of 48 healthy controls were recruited at the same hospital from the pool of individuals who came in for regular health screenings during the same period. Each control was given a non-structured clinical screening interview. Persons with any personal or familial history of psychiatric illness, autoimmune disease or chronic inflammatory disease and/or substance or alcohol abuse were excluded. All subjects were free of chronic and acute physical illness associated with abnormal immune changes within the 2 weeks before the study. This study was approved by the institutional ethical committee of Korea University and written informed consent was obtained from each patient or control. 2.2. Biochemical analysis
Fig. 1. Changes in the tryptophan catabolic pathway in schizophrenia are shown. The metabolites in blue colour indicated those discussed in this paper. The green arrows show metabolic processes. The respective thickness of each arrow indicates the degree or weight of the action or reaction. Dotted arrows signify inhibition. The grey arrow indicates the concentration increased or decreased.
Early morning venous blood was withdrawn following overnight fasting. Plasma was separated immediately and stored at 70 °C. Blood samples were taken from the patients again 6 weeks later. High performance liquid chromatography (HPLC) was used to
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measure plasma kynurenine and kynurenic acid (KYNA) levels, according to the method of Hervé et al. (1996) with some modifications. To measure 3-hydroxykynurenine (3HK) levels the recently published method using HPLC (Oades et al., 2010a,b) was used. Briefly, KYN was detected spectrophotometrically at 365 nm. KYNA was detected fluorimetrically at an excitation wavelength of 334 nm and an emission wavelength of 388 nm. KYNA was analysed in plasma that was deproteinised using perchloric acid. 3HK was measured at a wavelength of 365 nm by UV detection. All of the analyses were conducted using HPLC with a reverse phase c-18 column. The 3HK analysis method has been validated showing an absolute recovery of 85.8%, intra-day precision of 3.9%, and interday precision of 7.5%; time series demonstrated perfect stability of the analyte 3HK during our extraction and analysis steps; the result of the analysis was even stable up to three times repeated freezing/ thawing cycles. Thus, we could ensure the validity of the analysis in samples that had already been thawed and frozen again. All of these methods have been standardised by a quarterly External Quality Assurance Scheme (EQAS) amongst 6 universities (Ludwig-Maximilians University of Munich, University Medical Centre of Groningen, University of Antwerp, Trinity College Dublin, Illinois University and New South Wales University) under the European collaborative project MOODINFLAME and the Bundesministerium fuer Bildung und Forschung (BMBF) project, which contributed independently to this study. Standards were prepared for each calibration curve, and appropriate internal standards were used for analyses of all metabolites. Data are shown in nmol/l. The intra- and inter-assay coefficients of variation ranged from 5% to 7% for all of the metabolites. To avoid the effect of inter-assay variation, the samples from both schizophrenic patients and controls were placed alternately in each set of analyses. To avoid operator bias, the samples’ order was arranged by the investigator, and the laboratory technician was blind to the information of the sample. For every 20 samples and 3 standards, one quality control sample (pooled medication free EDTA plasma) was used.
admission to discharge on changes in clinical symptoms scores between admission and discharge. The ‘p’ values less than or equal to 0.05 were considered to be statistically significant, without adjusting for multiplicity. SPSS version 18.0 was used for data analyses. 3. Results The general characteristics of patients and controls are presented in Table 1. There was no relevant difference in age, BMI or gender ratio between the two diagnostic groups. 3.1. Comparison of tryptophan metabolites between diagnostic groups The biochemical data of the two diagnostic groups are shown in Table 2. In univariate regression analyses, the schizophrenic group had lower plasma concentrations of KYNA (nmol/l) (difference = 9.05 (13.22 to 3.65); p = 0.001) but higher plasma concentrations of 3HK (nmol/l) (difference = 9.02 (8.11–14.37); p < 0.001) compared to healthy controls. Moreover, the patients had lower KYNA/KYN (difference = 3.69 (5.115 to 0.336); p = 0.026) and KYNA/3HK ratios (difference = 1.07 (1.431 to 0.729); p < 0.001). 3.2. Comparison of tryptophan metabolites in patients on admission vs. discharge After 6 weeks of treatment, plasma KYNA levels (nmol/l) (difference = 3.85 (0.23 to 7.94); p = 0.064) showed a trend towards an increase, whereas plasma 3HK levels (nmol/l) decreased (difference = 22.41 (19.76–25.07); p < 0.001). Although there was no change in the KA/KYN ratio, the KYNA/3HK ratio was significantly reduced (difference = 4.41 (5.51 to 3.3); p < 0.001) (Table 3). The type of medication given during the treatment had no effect on the biochemical or clinical parameters. Table 1 Demographic data.
