Reduced Mismatch Negativity is Associated with Increased Plasma Level of Glutamate in First-episode Psychosis

Reduced amplitude of mismatch negativity (MMN) is one of the more promising biological markers of schizophrenia. This finding holds true in both early and chronic phases of the disorder, and is compatible with the glutamatergic dysfunction hypothesis. To further establish MMN as a biomarker of aberrant glutamatergic neurotransmission, an exploration for an association with blood levels of glutamatergic amino acids is an important next step. Despite a large body of work investigating MMN in schizophrenia, no previous studies have undertaken this endeavor. Nineteen patients with first-episode psychosis (FEP), 21 ultra-high risk individuals (UHR), and 16 healthy controls (HC) participated in the study. The MMNs in response to duration change (dMMN) and frequency change (fMMN) were measured. The fasting plasma levels of glutamate, glutamine, glycine, D-serine, and L-serine were measured. dMMN amplitudes were significantly reduced in FEP and UHR, compared to HC. The plasma levels of glutamate of FEP were significantly higher than those of HC. Higher plasma levels of glutamate were associated with smaller dMMN amplitudes in the FEP and HC groups. These findings are compatible with the hypothesis that MMN is a useful biological marker of aberrant glutamatergic neurotransmission in the early stages of schizophrenia.

a group, MMN amplitude reduction is only present in a subset of individuals with early stages of psychosis in these studies. Because MMN is thought to reflect NMDA receptor function, the MMN amplitude attenuation in the subset may be due to the pathophysiology of NMDA receptor hypofunction. Therefore, MMN may serve as a biomarker to identify the subset that has psychosis based on NMDA receptor hypofunction.
To further establish MMN as a useful biological markers of aberrant glutamatergic neurotransmission, especially in the early stages of psychosis, the next step should be to investigate the relationship between MMN amplitude and blood level of molecules that influence NMDA receptor function, such as glutamate and D-serine. Peripheral glutamatergic amino acids have been investigated in patients with schizophrenia 23,24 and a meta-analysis showed that peripheral glutamate levels in schizophrenia patients were significantly higher than those in controls 24 . This is consistent with a prevalent model of hyperglutamatergic state in schizophrenia. This model is the so-called disinhibition mechanisms whereby NMDA antagonists block the NMDA-medicated drive on GABA interneurons that normally inhibit pyramidal cells. This leads to enhanced activity of pyramidal cells, which then results in increased glutamate release in adjacent cortical or afferent regions 25 .
However, to the authors' knowledge, no studies have reported associations between MMN amplitude and peripheral glutamatergic amino acids in early stages of psychosis.
In this study, we measured MMN and the plasma levels of glutamatergic amino acids in subjects in the early stages of psychosis (UHR and first-episode psychosis [FEP]). We hypothesized the reduction of MMN amplitude, increased plasma level of glutamate, and an association between MMN amplitude and the plasma levels of glutamate in early stages of psychosis.

Results
Demographic and clinical variables. There were no significant differences in sex ratio, mean age, education, and premorbid IQ across the three groups. PANSS negative, general, or total were not significantly different between FEP and UHR. PANSS positive, GAF and antipsychotics dose were significantly different between FEP and UHR (Table 1).

Discussion
The current study found significantly reduced dMMN amplitude and relatively preserved fMMN amplitudes in FEP and UHR. We also observed increased plasma levels of glutamate in FEP. Importantly, smaller dMMN amplitudes were associated with higher plasma levels of glutamate in FEP. These findings are compatible with the glutamatergic dysfunction hypothesis of schizophrenia, and confirm that dMMN is a useful biomarker of aberrant glutamatergic neurotransmission in the early stages of schizophrenia.
FEP and UHR showed significantly smaller dMMN amplitude and relatively intact fMMN amplitude compared to HC. These results are in line with previous studies 16,26,27 . Previous studies have shown that dMMN amplitude predicts the onset of psychosis in individuals with ultra-high risk 21 , and that fMMN amplitude may be associated with chronicity 1 .
