Plasma Nervonic Acid Is a Potential Biomarker for Major Depressive Disorder: A Pilot Study

Plasma Nervonic Acid Is a Potential Biomarker for Major Depressive Disorder: A Pilot Study Background: Diagnostic biomarkers of major depressive disorder, bipolar disorder, and schizophrenia are urgently needed, because none are currently available. Methods: We performed a comprehensive metabolome analysis of plasma samples from drug-free patients with major depressive disorder (n = 9), bipolar disorder (n= 6), schizophrenia (n = 17), and matched healthy controls (n = 19) (cohort 1) using liquid chromatography time-of-flight mass spectrometry. A  significant effect of diagnosis was found for 2 metabolites: nervonic acid and cortisone, with nervonic acid being the most significantly altered. The reproducibility of the results and effects of psychotropic medication on nervonic acid were verified in cohort 2, an independent sample set of medicated patients [major depressive disorder (n= 45), bipolar disorder (n= 71), schizophrenia (n = 115)], and controls (n = 90) using gas chromatography time-of-flight mass spectrometry. Results: The increased levels of nervonic acid in patients with major depressive disorder compared with controls and patients with bipolar disorder in cohort 1 were replicated in the independent sample set (cohort 2). In cohort 2, plasma nervonic acid levels were also increased in the patients with major depressive disorder compared with the patients with schizophrenia. Significance Statement Box Sample In cohort 2, nervonic acid levels were increased in the depressive state in patients with major depressive disorder compared with the levels in the remission state in patients with major depressive disorder and the depressive state in patients with bipolar disorder. Conclusion: These results suggested that plasma nervonic acid is a good candidate biomarker for the depressive state of major depressive disorder. Keywords: biomarker, bipolar disorder, major depressive disorder, metabolomics, nervonic acid Received: June 19, 2017; Revised: September 21, 2017; Accepted: October 2, 2017 © The Author(s) 2017. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 207 provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/207/4356809 by Ed 'DeepDyve' Gillespie user on 16 March 2018 208 | International Journal of Neuropsychopharmacology, 2018 Significance Statement Plasma nervonic acid levels were increased in patients with major depressive disorder (MDD) compared with those in patients with bipolar disorder and healthy controls. Nervonic acid levels were higher in the depressive state in the patients with MDD compared with the remission state in the patients with MDD and the depressive state of patients with bipolar disorder, suggesting state-dependent alterations. Therefore, plasma nervonic acid is a good candidate diagnostic biomarker for MDD. Introduction Although a proper diagnosis and medical treatment are import- City University Hospital and Hannan Hospital, where controls ant for improving the prognosis of patients with mental illness were recruited from among the healthy spouses of the patients. (Post et al., 2010; Gaebel and Zielasek, 2015), diagnoses are diffi- The control samples in the NCNP Biobank were collected by cult to make, especially when major depressive disorder (MDD) advertising in local free magazines and on websites. needs to be distinguished from the depressive state of bipolar The sex ratios and ages did not significantly differ among the disorder (BD). In addition, patients with schizophrenia (SZ) can groups in cohort 1 (Table 1 ). Among the groups in cohort 2, the also show depressive symptoms. Because treatment approaches sex ratios did not significantly differ, while age did (Table ). 