2.3. Calculations The ratio between plasma KYNA and KYN concentrations enabled the determination of how much KYN would be catabolised into KYNA (Myint et al., 2007):
KYNA=KYN ratio ¼
1000 plasma KYNA ðnmol=lÞ plasma KYN ðnmol=lÞ
The ratio between plasma KYNA and 3HK levels was calculated to determine the balance between the two arms of kynurenine pathway (Oades et al., 2010a,b):
KYNA=OHK ratio ¼
plasma KYNA ðnmol=lÞ plasma OHKðnmol=lÞ
2.4. Statistical analysis The comparison of the general characteristics between the two groups was carried out using Student’s ‘t’ test and Fisher’s exact test. Student’s ‘t’ test was applied to detect differences in the biochemical parameters between the two groups when age, gender and body mass index (BMI) were controlled using univariate regression analysis. Paired ‘t’ tests were applied to calculate the differences between patients’ admission data and their data at the time of discharge. The univariate regression analysis was also applied to analyse the effects of the initial biochemical parameters at admission and changes in these biochemical parameters from
Sex (male/female) Age (years) BMI (kg/m2) Age at onset (years) Duration of total illness (months) Duration of current illness (months) Number of previous admissions (mode and range) Family history of psychiatric illness Medication status on admission Medication-naïve Medication-free (P4 months) Subtypes Paranoid Undifferentiated Disorganised Psychopathology scores PANSS at admission PANSS positive score PANSS negative score PANSS at 6 weeks PANSS positive score PANSS negative score K-CDSS at admission K-CDSS at 6 weeks General symptoms score at admission General symptoms score At 6 weeks
Schizophrenia (n = 53)
Healthy controls (n = 48)
23/30 33.3 ± 12.2 23.19 ± 3.8 27.7 ± 11.2 74.9 ± 88.7 3.6 ± 5.3 0 (0–7)
21/27 32.56 ± 10.32 22.25 ± 3.08
9 35 18 38 14 1
26.16 ± 6.75 22.08 ± 7.55 14.04 ± 4.43 14.02 ± 0.87 8.4 ± 5.8 3.0 ± 3.2 49.23 ± 11.12 29.74 ± 7.45
PANSS, the positive and negative syndrome scale; K-CDSS, the Korean version of the calgary depression scale for schizophrenia.
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Kynurenic acid (nmol/l) 3-Hydroxykynurenine (nmol/l) KYNA/KYN ratio KYNA/3HK ratio
Schizophrenia (n = 53)
Healthy controls (n = 48)
‘p’ Value
26.90 ± 2.25 28.60 ± 1.49
35.95 ± 1.37 19.58 ± 0.77
0.001 <0.0001
15.67 ± 1.20 0.96 ± 0.13
19.36 ± 0.61 2.03 ± 0.15
0.026 <0.0001
‘p’ Values were generated using Student’s ‘t’ test when age, gender and BMI were controlled using an univariate regression analysis.
Table 3 Plasma tryptophan metabolites in schizophrenia patients on admission and at the time of discharge.
Kynurenic acid (nmol/l) 3-Hydroxykynurenine (nmol/l) KYNA/KYN ratio KYNA/3HK ratio
On admission (n = 53)
At discharge (n = 53)
‘p’ value
26.90 ± 2.25 28.60 ± 1.49
30.75 ± 2.27 6.19 ± 0.37
0.064 <0.0001
15.67 ± 1.20 0.96 ± 0.13
15.72 ± 0.93 5.37 ± 0.64
0.968 <0.0001
‘p’ Values were generated using a paired ‘t’ test.
3.3. Associations between tryptophan metabolites at admission and changes in clinical parameters The initial plasma kynurenic acid concentrations were associated with a reduced PANSS positive symptoms score and a reduced K-CDSS depressive symptoms score upon discharge. In contrast, the higher the KYNA/KYN ratio on admission, the higher the PANNS positive symptoms score on admission. However, the higher the KYNA/KYN ratio on admission the higher the reduction in K-CDSS scores at discharge. Moreover, the increase in this ratio after 6-week medication is associated with higher reduction in PANNS positive symptoms, general symptoms and K-CDSS symptoms scores. Initial plasma 3HK concentrations is negatively associated to the admission PANNS positive symptoms score. However, the higher the plasma 3HK on admission the lower the reduction in general symptom scores at the time of discharge. The quantitative results of the regression analyses are presented in Table 4. 4. Discussion The results of this study demonstrate that 3HK concentrations are increased while KYNA concentrations are decreased in the plasma of medication naïve and medication free patients with schizophrenia. This finding indirectly demonstrates the imbalance between the peripheral KYNA and 3HK arms of the KYN pathway. These results also show that the imbalance is corrected following
effective antipsychotic treatment, mainly due to decreases in plasma 3HK levels. In addition, the KYNA/KYN ratio and the KYNA/3HK ratio were lower in the patients. These findings indicate that a greater quantity of KYN was catabolised into 3HK than into KYNA. These data lend credibility to our proposed hypothesis that imbalanced kynurenine metabolites play a role in pathophysiology of schizophrenia. While most of the previous studies on the role of kynurenine metabolites in schizophrenia concentrated mainly on changes in KYNA levels, specifically increases in KYNA, and described the pathophysiology in schizophrenia as a result of NMDA-R hypofunctioning (Olney et al., 1999), our data demonstrated a significant reduction in KYNA and significant increases in 3HK in the plasma. These findings are in accordance with studies that demonstrated increased pro-inflammatory cytokines, such as interferon-c (Avgustin et al., 2005; Kim et al., 2004, 2009), in the peripheral blood of patients, as this cytokine can enhance both IDO and KMO activity, resulting in high 3HK formation (Yasui et al., 1986). Increases in 3HK formation would shift metabolism more to the 3HK arm, causing an increase in QUINA formation. There is evidence of a pro-inflammatory state in the periphery (Kim et al., 2004) and in the brain of patients with schizophrenia (Doorduin et al., 2009). The pro-inflammatory state, leading to production of pro-inflammatory cytokines, may enhance tryptophan breakdown and formation of 3HK and QUINA both in the periphery and in the brain. Both 3HK and QUINA are neurotoxic metabolites. This hypothesis is