The plasma level of glutamate of FEP was significantly higher than that of HC. The result is compatible with previous reports, which showed increased serum levels of glutamate in chronic schizophrenia patients 28,29 . A couple of reports have attempted and failed to replicate these findings 30,31 ; however, a recent meta-analysis confirmed that the peripheral glutamate level of schizophrenia subjects was significantly higher than that of HC 24 . While these are findings pertain to chronic schizophrenia, we found increased glutamate levels in FEP. Since the present study used peripheral blood samples, it is uncertain whether the concentration of glutamate in the brain was also elevated. However, previous studies reported the peripheral glutamate levels were significantly correlated with cerebrospinal fluid (CSF) levels of glutamate 32,33 . Furthermore, a recent meta-analysis reported that in schizophrenia, there were significant elevations in glutamate in the basal ganglia and no region showed a reduction in glutamate metabolites 34 . Therefore, the higher plasma glutamate concentration established in the present study might, at least in part, reflect an elevated glutamate level in the brain of FEP subjects.
Plasma levels of glutamine, glycine, D-serine, and L-serine showed no significant differences between FEP, UHR, and HC. Previous studies of peripheral levels of these glutamatergic amino acids in schizophrenia have yielded mixed findings: glutamine: significantly lower than HC 35 , comparable with HC 31 ; glycine: higher 36 , lower 37 , comparable 38 ; D-serine: higher 36 , lower 31,38,39 ; L-serine: higher 40 , comparable 39 . Additionally, Hashimoto et al. 41 reported that CSF glutamine levels were not different between schizophrenia subjects and HC. A recent meta-analysis showed the peripheral and CSF levels of glycine, D-serine, and L-serine were not significantly different between schizophrenia subjects and HC 23 , which corroborates our findings.
The most important finding of this study is that lower dMMN amplitudes were associated with higher plasma glutamate levels in FEP, although this inverse correlation did not appear to be specific to FEP; it was also observed in HC. There was no such association in UHR. To the authors' knowledge, this is the first study that has reported the correlation between MMN amplitude and peripheral glutamate level in psychotic disorders. Although the molecular mechanism underlying the increased plasma level of glutamate in schizophrenia patients remains elusive, administration of NMDA receptor antagonists increased the concentration of glutamate in the prefrontal cortex in animals 42,43 . Furthermore, administration of NMDA receptor antagonists attenuates MMN amplitude 6,7,44 . Therefore, hypofunction of NMDA receptors might cause increased plasma levels of glutamate and reduced MMN amplitude in FEP. The plasma level of glycine, D-serine and L-serine neither decrease nor correlate with MMN amplitude in FEP or UHR. These results suggest that NMDA hypofunction in schizophrenia may not due to the decrease of NMDA receptor co-agonists. The plasma level of glutamine also showed no significant correlation with MMN amplitude in FEP or UHR. Because glutamine has no agonistic activity at NMDA receptors, the plasma level of glutamine may not reflect NMDA receptor function.
Although dMMN amplitude was lower and the plasma level of glutamate was higher in FEP compared to HC, not all patients with FEP had reduced amplitude of dMMN and increased level of glutamate (Fig. 3). These results suggest that not all patients with FEP but a subset of FEP may have hypofunction of NMDA receptors. Therefore, dMMN and the plasma level of glutamate may serve as biomarkers to differentiate individuals with hypofunction of NMDA receptors from individuals without hypofunction of NMDA receptors in patients with FEP. According to a recent meta-analysis, glutamate positive modulators may not be effective in reversing overall cognitive impairments in patients with schizophrenia 45 . However, glutamate positive modulators may be effective only in a subset of the patients who has NMDA receptor hypofunction. The plasma level of glutamate and dMMN may be useful for identifying this subset. Future clinical trials using dMMN and the plasma level of glutamate as biomarkers will be useful for developing new treatments that modulate NMDA receptor function. These stratified clinical trials based on biomarkers will lead to precision medicine for early stages of psychosis that is the objective of the Research Domain Criteria (RDoC) 46 .