2 The differ among patients with MDD, BD, and SZ, diagnostic bio- age of participants in the MDD and control groups was higher markers that distinguish MDD, BD, and SZ are required to avoid than that of SZ (P = .000046 and .000004, respectively). ANCOVA misdiagnosis (Altamura et al., 2015) and obtain a better progno- was used for the analysis of age. sis (Drancourt et al., 2013). Although candidate biomarkers have All participants provided written informed consent after the been studied in various ways (Singh and Rose, 2009), the results entire study was explained to them. This study was conducted in of previous studies lack reproducibility, sensitivity, and selectiv- accordance with the Declaration of Helsinki and approved by the ity (Kunugi et al., 2015; Buoli et al., 2016). following: the Ethics Committee of NCNP, the Ethics Committee The plasma levels of hydrophobic molecules, such as ster - of Hannan Hospital, the Research Ethics Committee of Osaka oids and unsaturated fatty acids, have been studied as candi- City University, and Wako First Research Ethics Committee of date biomarkers of MDD (Halbreich et al., 1985Lin et  ; al., 2010), RIKEN. BD (Chiu et al., 2003; Marx et al., 2006; Sublette et al., 2007), and/ or SZ (Peet et al., 2004; Reddy et al., 2004; Freeman et al., 2006; Diagnosis and Assessment of Symptoms Marx et al., 2006; Amminger et al., 2015). However, these candi- Trained psychiatrists or psychologists conducted structured date molecules were examined in patients taking medication, interviews of the participants using a Japanese version of and the potential effects of medication cannot therefore be the Mini-International Neuropsychiatric Interview (M.I.N.I.) excluded. (Sheehan et  al., 1998), and the results were used to make the In the present study, we performed a comprehensive metab- diagnoses according to the criteria of the DSM-IV (American olome analysis using liquid chromatography time-of-flight mass Psychiatric Association, 1994). In cohort 2, we divided the spectrometry (LC-TOFMS) to investigate the use of nonpolar or patients with MDD into remitted and depressive state based medium-polar metabolites as biomarkers in plasma samples of on M.I.N.I. We also divided the patients with BD into manic, drug-free patients with MDD, BD, and SZ. The reproducibility of remitted, and depressive states based on M.I.N.I. Patients with the results and the effects of psychotropic medication on ner - any comorbid axis I disorders, histories of central nervous sys- vonic acid, which was the most significantly altered metabolite, tem diseases, substance abuse/dependence, or severe head were verified in an independent sample set of mostly medicated trauma were excluded. Depressive symptoms were assessed patients. Here, we report that the levels of nervonic acid were using the 21-item Hamilton Depression Rating Scale (HAMD- increased in patients with MDD in 2 independent sample sets. 21) (Hamilton, 1960). The Positive and Negative Syndrome Scale (PANSS) (Kay and Fiszbein, 1987) was used to measure the symp- Materials and Methods tom severity of the patients with SZ. The Young Mania Rating Scale (YMRS) (Young et  al., 1978) was utilized to measure the Participants severity of the manic episodes in patients with BD. Cohort 1 of the samples used for LC-TOFMS [MDD (n= 9), BD (n = 6), SZ (n = 17)] and matched healthy controls (n = 19) w as Plasma Sampling derived from the National Center of Neurology and Psychiatry Blood samples were collected between 9:00 am and 4:00 pm (NCNP) Biobank (project no. NCNPBB-0002). The patients in with or without overnight fasting. Information on meals was cohort 1 had not taken any antipsychotic or antidepressant not available and not considered in the statistical analyses. medications for at least 2 weeks. To measure the absolute con- Blood samples were drawn from a peripheral vein, collected centrations of nervonic acid in cohort 1, gas chromatography in ethylenediaminetetraacetic acid-2Na-containing vacuum TOFMS (GC-TOFMS) was performed, but only 30 samples [MDD blood collection tubes (VENOJECT II, Terumo Corporation), (n = 6), BD (n = 4), SZ (n = 13)] and controls (n= 7) of the 51 sam- and immediately placed on ice. Within 30 minutes of the ples of cohort 1 from the NCNP Biobank were available. A second blood collection, plasma samples were isolated via centrifu- cohort of samples [MDD (n= 45), BD (n = 71), SZ (n = 115)] and con- gation at 2500× g at 4°C for 10 minutes and stored at –80°C trols (n= 90) was obtained from the NCNP Biobank (project no. until use. NCNPBB-0002-1). Cohort 2 also contained samples from Osaka Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/207/4356809 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Kageyama et al. | 209 Table 1. Demographic and Clinical Data for the Participants in Cohort 1 SZ MDD BD Control Statistics Number (male/female) 17 (8/9) 9 (3/6) 6 (1/5) 19 (10/9) P = .41 Age, years (mean ± SD) 33.6 ± 15.7 39.1 ± 10.2 41.8 ± 13.3 36.1 ± 12.9 P = .57 c b Duration of illness, years (mean ± SD) 9.1 ± 13.7 4.8 ± 2.9 7.8 ± 6.3 N.A. P = .81 HAMD-21, score (mean ± SD) N.A. 11.2 ± 8.5 14.4 ± 5.9 N.A. N.A PANSS positive, score (mean ± SD) 21.4 ± 5.4 N.A. N.A. N.A. N.A. PANSS negative, score (mean ± SD) 22.4 ± 6.4 N.A. N.A. N.A. N.A. PANSS general pathology, score (mean ± SD) 47.3 ± 11.4 N.A. N.A. N.A. N.A. YMRS, score (mean ± SD) N.A. N.A. 6.3 ± 4.0 N.A. N.A. Abbreviations: BD, bipolar disorder; HAMD-21, 21-item Hamilton Depression Rating Scale; MDD, major depressive disorder; N.A., not applicable; PANSS, Positive and Negative Symptom Scale; SZ, schizophrenia; YMRS, Young Mania Rating Scale. Chi-squared test. ANOVA. Missing data for 5 individuals in the MDD patient group and 1 individual in the BD patient group. Missing data for 4 individuals in the SZ patient group. Table 2. Demographic and Clinical Data for the Participants in Cohort 2 SZ MDD BD Control Statistics Number (male/female) 115 (59/56) 45 (19/26) 71 (37/34) 90 (46/54) P = .72 Age, years (mean ± SD) 39.0 ± 13.6 50.4 ± 16.0 45.2 ± 13.3 49.2 ± 15.0 P = .000046 Number of drug-free patients (male/female) 7 (5/2) 4 (3/1) 0 N.A. N.A. Number of patients with depressed state N.A. 34 25 N.A. N.A. Number of patients with remitted state N.A. 11 34 N.A. N.A. Number of patients with manic state N.A. N.A. 12 N.A. N.A. HAMD-21, score (mean ± SD) N.A. 17.0 ± 8.6 9.2 ± 8.1 N.A. N.A PANSS positive, score (mean ± SD) 17.7 ± 5.6 N.A. N.A. N.A. N.A. PANSS negative, score (mean ± SD) 18.7 ± 6.2 N.A. N.A. N.A. N.A. PANSS general pathology, score (mean ± SD) 36.0 ± 8.7 N.A. N.A. N.A. N.A. YMRS, score (mean ± SD) N.A. N.A. 5.7 ± 7.4 N.A. N.A. Abbreviations: BD, bipolar disorder; HAMD-21, 21-item Hamilton Depression Rating Scale; MDD, major depressive disorder; N.A., not applicable; PANSS, Positive and Negative Symptom Scale; SZ, schizophrenia; YMRS, Young Mania Rating Scale. Chi-squared test. ANOVA. at 800× g for 2 minutes at room temperature. Next, 920 µL of LC-TOFMS Analysis supernatant (upper organic phase) was dispensed in a conical Plasma samples (500 µL) were added to 1200 µL of 1% formic acid/ glass insert for autosampler vials, dried in vacuo, and added to a acetonitrile containing an internal standard solution (Solution mixture of 40 µL of pyridine, 40 µL of N-(tert-butyldimethylsilyl)- ID: H3304-1002, Human Metabolome Technologies, Inc.) at 0°C. N-methyl trifluoroacetamide, and 30  µL of acetonitrile to obtain The solution was thoroughly mixed and centrifuged at 2300 × g tert-butyldimethylsilyl (TBDMS) esters from the free fatty acids. and 4°C for 5 minutes. The supernatant was filtered through The sample solutions were placed in sealed autosampler vials, Hybrid SPE-Phospholipid (product no.  55261-U; Sigma-Aldrich vortexed, and maintained at 60°C for at least 60 minutes before Corporation). The filtrate was desiccated and then dissolved the analysis. Absolute concentrations were quantified using a with 80 µL of isopropanol and Milli-Q (1:1, v/v) for the LC-TOFMS series of standards of known concentrations of nervonic acid analysis. The detection limit was determined at a signal-to- (Sigma-Aldrich, Japan K.K.) mixed with the internal stand- noise ratio>3. The candidate peaks were assigned a specific ard. All other regents were obtained from Wako Pure Chemical metabolite identity based on their m/z (±10 ppm) and retention Industries, Ltd. time (±0.5 minutes) determined by TOFMS. The absolute concen- We used a JMS-T100GCV time-of-flight mass spectrometer trations were not determined, and the relative peak areas were (JEOL Ltd) equipped with a 7890A GC (Agilent Technologies, used in the comparisons. The metabolome measurements were Inc.) and 7693 autosampler (Agilent Technologies, Inc.). Each conducted at Human Metabolome Technologies, Inc. TBDMS-derivatized sample solution was injected (2 µL) in the splitless injection mode up to a temperature of 320°C. We used a Zebron ZB-1 MS column (30 m × 0.32  mm id; film thickness GC-TOFMS Analysis 0.25 µm, Phenomenex, Inc.). The carrier gas was helium, and it We measured the levels of nervonic acid using GC-TOFMS was applied at a constant flow-rate of 1.5 mL/min. The GC oven according to a protocol described in a previous report temperature was maintained at 120°C for 4 minutes, ramped up (Quehenberger et al., 2011) with slight modifications. Oleic acid linearly from 120°C to 360°C at 30°C/min, and then maintained d9 (2.5 ng) (Avanti Polar