supported by the findings of Condray et al. (2011) who demonstrated that higher 3HK correlated with lower change in clinical symptoms after 4-weeks treatment in 25 medication-naive and medication-free schizophrenia patients. Our data also showed similar trend in terms of initial 3HK and change in general symptoms score. Loss of brain volume and neuronal density has been well documented in schizophrenia (Theberge et al., 2007; Perez-Neri et al., 2006; Byne et al., 2006, 2007, Seok Jeong et al., 2005; Blennow et al., 1996). As 3HK is neurotoxic and apoptotic (Okuda et al., 1998), it may be hypothesised that high 3HK concentrations could contribute to a loss of brain volume in schizophrenia. The increased 3HK concentrations in patients may be due to (1) enhanced production of both 3HK and QUINA or (2) the accumulation of 3HK due to the blockade of 3HK breakdown into further metabolites, such as 3-hydroxyanthranilic acid (HAA), because of reduced activity of the kynureninase enzyme, which degrades 3HK to HAA (Price et al., 1972) while KMO activity would be enhanced by the pro-inflammatory state. In the latter case, reduction of the breakdown of 3HK may result in reduced formation of the NMDA-R agonist QUINA and would be in line with NMDA hypofunctioning hypothesis. Determination of plasma and brain QUINA concentrations is crucial to determine the exact mechanism. Our data suggest that initial plasma KYNA levels have predictive value on positive symptoms score and depressive symptoms score reductions after 6 weeks of medication. This may be due to higher neuroprotective KYNA availability in the periphery and in the
Table 4 Association between plasma tryptophan metabolites and clinical parameters in schizophrenia patients. Biochemical parameter
Dependent Clinical variable
b
95% CI for b
‘p’ Value
KYNA admission
PANSS positive change CDSS change PANSS positive on admission General symptoms change PANSS positive on admission CDSS change PANSS positive change CDSS change General symptoms change
0.44 0.68 0.258 0.398 0.249 0.204 0.381 0.225 0.607
0.68 to 0.19 1.23 to 0.36 0.443 to 0.073 0.018 to 0.778 0.042 to 0.456 0.4 to 0.008 0.623 to 0.138 0.449 to 0.001 1.005 to 0.159
0.001 0.016 0.007 0.041 0.019 0.041 0.003 0.049 0.009
3HK admission KYNA/KYN admission KYNA/KYN discharge – KYNA/KYN admission
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brain, resulting in greater protection against the neurotoxic effects of 3HK on the glial-neuronal network, and thus enables better responses to treatment. This is again supported by the associations between increase in KYNA/KYN ratio after medication and reduction in clinical symptoms scores after medication. In experimental studies, antipsychotic treatment has been reported to reduce KYNA concentrations in the brain (Olney and Farber, 1994; Ceresoli-Borroni et al., 2006). However, in our study, 6 weeks of antipsychotic treatment slightly increased plasma KYNA levels and dramatically reduced plasma 3HK concentrations. This response to treatment may be associated with a reduction in KMO activity through correction of the pro-inflammatory state following antipsychotic treatment in the same group of patients (Kim et al., 2009), which significantly reduced the plasma 3HK levels. The changes in the brain after medication are still necessary to be investigated. While these findings supported our hypothesis that in schizophrenia with pro-inflammatory state the kynurenine pathway will shift to the 3HK arm, reports from some investigators have suggested the involvement of increased KYNA levels in the pathophysiology of schizophrenia (Schwarcz et al., 2001). Somehow, our data supports those findings since we observed the association between higher KYNA/KYN ratios or the lower 3HK level and higher initial positive symptoms score. Condray et al., 2011 had also observed similar result. Taken together with the findings that higher initial 3HK is associated with lower reduction of symptoms scores, in other words, poorer clinical outcome, it can be concluded that the imbalance between KYNA arm and 3HK arm in both directions might induce negative impact in terms of positive symptoms development and impaired response to treatment. We did not observe the association between change in 3HK after treatment and clinical symptoms scores changes. The reason could be the fact that the reduction in neurotoxic metabolite would result only in slowing down the further neuronal-glial network damage but not in immediate change of clinical symptoms. To clarify these points, further studies are necessary to explore (1) the association between changes in concentrations of kynurenines in the plasma and CSF and the detailed clinical symptoms and relapse or remission in patients with schizophrenia and (2) the detailed postmortem morphological evidence of kynurenine metabolites in different brain areas. For the ethical reasons, it was not possible to obtain CSF from the same patients in this study. Furthermore, we have neither the facilities nor sufficient plasma samples to determine QUINA concentrations. In future studies, we plan to analyse the complete profile of metabolites from this kynurenine pathway in the plasma or serum and CSF. Nevertheless, this study has demonstrated the imbalance between KYNA and 3HK arms in kynurenine metabolism in medication-naïve and medication-free patients with schizophrenia. Our data indicated the involvement of imbalance in kynurenine metabolism in pathophysiology of schizophrenia. Moreover, we have also demonstrated a possible role of plasma kynurenines in predicting responses to treatment. Acknowledgments This study was funded by Advanced Practical Diagnostics n.v., Belgium for the laboratory analyses and Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A040042) for the clinical sample and data collection. References APA, 1994. Diagnostic and statistical manual of mental disorders, American Psychiatric Press, Washington, DC.