We investigated association between MMN and glutamatergic amino acids in psychosis based on glutamatergic hypothesis of schizophrenia. However, previous studies reported the reduced amplitude of MMN in other mental disorders such as bipolar disorder 47 . In addition, bipolar disorder also shows aberrant glutamate transmission 48 . Therefore, the association between reduced amplitude of MMN and increased plasma level of glutamate may not be specific to psychosis. However, future studies with a sample including various mental disorders will be needed to investigate the specificity of the association between reduced amplitude of MMN and increased plasma level of glutamate.
We have several limitations in this study. First, although the antipsychotics dose was not significantly correlated with dMMN amplitude, nor with the plasma level of glutamate, correlational analyses may be insufficient to clarify potential medication effects. Further studies with drug-naïve participants would be necessary to investigate whether the association between dMMN and plasma level of glutamate reflect NMDA pathology of schizophrenia or potential medication effects. Second, since the present study is a cross-sectional one, the association between dMMN amplitude and plasma level of glutamate is correlative. A longitudinal study, currently underway in our laboratory, will clarify the association during the early stages of schizophrenia. Third, a sample size was small in this study. Further studies with a large sample size will be needed to confirm the correlation between MMN and the plasma level of glutamate.
In conclusion, we found reduced dMMN amplitude and increased plasma level of glutamate in FEP, and a significant association between the two indices. These findings suggest that dMMN and plasma glutamate levels may be useful biological markers for altered glutamatergic neurotransmission in the early stages of schizophrenia. If replicable in larger-scale studies, they may represent promising candidates for biomarkers to be utilized in real-world clinical settings, which has, thus far, been a great challenge in psychiatry.

Methods
Participants. Nineteen patients with FEP, 21 individuals with UHR and 16 healthy controls (HC) participated in this study (Table 1). MMNs from a subset of these participants were previously published 16 . Thirteen FEP, 7 UHR and 10 HC were included in the previous study who agreed to blood drawing and the examination of the plasma level of amino acids, and the rest were newly recruited for the present study. All participants with FEP or UHR were recruited from The University of Tokyo Hospital. All individuals with UHR, and most patients with FEP, were help-seeking individuals registered at the University of Tokyo Hospital outpatient unit specialized for early intervention. HC were recruited through advertisements placed at several universities near The University of Tokyo. This study was conducted as a part of our large-scale, multimodal, neuroimaging and psychophysiological studies in the early stages of psychosis (Integrative Neuroimaging Studies for Schizophrenia Targeting Early Intervention and Prevention; IN-STEP). Koike et al. 49 reported the detail of the recruitment protocol. All subjects gave written informed consent to the Research Ethics Committee of the Faculty of Medicine, The University of Tokyo (approval Nos 629-8, 2094-7, 2226-4) following a complete explanation of this study, and in accordance with the Declaration of Helsinki. Inclusion and exclusion criteria for each group are shown in Table 2. We used the Structured Interview for Prodromal Symptoms (SIPS) as the UHR criteria, which consists of 3 criteria: attenuated psychotic symptoms (APS), brief intermittent psychotic symptoms (BIPS), or genetic risk and deterioration (GRD) 50,51 . APS correspond to individuals who exhibited onset or worsened subthreshold psychotic symptoms within 12 months but not psychotic severity. BIPS correspond to individuals who had psychotic symptoms within 3 months but with a limited duration and frequency such that they were not at all or only slightly influenced by their symptoms and did not meet the psychotic episode criteria according to the DSM-IV criteria 52 . GRD corresponds to individuals whose functioning had deteriorated in the previous 12 months as defined by a 30% or more decrease in the GAF score 52 as well as those who also had one or more first-degree relatives diagnosed with psychosis and/or schizotypal personality disorder according to the DSM-IV criteria.