Lipids, Inc) was added to each plasma at 360°C for 0.5 minutes. The outlet of the column was directly sample (100  µL) as an internal standard. The plasma sample was connected to a 70-eV electron ionization source of the mass added to a mixture of 400 µL methanol, 25 µL 1N HCl, and 1000 µL spectrometer, with a resolving power >6000 (typical full width isooctane. The solution was thoroughly mixed and centrifuged at half-maximum mass resolution, 9000). The typical mass Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/207/4356809 by Ed 'DeepDyve' Gillespie user on 16 March 2018 210 | International Journal of Neuropsychopharmacology, 2018 accuracies were <5  ppm after the application of single-point (χ = 13.41, P = .0038). A nominally significant increase was found internal mass drift compensation using the column bleed peak in cortisone, a precursor of cortisol, in patients with SZ com- (m/z, 281.0517). Peak areas of high-resolution accurate-mass pared with controls (P = .048) (Figure 1a). None of these differ - extracted ion chromatograms for the characteristic fragment ences were statistically significant after correcting for multiple ions at [M -  57] from TBDMS-derivatized analyte and internal comparisons. For nervonic acid, a Kruskal-Wallis test showed standards were used for quantitation. Examples of chromato- nominally significant effect of diagnosis. However, over one-half grams of nervonic acid (m/z, 423.3658± 0.005; retention time, of the nervonic acid measures were below the detection limit of 11.50 minutes) and oleic acid d9 (m/z, 348.32842 ± 0.005; reten- LC-TOFMS. To determine the groups that showed significantly tion time, 10.00 minutes) are shown in supplementary Figure 1. different levels of nervonic acid, we reanalyzed the samples for nervonic acid using GC-TOFMS, which is more sensitive than LC-TOFMS, to obtain more precise data. We measured the abso- Statistical Analysis lute concentrations of plasma nervonic acid in the 30 available The data are presented as mean± SD. The means were compared samples [MDD (n = 6), BD (n = 4), SZ (n = 13)] and controls (n= 7) using Welch t test, 1-way ANOVA followed by posthoc Tukey’s of the 51 subjects of cohort 1. We were not able to measure the test, or Kruskal-Wallis test. Any missing values for the relative nervonic acid in the remaining 21 subjects, because sufficient area were replaced with machine epsilon (≃ 0). In the full Institute sample volumes were not available. We detected plasma ner - of Electrical and Electronics Engineers system, the spacing was vonic acid in all 30 samples. The inter-assay CV for nervonic acid - 52 2 × 10 . ANCOVA that controlled for age and sex was used to (n = 12) was 9.2%. Patients with MDD had significantly increased evaluate the effects of age and sex on the nervonic acid levels. levels of nervonic acid (0.62± 0.20 µM) compared with controls Categorical variables were compared using chi-squared test. The (0.29 ± 0.053 µM, P = .005) and patients with BD (0.35± 0.17 µM, threshold for statistical significance was set at <P .05 for all anal - P = .049) (Figure 1b). To evaluate the effects of potential con- yses, and multiple comparisons were corrected for using the founding factors on the plasma levels of nervonic acid, we false discovery rate. Statistical power and effect size were calcu- applied an ANCOVA with a dependent variable of the metabolite lated using G-Power version 3.1 (Faul et al., 2007). The statistical levels, an independent variable of diagnosis, and the covariates analyses were conducted using R version 3.2.5 (https://www . of age and sex. The effects of diagnosis on the nervonic acid lev- r-project.org/) and IBM Statistical Package for Social Sciences els remained significant (P = .009). The nervonic acid levels were Statistics 23, Japanese version (IBM Japan). not significantly correlated with age (P = .082) or sex (P = .42). Results Increased Levels of Nervonic Acid Level in Patients with MDD Metabolome Analysis in Cohort 1 We focused on nervonic acid, because it was the most signifi- The LC-TOFMS metabolome analysis detected 126 candidate cantly altered metabolite (P = .005, f = 0.78). We confirmed the peaks in the cohort 1 plasma samples (supplementary Table 1). reproducibility of the results in the medicated condition and the We used an ANOVA or Kruskal-Wallis test to identify the peaks effects of psychotropic medication on