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Brain, Behavior, and Immunity journal homepage: www.elsevier.com/locate/ybrbi
Reversal of imbalance between kynurenic acid and 3-hydroxykynurenine by antipsychotics in medication-naïve and medication-free schizophrenic patients A.M. Myint a,b,⇑, M.J. Schwarz b, R. Verkerk c, H.H. Mueller d, J. Zach b, S. Scharpé c, H.W.M. Steinbusch a, B.E. Leonard a,b, Y.K. Kim e a
Division of Cellular Neuroscience, Department of Psychiatry and Neuropsychology, University of Maastricht, 6200 MD Maastricht, The Netherlands Laboratory of Psychoneuroimmunology, University Psychiatric Hospital, Ludwig-Maximilians University, D-80336 Munich, Germany Department of Clinical Biochemistry, Institute of Pharmaceutical Sciences, University of Antwerp, 2610 Antwerpen-Wilrijk, Belgium d Institute of Medical Informatics, Biometry and Epidemiology, Ludwig-Maximilians University, D-81377 Munich, Germany e Department of Psychiatry, Ansan Korea University Medical Centre, Korea University, 425-020 Ansan, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 7 March 2011 Received in revised form 6 May 2011 Accepted 12 May 2011 Available online 17 May 2011 Keywords: Schizophrenia Kynurenine Kynurenic acid 3-Hydroxykynurenine Antipsychotics
a b s t r a c t The association between the pro-inflammatory state of schizophrenia and increased tryptophan degradation into kynurenine has been reported. However, the relationship between metabolites from subdivisions of the kynurenine pathway, kynurenic acid and 3-hydroxykynurenine, remains unknown. The present study tested the relationship between these kynurenine metabolites in the plasma of medication-naïve (n = 35) or medication-free (n = 18) patients with schizophrenia at admission and following 6-week antipsychotic treatment compared to healthy controls (n = 48). The plasma concentrations of kynurenic acid (nmol/l) were lower (difference = 8.44 (13.22 to 3.65); p = 0.001) and of 3-hydroxykynurenine (nmol/l) were higher (difference = 11.24 (8.11–14.37); p < 0.001) in the patients compared with the healthy controls. The kynurenic acid/kynurenine (difference = 2.75 (5.115 to 0.336); p = 0.026) and kynurenic acid/3-hydroxykynurenine (difference = 1.08 (1.431 to 0.729); p < 0.001) ratios were also lower in the patients. After the 6-week treatment, the patients’ plasma kynurenic acid levels (difference = 3.85 (0.23 to 7.94); p = 0.064) showed a trend towards an increase, whereas plasma 3-hydroxykynurenine levels (difference = 22.41 (19.76–25.07); p < 0.001) decreased. As a consequence, the kynurenic acid/3-hydroxykynurenine ratio (difference = 4.41 (5.51 to 3.3); p < 0.001) increased. Higher initial plasma kynurenic acid levels on admission or increased kynurenic acid/kynurenine ratio after treatment were associated with reduction of clinical symptoms scores upon discharge although higher kynurenic acid/kynurenine on admission may induce higher positive symptoms score. In contrast, higher 3-hydroxykynurenine is associated with lower positive symptoms score. These results indicate that there is an imbalance in the kynurenine pathway in schizophrenia. The 6-week antipsychotic treatment may partially reverse the imbalance in kynurenine metabolism and that in turn induces clinical response. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction The involvement of kynurenine metabolites, especially kynurenic acid (KYNA), in the pathophysiology of schizophrenia has been widely reported (Schwarcz et al., 2001; Erhardt et al., 2001). The role of 3-hydroxykynurenine (3HK) in schizophrenia has also been reported (Yao et al., 2010; Condray et al., 2011). However, the balance between these neuroactive metabolites of the tryptophan breakdown pathway, called the kynurenine (KYN) pathway, remains unknown. ⇑ Corresponding author at: Laboratory of Psychoneuroimmunology, University Psychiatric Hospital, Ludwig-Maximilians University, Nussbaumstrasse 7, D-80336 Munich, Germany. Fax: +49 89 5160 5890. E-mail address: [email protected] (A.M. Myint). 0889-1591/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2011.05.005
Tryptophan metabolism occurs in the liver and other sites, including immune cells in the blood, lungs and astrocytes and microglia in the brain. Pro-inflammatory cytokines, such as interferon-c (Carlin et al., 1989; Yasui et al., 1986), stimulate indoleamine 2,3-dioxygenase (IDO), the key enzyme that degrades tryptophan into KYN in the lungs, placenta, blood and brain (Heyes et al., 1993; Mellor and Munn, 1999). Tryptophan breakdown to kynurenine has been shown to increase following peripheral inflammation. As kynurenine can pass through the blood– brain-barrier and 60% of the brain KYN comes from the periphery (Gal and Sherman, 1980), peripheral inflammation is likely to increase central KYN concentrations. After tryptophan is degraded to KYN in the brain, the metabolites from this KYN pathway contribute to neuroprotective and neurotoxic/neurodegenerative changes. KYN is further catabolised