Audiometer testing was used to ensure that all participants had normal hearing in both ears and could detect 30-dB sound pressure level tone at 1000 Hz and 40-dB at 4000 Hz. Eighteen FEP and 9 UHR subjects were prescribed second-generation antipsychotic medications at the time of testing. No other antipsychotic was prescribed. All patients with FEP were diagnosed by experienced psychiatrists according to the DSM-IV criteria. Out of 19 FEP patients, 17 patients had first-episode schizophrenia, and 2 patients had schizophreniform disorder. Estimated premorbid intelligence quotient (IQ) was assessed with the Japanese version of the National Adult Reading Test 53, 54 FEP and UHR participants were assessed with regards to their functioning and symptoms, using the Global Assessment of Functioning (GAF) 52 and the Positive and Negative Syndrome Scale (PANSS) 55 . These clinical ratings were measured by experienced psychiatrists (T.N., M.T., and D.K.). To control the quality of data assessment, we used video interviews to ensure the scoring of the clinical ratings and calculated the inter-rater reliability of the clinical ratings (Cronbach alpha = 0.86) 49 .

Measurement of the plasma levels of amino acids.
We measured plasma levels of the peripheral glutamatergic amino acids glutamate, glutamine, glycine, D-serine, and L-serine. Measurement of the plasma levels of glutamate, glutamine, and glycine was carried out using a column-switching high performance liquid chromatography (HPLC) system (Shimadzu Corporation, Kyoto, Japan) with fluorescence detection, as has been previously reported 41 . Measurement of D-serine and L-serine was carried out using a HPLC system, as has been previously reported 39,56 . In order to minimize the effect of amino acids originating from food sources, we obtained fastening (>3 h without any meals and/or nutritious drinks) blood samples, as has been previously reported 57 . Stimuli and Procedure. For duration MMN (dMMN), a two-tone auditory oddball paradigm with 2000 stimuli was used. Ninety percent of the stimuli were standard tones (1000 Hz, 50 ms) and 10% of the stimuli were deviant tones (1000 Hz, 100 ms). For frequency MMN (fMMN), another two-tone auditory oddball paradigm with 2000 stimuli was used. Ninety percent of the stimuli were standard tones (1000 Hz, 50 ms) and 10% of the stimuli were deviant tones (1200 Hz, 50 ms). All the stimuli were of 80 dB sound pressure level and 1 ms rise/fall time. Stimulus onset asynchrony was 500 ms. These oddball paradigms were counter-balanced. Tones were presented binaurally through earphones while participants watched a silent cartoon.
Electroencephalography recording and analyses. Electroencephalography (EEG) data were acquired with a 64-channel Geodesic EEG System (Electrical Geodesics Inc., Eugene, OR). Electrodes were referenced to the vertex, and impedances were kept below 50 kΩ. The sampling rate was 500 Hz. The analog filter bandpass was set at 0.1-100 Hz. EEGLAB 58 was used for EEG processing and data analysis. The continuous EEG data were re-referenced to an average reference, digitally filtered at 0.1-20 Hz, and segmented from −100 to 500 ms relative to the stimulus onset. The mean of the pre-stimulus baseline was subtracted for baseline correction. Independent component analysis was used for eye blink correction. Epochs exceeding ±100 μV at any electrode were rejected. After averaging across trials, the ERP waveform in response to standard stimuli was subtracted from the ERP waveform in response to deviant stimuli.
For MMN analysis, a front-central electrode (FCz) was selected (the largest amplitude was obtained with this FCz). The dMMN amplitude was measured as the mean voltage from 135 to 205 ms post stimuli, in accordance with previous studies 16  Statistical Analyses. We performed Shapiro-Wilk tests to examine whether each variable follows a normal distribution. When the variable followed a normal distribution, we employed independent t-test for comparison between 2 groups and one-way analysis of variance (ANOVA) for comparison among 3 groups. When the variable did not follow a normal distribution, we employed Mann-Whitney test for comparison between 2 groups and Kruskal-Wallis test for comparison among 3 groups. We employed a χ 2 test for sex ratio.
When we found significant difference in one-way ANOVA, Post hoc tests with the Bonferroni correction were performed to examine group difference. When we found significant difference in Kruskal-Wallis test, Mann-Whitney tests with the Bonferroni correction were performed to examine group differences.
We reported effect sizes using Cohen's d for variables with normal distribution, r for variables with non-normal distribution, and η 2 for ANOVA.
To assess the association of MMN amplitude with the plasma levels of glutamatergic amino acids, Spearman correlation coefficients were calculated. The significance level was set at p < 0.05.