nervonic acid by measur - that significantly differed among the 4 sample groups (MDD, ing the levels of nervonic acid using GC-TOFMS in an independ- BD, SZ, and controls). A nominally significant effect of diagno- ent sample set. We performed a power analysis to determine the sis on the relative peak area was found for 2 peaks, which were sample size required to replicate the initial nervonic acid find- assigned to cortisone (F = 2.851, P = .047, f = 0.42) and nervonic acid ing with reasonable statistical power at an alpha value of 0.05, Cortisone a Nervonic acid 0.00004 2.5 P = 0.048 P = 0.005 2.0 0.00003 P = 0.049 1.5 0.00002 1.0 0.00001 0.5 0.0 0.00000 Figure  1. Plasma level of metabolites with significant differences in the cohort 1 samples. The bars indicate the mean of each group. The error bars represent the SDs. Comparison of the plasma levels of metabolites among the patients with schizophrenia (SZ), major depressive disorder (MDD), and bipolar disorder (BD), and healthy controls (1-way ANOVA with posthoc Tukey’s test). (a) Cortisone was measured using liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) in 51 samples. The Y-axis shows the relative concentrations. (b) The levels of nervonic acid were measured using gas chromatography time-of-flight mass spectrometry (GC-TOFMS) in the 30 available samples of cohort 1. The y-axis shows the absolute concentrations. The data on the relative concentrations of nervonic acid are listed in supplementary Table 1. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/207/4356809 by Ed 'DeepDyve' Gillespie user on 16 March 2018 SZ MDD BD control SZ MDD BD control Relative area μM Kageyama et al. | 211 (1  – β) of 0.80, and an effect size of f= 0.2. Because the sample M.I.N.I. results: depressed (dMDD) patients (n = 34) and remitted conditions differed between the sets (cohort 1, drug-free; cohort (rMDD) patients (n= 11) (supplementary Table  2). The levels of 2, mostly medicated), we estimated that the effect size would be nervonic acid were higher in the dMDD patients (0.65 ± 0.45 µM) relatively small. The total number of samples required to attain compared with the rMDD patients (0.40± 0.17 µM; effect size of sufficient statistical power to detect a difference was = n 280. We Cohen’s d = 0.75; P = .0091) (Figure 3a). measured 321 samples of mostly medicated patients or con- The patients with BD were divided into the following 3 trols (MDD, n = 45; BD, n = 71; SZ, n = 115; control, n = 90) (Table 2). groups based on the M.I.N.I.  results: depressed (dBD) patients Similar to the results obtained for cohort 1, patients with MDD in (n = 25), remitted (rBD) patients (n= 34), and manic (mBD) cohort 2 had significantly increased plasma nervonic acid levels patients (n= 12) (supplementary Table  3). No significant state- (0.59 ± 0.41 µM) compared with controls (0.40 ± 0.29 µM; P = .0022), dependent difference were found in the patients with BD (dBD, patients with BD (0.39± 0.25 µM; P = .0013), and patients with SZ 0.44 ± 0.30  µM; rBD, 0.32 ± 0.17 µM; mBD, 0.46 ± 0.31  µM; f = 0.57, (0.44 ± 0.24 µM; P = .021) (Figure 2). The effect size was f = 0.26. The P = .09) (Figure  3b). We compared the plasma levels of nervonic sensitivity and specificity of the plasma nervonic acid levels acid in the dMDD and dBD patients (supplementary Table 4) and that discriminated between patients with MDD and controls found that the dMDD patients had increased plasma nervonic were 67.1% and 62.2%, respectively. To evaluate the effects of the acid levels compared with those in the dBD patients (d = 0.54, confounding factors on the plasma levels of nervonic acid, we P = .038) (Figure 3c). The plasma nervonic acid levels did not sig- applied an ANCOVA with a dependent variable of the metabolite nificantly differ between the rMDD and rBD patients (d = 0.47, levels, an independent variable of diagnosis, and the covariates P = .20) (Figure 3d; supplementary Table 5). of age and sex. The effects of diagnosis on the nervonic acid lev- We then investigated the relationships among the symp- els remained significant (P = .0016), although the levels of ner - toms of the patients (PANSS positive, negative, or general path- vonic acid were affected by age (P = .010). ology scores; YMRS scores; and HAM-D21 scores), duration of illness, and nervonic acid levels with correlation analyses using Spearman’s rank correlation coefficients. There