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into 3-hydroxykynurenine (3HK) and the NMDA receptor agonist quinolinic acid (QUINA) (Chiarugi et al., 2001; Bender and McCreanor, 1985) (Fig. 1) HK causes neuronal apoptosis (Okuda et al., 1998), while QUINA causes excitotoxic neurodegenerative changes (Schwarcz et al., 1983). However, KYN can also be catabolised into kynurenic acid (KYNA) (Fig. 1), which is an NMDA receptor antagonist (Perkins and Stone, 1982) and is therefore protective against the excitotoxic action of QUINA (Kim and Choi, 1987; Stone and Darlington, 2002). In addition to enhanced IDO activity, proinflammatory cytokines also enhance the activity of kynurenine3-monooxygenase (KMO), the enzyme that converts KYN into 3HK, which in turn enhances the excitotoxic degenerative arm of the pathway. The involvement of immune changes in schizophrenia with respect to the imbalance between T-helper type-1 (Th1) cytokines, most of which are pro-inflammatory, and Th2 cytokines, most of which are anti-inflammatory, has been reported, although the findings are controversial (Avgustin et al., 2005; Kim et al., 2004; Pae et al., 2006; Schwarz et al., 2001b; Arolt et al., 2000; Rothermundt et al., 1998; Sperner-Unterweger et al., 1999; Schwarz et al., 2001a). The involvement of inflammation in schizophrenia was also supported by the increase in the inflammatory fingerprint at the transcriptome level in schizophrenic patients (Drexhage et al., 2010). Moreover, the pro-inflammatory state and enhanced tryptophan breakdown in schizophrenia has also been reported (Kim et al., 2009). Therefore, it is possible that the high IDO and KMO activities induced by the pro-inflammatory state in schizophrenia may result in the high formation of toxic 3HK and QUINA syntheses, while KYNA formation would be decreased. However, the KYNA can also induce abnormal behaviour by interfering with glutamatergic transmission if it is present in excess. Micro-molar brain levels of KYNA have been reported to be associated with disruption of auditory sensory gating in rats, and this association was due to down-regulation of the permissive action of 5-hydroxy indole (5HI) at a-7 nicotinic acetylcholine receptors (a-7nChR) caused by high KYNA concentrations (Shepard et al., 2003). KYNA has also been shown to induce psychotic symptoms through antagonism at a-7nChR (Schwieler and Erhardt, 2003). In addition, KYNA concentrations have been found to be increased in Brodmann area 9 (Schwarcz et al., 2001) and the anterior cingulate area (Miller et al., 2006) of post-mortem schizophrenic brains. Moreover, increased KYNA levels were de-
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tected in the CSF of medication naïve and medicated male schizophrenic patients, but not in medication-free male schizophrenic patients (Nilsson et al., 2005). The role of KYNA as an endogenous NMDA-R antagonist supports the hypothesis of NMDA hypofunctioning in schizophrenia (Olney et al., 1999; Olney and Farber, 1995; Muller and Schwarz, 2006). However, Ravikumar et al. (2000) have reported increases in both QUINA and KYNA in the plasma of small groups of patients with multiple sclerosis, Parkinson’s disease, generalised epilepsy, schizophrenia, gliomas and syndrome X compared with healthy controls. Although the sample sizes were small, this study suggested that both neuroprotective and neurotoxic/neurodegenerative arms of the kynurenine pathway can be increased in those diseases. The above findings raise questions of whether (i) the balance between the two arms of KYN metabolism is disturbed in schizophrenic patients, (ii) there is any effect of KYN metabolites on clinical parameters and (iii) there is any effect of antipsychotic treatment on kynurenine metabolites in the periphery. These questions have been addressed in the present study. 2. Methods 2.1. Subjects Amongst the patients admitted to Korea University Medical Centre from 2003 to 2005, 53 medication-naïve or medication-free patients who met the DSM-IV criteria (APA, 1994) for schizophrenia and completed 6-week antipsychotic therapy as inpatients were recruited. Diagnoses were made using the DSM-IV Structured Clinical Interview (First, 1998). Patients with a history of any concomitant psychiatric illness, such as substance or alcohol abuse, infection or a known autoimmune disease, were excluded. Patients had normal values in routine blood and urine tests, electrocardiograms and electroencephalograms. All patients had acute psychotic symptoms at the time of recruitment. All patients were either medication-naive (n = 35) or medication-free for at least 4 months (n = 18). The psychopathological status of the patients was assessed by a trained psychiatrist using the Positive and Negative Syndrome Scale (PANSS) (Kay et al., 1987) and the Korean version of the Calgary Depression Scale for Schizophrenia (K-CDSS) (Kim et al., 2005). Each patient’s symptoms were assessed again 6 weeks later by the same physician. During the 6-week treatment, 20 patients were medicated with risperidone (4–12 mg), 19 patients were given amisulpride (200– 900 mg), 10 patients were administered olanzapine (10–30 mg) and 4 patients were medicated with aripiprazole (15–30 mg). A total of 48 healthy controls were recruited at the same hospital from the pool of individuals who came in for regular health screenings during the same period. Each control was given a non-structured clinical screening interview. Persons with any personal or familial history of psychiatric illness, autoimmune disease or chronic inflammatory disease and/or substance or alcohol abuse were excluded. All subjects were free of chronic and acute physical illness associated with abnormal immune changes within the 2 weeks before the study. This study was approved by the institutional ethical committee of Korea University and written informed consent was obtained from each patient or control. 2.2. Biochemical analysis
Fig. 1. Changes in the tryptophan catabolic pathway in schizophrenia are shown. The metabolites in blue colour indicated those discussed in this paper. The green arrows show metabolic processes. The respective thickness of each arrow indicates the degree or weight of the action or reaction. Dotted arrows signify inhibition. The grey arrow indicates the concentration increased or decreased.
Early morning venous blood was withdrawn following overnight fasting. Plasma was separated immediately and stored at 70 °C. Blood samples were taken from the patients again 6 weeks later. High performance liquid chromatography (HPLC) was used to
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measure plasma kynurenine and kynurenic acid (KYNA) levels, according to the method of Hervé et al. (1996) with some modifications. To measure 3-hydroxykynurenine (3HK) levels the recently published method using HPLC (Oades et al., 2010a,b) was used. Briefly, KYN was detected spectrophotometrically at 365 nm. KYNA was detected fluorimetrically at an excitation wavelength of 334 nm and an emission wavelength of 388 nm. KYNA was analysed in plasma that was deproteinised using perchloric acid. 3HK was measured at a wavelength of 365 nm by UV detection. All of the analyses were conducted using HPLC with a reverse phase c-18 column. The 3HK analysis method has been validated showing an absolute recovery of 85.8%, intra-day precision of 3.9%, and interday precision of 7.5%; time series demonstrated perfect stability of the analyte 3HK during our extraction and analysis steps; the result of the analysis was even stable up to three times repeated freezing/ thawing cycles. Thus, we could ensure the validity of the analysis in samples that had already been thawed and frozen again. All of these methods have been standardised by a quarterly External Quality Assurance Scheme (EQAS) amongst 6 universities (Ludwig-Maximilians University of Munich, University Medical Centre of Groningen, University of Antwerp, Trinity College Dublin, Illinois University and New South Wales University) under the European collaborative project MOODINFLAME and the Bundesministerium fuer Bildung und Forschung (BMBF) project, which contributed independently to this study. Standards were prepared for each calibration curve, and appropriate internal standards were used for analyses of all metabolites. Data are shown in nmol/l. The intra- and inter-assay coefficients of variation ranged from 5% to 7% for all of the metabolites. To avoid the effect of inter-assay variation, the samples from both schizophrenic patients and controls were placed alternately in each set of analyses. To avoid operator bias, the samples’ order was arranged by the investigator, and the laboratory technician was blind to the information of the sample. For every 20 samples and 3 standards, one quality control sample (pooled medication free EDTA plasma) was used.
admission to discharge on changes in clinical symptoms scores between admission and discharge. The ‘p’ values less than or equal to 0.05 were considered to be statistically significant, without adjusting for multiplicity. SPSS version 18.0 was used for data analyses. 3. Results The general characteristics of patients and controls are presented in Table 1. There was no relevant difference in age, BMI or gender ratio between the two diagnostic groups. 3.1. Comparison of tryptophan metabolites between diagnostic groups The biochemical data of the two diagnostic groups are shown in Table 2. In univariate regression analyses, the schizophrenic group had lower plasma concentrations of KYNA (nmol/l) (difference = 9.05 (13.22 to 3.65); p = 0.001) but higher plasma concentrations of 3HK (nmol/l) (difference = 9.02 (8.11–14.37); p < 0.001) compared to healthy controls. Moreover, the patients had lower KYNA/KYN (difference = 3.69 (5.115 to 0.336); p = 0.026) and KYNA/3HK ratios (difference = 1.07 (1.431 to 0.729); p < 0.001). 3.2. Comparison of tryptophan metabolites in patients on admission vs. discharge After 6 weeks of treatment, plasma KYNA levels (nmol/l) (difference = 3.85 (0.23 to 7.94); p = 0.064) showed a trend towards an increase, whereas plasma 3HK levels (nmol/l) decreased (difference = 22.41 (19.76–25.07); p < 0.001). Although there was no change in the KA/KYN ratio, the KYNA/3HK ratio was significantly reduced (difference = 4.41 (5.51 to 3.3); p < 0.001) (Table 3). The type of medication given during the treatment had no effect on the biochemical or clinical parameters. Table 1 Demographic data.