were no signifi- Relationship between Nervonic Acid Levels and the cant correlations between nervonic acid levels and the clinical Clinical Assessments scores. However, the nervonic acid concentrations positively To determine whether the levels of nervonic acid changed in correlated with the duration of illness in the patients with MDD a state-dependent manner in patients with MDD, we divided (ρ = 0.55, P = .0011). the patients with MDD into the following 2 groups based on the Discussion P = 0.0022 To the best of our knowledge, this is the first study to examine plasma nervonic acid as a potential biomarker of MDD. Because P = 0.0013 the profile of plasma metabolites might be potentially affected 2.5 by antipsychotic (Cai et  al., 2012) and antidepressant drugs P = 0.021 (Webhofer et  al., 2011), we investigated candidate metabolites in a drug-free sample set of patients, who were not currently on medication, to avoid detection of metabolites that are affected by medication. Subsequently, we confirmed that the levels of 2.0 plasma nervonic acid were altered in an independent sample set of patients who were mostly medicated. Nervonic acid [(Z)-Tetracos-15-enoic acid or 24:1, n-9 by the International Union of Pure and Applied Chemistry nomencla- 1.5 ture] is a long chain of monounsaturated omega-9 fatty acids that are particularly abundant in the white matter of the brain. Nervonic acid is an essential molecule for the growth and main- tenance of the brain and peripheral nervous tissue enriched in 1.0 sphingomyelin (Martínez and Mougan, 2002) and related to psy- chiatric disorders (Amminger et al., 2012). Because sphingomye- lin is a key constituent of myelin, it is abundant in the white matter of the brain. Our results showed that plasma nervonic 0.5 acid levels were increased in patients with MDD, in whom the integrity of the white matter has been reported as impaired (Nobuhara et al., 2006; Li et al., 2007). Thus, increased levels of plasma nervonic acid might reflect white matter dysfunction in patients with MDD. 0.0 White matter dysfunction has also been reported in patients with BD and SZ (Sussmann et al., 2009; Skudlarski et al., 2013). Why were the levels of plasma nervonic acid increased only in the patients with MDD? One possibility is that the cause of white matter dysfunction might be different in the patients with Figure  2. Absolute plasma levels of nervonic acid in the cohort 2 samples. MDD compared with the patients with BD and SZ (Johnston- The bars indicate the mean of each group. The error bars represent the SDs. Wilson et  al., 2000). Whereas sphingomyelin is abundant in Comparison of the absolute plasma levels of nervonic acid among the patients myelin-forming oligodendrocytes, it also exists in lipid rafts with schizophrenia (SZ), major depressive disorder (MDD), bipolar disorder (BD), and healthy controls (1-way ANOVA with posthoc Tukey’s test). of neuronal cells, which influence the potency and efficacy of Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/207/4356809 by Ed 'DeepDyve' Gillespie user on 16 March 2018 SZ MDD BD control Nervonicacid(μM) 212 | International Journal of Neuropsychopharmacology, 2018 a b P = 0.0091 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 P = 0.038 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 Figure 3. State-dependent changes in plasma levels of nervonic acid in patients with MDD and BD in cohort 2. The bars indicate the mean for each group. The error bars represent the SDs. (a) Comparison between patients in the depressive state of major depressive disorder (dMDD) and the remitted state of major depressive dis- order (rMDD) (Welch t test). (b) Comparison among patients in the depressive state of bipolar disorder (dBD), the remitted state of bipolar disorder (rBD), and the manic state of bipolar disorder (mBD) (1-way ANOVA with posthoc Tukey’s test). (c) Comparison between patients with dMDD and dBD (Welch t test). (d) Comparison between patients with rMDD and rBD (Welch t test). neurotransmitter receptors and transporters. The effects of types of samples (plasma vs erythrocyte membrane) (Hayashi- lipid rafts on neurotransmitter signaling have been implicated Takagi et  al., 2014). In addition, previous studies (Assies et  al., in neurological and psychiatric diseases (Allen et  al., 2007). 