2.3. Calculations The ratio between plasma KYNA and KYN concentrations enabled the determination of how much KYN would be catabolised into KYNA (Myint et al., 2007):
KYNA=KYN ratio ¼
1000 plasma KYNA ðnmol=lÞ plasma KYN ðnmol=lÞ
The ratio between plasma KYNA and 3HK levels was calculated to determine the balance between the two arms of kynurenine pathway (Oades et al., 2010a,b):
KYNA=OHK ratio ¼
plasma KYNA ðnmol=lÞ plasma OHKðnmol=lÞ
2.4. Statistical analysis The comparison of the general characteristics between the two groups was carried out using Student’s ‘t’ test and Fisher’s exact test. Student’s ‘t’ test was applied to detect differences in the biochemical parameters between the two groups when age, gender and body mass index (BMI) were controlled using univariate regression analysis. Paired ‘t’ tests were applied to calculate the differences between patients’ admission data and their data at the time of discharge. The univariate regression analysis was also applied to analyse the effects of the initial biochemical parameters at admission and changes in these biochemical parameters from
Sex (male/female) Age (years) BMI (kg/m2) Age at onset (years) Duration of total illness (months) Duration of current illness (months) Number of previous admissions (mode and range) Family history of psychiatric illness Medication status on admission Medication-naïve Medication-free (P4 months) Subtypes Paranoid Undifferentiated Disorganised Psychopathology scores PANSS at admission PANSS positive score PANSS negative score PANSS at 6 weeks PANSS positive score PANSS negative score K-CDSS at admission K-CDSS at 6 weeks General symptoms score at admission General symptoms score At 6 weeks
Schizophrenia (n = 53)
Healthy controls (n = 48)
23/30 33.3 ± 12.2 23.19 ± 3.8 27.7 ± 11.2 74.9 ± 88.7 3.6 ± 5.3 0 (0–7)
21/27 32.56 ± 10.32 22.25 ± 3.08
9 35 18 38 14 1
26.16 ± 6.75 22.08 ± 7.55 14.04 ± 4.43 14.02 ± 0.87 8.4 ± 5.8 3.0 ± 3.2 49.23 ± 11.12 29.74 ± 7.45
PANSS, the positive and negative syndrome scale; K-CDSS, the Korean version of the calgary depression scale for schizophrenia.
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A.M. Myint et al. / Brain, Behavior, and Immunity 25 (2011) 1576–1581 Table 2 Plasma tryptophan metabolites in schizophrenia patients and healthy controls.
Kynurenic acid (nmol/l) 3-Hydroxykynurenine (nmol/l) KYNA/KYN ratio KYNA/3HK ratio
Schizophrenia (n = 53)
Healthy controls (n = 48)
‘p’ Value
26.90 ± 2.25 28.60 ± 1.49
35.95 ± 1.37 19.58 ± 0.77
0.001 <0.0001
15.67 ± 1.20 0.96 ± 0.13
19.36 ± 0.61 2.03 ± 0.15
0.026 <0.0001
‘p’ Values were generated using Student’s ‘t’ test when age, gender and BMI were controlled using an univariate regression analysis.
Table 3 Plasma tryptophan metabolites in schizophrenia patients on admission and at the time of discharge.
Kynurenic acid (nmol/l) 3-Hydroxykynurenine (nmol/l) KYNA/KYN ratio KYNA/3HK ratio
On admission (n = 53)
At discharge (n = 53)
‘p’ value
26.90 ± 2.25 28.60 ± 1.49
30.75 ± 2.27 6.19 ± 0.37
0.064 <0.0001
15.67 ± 1.20 0.96 ± 0.13
15.72 ± 0.93 5.37 ± 0.64
0.968 <0.0001
‘p’ Values were generated using a paired ‘t’ test.
3.3. Associations between tryptophan metabolites at admission and changes in clinical parameters The initial plasma kynurenic acid concentrations were associated with a reduced PANSS positive symptoms score and a reduced K-CDSS depressive symptoms score upon discharge. In contrast, the higher the KYNA/KYN ratio on admission, the higher the PANNS positive symptoms score on admission. However, the higher the KYNA/KYN ratio on admission the higher the reduction in K-CDSS scores at discharge. Moreover, the increase in this ratio after 6-week medication is associated with higher reduction in PANNS positive symptoms, general symptoms and K-CDSS symptoms scores. Initial plasma 3HK concentrations is negatively associated to the admission PANNS positive symptoms score. However, the higher the plasma 3HK on admission the lower the reduction in general symptom scores at the time of discharge. The quantitative results of the regression analyses are presented in Table 4. 4. Discussion The results of this study demonstrate that 3HK concentrations are increased while KYNA concentrations are decreased in the plasma of medication naïve and medication free patients with schizophrenia. This finding indirectly demonstrates the imbalance between the peripheral KYNA and 3HK arms of the KYN pathway. These results also show that the imbalance is corrected following
effective antipsychotic treatment, mainly due to decreases in plasma 3HK levels. In addition, the KYNA/KYN ratio and the KYNA/3HK ratio were lower in the patients. These findings indicate that a greater quantity of KYN was catabolised into 3HK than into KYNA. These data lend credibility to our proposed hypothesis that imbalanced kynurenine metabolites play a role in pathophysiology of schizophrenia. While most of the previous studies on the role of kynurenine metabolites in schizophrenia concentrated mainly on changes in KYNA levels, specifically increases in KYNA, and described the pathophysiology in schizophrenia as a result of NMDA-R hypofunctioning (Olney et al., 1999), our data demonstrated a significant reduction in KYNA and significant increases in 3HK in the plasma. These findings are in accordance with studies that demonstrated increased pro-inflammatory cytokines, such as interferon-c (Avgustin et al., 2005; Kim et al., 2004, 2009), in the peripheral blood of patients, as this cytokine can enhance both IDO and KMO activity, resulting in high 3HK formation (Yasui et al., 1986). Increases in 3HK formation would shift metabolism more to the 3HK arm, causing an increase in QUINA formation. There is evidence of a pro-inflammatory state in the periphery (Kim et al., 2004) and in the brain of patients with schizophrenia (Doorduin et al., 2009). The pro-inflammatory state, leading to production of pro-inflammatory cytokines, may enhance tryptophan breakdown and formation of 3HK and QUINA both in the periphery and in the brain. Both 3HK and QUINA are neurotoxic metabolites. This hypothesis is supported by the findings of Condray et al. (2011) who demonstrated that higher 3HK correlated with lower change in clinical symptoms after 4-weeks treatment in 25 medication-naive and medication-free schizophrenia patients. Our data also showed similar trend in terms of initial 3HK and change in general symptoms score. Loss of brain volume and neuronal density has been well documented in schizophrenia (Theberge et al., 2007; Perez-Neri et al., 2006; Byne et al., 2006, 2007, Seok Jeong et al., 2005; Blennow et al., 1996). As 3HK is neurotoxic and apoptotic (Okuda et al., 1998), it may be hypothesised that high 3HK concentrations could contribute to a loss of brain volume in schizophrenia. The increased 3HK concentrations in patients may be due to (1) enhanced production of both 3HK and QUINA or (2) the accumulation of 3HK due to the blockade of 3HK breakdown into further metabolites, such as 3-hydroxyanthranilic acid (HAA), because of reduced activity of the kynureninase enzyme, which degrades 3HK to HAA (Price et al., 1972) while KMO activity would be enhanced by the pro-inflammatory state. In the latter case, reduction of the breakdown of 3HK may result in reduced formation of the NMDA-R agonist QUINA and would be in line with NMDA hypofunctioning hypothesis. Determination of plasma and brain QUINA concentrations is crucial to determine the exact mechanism. Our data suggest that initial plasma KYNA levels have predictive value on positive symptoms score and depressive symptoms score reductions after 6 weeks of medication. This may be due to higher neuroprotective KYNA availability in the periphery and in the
Table 4 Association between plasma tryptophan metabolites and clinical parameters in schizophrenia patients. Biochemical parameter
Dependent Clinical variable
b
95% CI for b
‘p’ Value
KYNA admission
PANSS positive change CDSS change PANSS positive on admission General symptoms change PANSS positive on admission CDSS change PANSS positive change CDSS change General symptoms change
0.44 0.68 0.258 0.398 0.249 0.204 0.381 0.225 0.607
0.68 to 0.19 1.23 to 0.36 0.443 to 0.073 0.018 to 0.778 0.042 to 0.456 0.4 to 0.008 0.623 to 0.138 0.449 to 0.001 1.005 to 0.159
0.001 0.016 0.007 0.041 0.019 0.041 0.003 0.049 0.009
3HK admission KYNA/KYN admission KYNA/KYN discharge – KYNA/KYN admission
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brain, resulting in greater protection against the neurotoxic effects of 3HK on the glial-neuronal network, and thus enables better responses to treatment. This is again supported by the associations between increase in KYNA/KYN ratio after medication and reduction in clinical symptoms scores after medication. In experimental studies, antipsychotic treatment has been reported to reduce KYNA concentrations in the brain (Olney and Farber, 1994; Ceresoli-Borroni et al., 2006). However, in our study, 6 weeks of antipsychotic treatment slightly increased plasma KYNA levels and dramatically reduced plasma 3HK concentrations. This response to treatment may be associated with a reduction in KMO activity through correction of the pro-inflammatory state following antipsychotic treatment in the same group of patients (Kim et al., 2009), which significantly reduced the plasma 3HK levels. The changes in the brain after medication are still necessary to be investigated. While these findings supported our hypothesis that in schizophrenia with pro-inflammatory state the kynurenine pathway will shift to the 3HK arm, reports from some investigators have suggested the involvement of increased KYNA levels in the pathophysiology of schizophrenia (Schwarcz et al., 2001). Somehow, our data supports those findings since we observed the association between higher KYNA/KYN ratios or the lower 3HK level and higher initial positive symptoms score. Condray et al., 2011 had also observed similar result. Taken together with the findings that higher initial 3HK is associated with lower reduction of symptoms scores, in other words, poorer clinical outcome, it can be concluded that the imbalance between KYNA arm and 3HK arm in both directions might induce negative impact in terms of positive symptoms development and impaired response to treatment. We did not observe the association between change in 3HK after treatment and clinical symptoms scores changes. The reason could be the fact that the reduction in neurotoxic metabolite would result only in slowing down the further neuronal-glial network damage but not in immediate change of clinical symptoms. To clarify these points, further studies are necessary to explore (1) the association between changes in concentrations of kynurenines in the plasma and CSF and the detailed clinical symptoms and relapse or remission in patients with schizophrenia and (2) the detailed postmortem morphological evidence of kynurenine metabolites in different brain areas. For the ethical reasons, it was not possible to obtain CSF from the same patients in this study. Furthermore, we have neither the facilities nor sufficient plasma samples to determine QUINA concentrations. In future studies, we plan to analyse the complete profile of metabolites from this kynurenine pathway in the plasma or serum and CSF. Nevertheless, this study has demonstrated the imbalance between KYNA and 3HK arms in kynurenine metabolism in medication-naïve and medication-free patients with schizophrenia. Our data indicated the involvement of imbalance in kynurenine metabolism in pathophysiology of schizophrenia. Moreover, we have also demonstrated a possible role of plasma kynurenines in predicting responses to treatment. Acknowledgments This study was funded by Advanced Practical Diagnostics n.v., Belgium for the laboratory analyses and Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A040042) for the clinical sample and data collection. References APA, 1994. Diagnostic and statistical manual of mental disorders, American Psychiatric Press, Washington, DC.
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