2010; Lin et  al., 2010) have shown that omega-3 and omega-6 Sphingomyelin has also been reported to be associated with polyunsaturated fatty acids are altered in patients with MDD the regulation of neurogenesis. An association of decreased compared with controls. We detected omega-3 and omega-6 neurogenesis in the hippocampus and the dysregulation of polyunsaturated fatty acids (supplemental Table 1), but no sig- sphingomyelin metabolic pathway has been suggested to play nificant differences were found between patients with MDD and a role in depression (Gulbins et  al., 2015), and antidepressants controls. Because nervonic acid does not cross the blood-brain have been postulated to enhance neurogenesis by inhibiting the barrier (Coupland et al., 2003), it is not clear whether there is a acid sphingomyelinase/ceramide system, which is part of the relationship between peripheral nervonic acid levels and its lev- sphingomyelin metabolic pathway (Gulbins et  al., 2013). Thus, els in the brain. More studies, including studies of postmortem increased levels of plasma nervonic acid might reflect dysregu- brains or cerebrospinal fluid, are necessary to fully elucidate the lation of oligodendrocytes, sphingomyelin-rich lipid rafts, and/ relationship between MDD and nervonic acid. or the sphingomyelin metabolic pathway in patients with MDD. A previous study (Kim et al., 2016) has reported a correlation A previous report has shown that the nervonic acid levels between erythrocyte nervonic acid and positive symptoms on that were measured in erythrocyte membranes from patients PANSS in ultra-high-risk subjects. However, plasma nervonic with recurrent MDD were decreased (Assies et  al., 2010). This acid levels and PANSS scores were not correlated in the results finding might be due to differences in MDD subtype (recurrent of the present study, and the difference between the previous vs not recurrent), methodology (LC-TOFMS vs GC-TOFMS), or the findings and our findings might have been due to the differences Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/207/4356809 by Ed 'DeepDyve' Gillespie user on 16 March 2018 dMDD rMDD dBD rBD dMDD mBD dBD rMDD rBD Nervonic acid (μM) Nervonic acid (μM) Nervonic acid (μM) Nervonic acid (μM) Kageyama et al. | 213 in the subjects (SZ vs ultra-high-risk subjects) or types of sam- Acknowledgments ples (plasma vs erythrocyte membrane). We thank Drs Taku Doi and Mariko Yamada for their help during We calculated the effect sizes to evaluate if plasma nervonic sample collection. We are grateful to the members of our labora- acid levels would be useful as a diagnostic biomarker. The effect tory for their valuable discussions and technical assistance and size of the nervonic acid level comparisons between the dMDD to the patients and controls who participated in this study. and rMDD patients (d = 0.75, Figure  3a) and dMDD and dBD patients (d = 0.54, Figure  3c) was medium, while the effect size was small in the comparisons among MDD, BD, SZ, and controls Author Contributions (f = 0.26, Figure 2). These results suggested that nervonic acid lev- T. Kasahara, T. Kato, and Y. Kageyama conceived and designed els can be used as a biomarker to discriminate the depressive the study. Y. Kageyama, T. Nakamura, T. Kasahara, and T. Kato state of patients with MDD from the euthymic state of patients acquired and analyzed the data. K. Hattori, S. Yoshida, Y. Goto , with MDD or the depressive state of patients with BD. On the K. Inoue, M. Tani, Y. Deguchi, K. Kuroda, and Y. Kageyama other hand, nervonic acid levels would not be useful to discrim- collected the human samples and drafted the manuscript. inate euthymic patients with MDD from controls or patients Y. Kageyama, T. Kasahara, and T. Kato wrote the paper. with other mental disorders (SZ or BD). Additional longitu- dinal measurements of plasma nervonic acid levels along with detailed information on clinical symptoms in the same individ- Funding ual are required to determine whether nervonic acid levels show This work was supported by the Japan Agency for Medical state or trait-dependent changes. Research and Development (T. Kato: 16815678), MEXT/JSPS Cortisone is a 21-carbon steroid that is one of the main KAKENHI grants (T. Kato: 24249063 and 17H01573), and a grant- hormones released by the adrenal gland in response to stress in-aid from the Japanese Ministry of Health and Labor (T. Kato: (Tacker et  al., 1978). It is converted to the active metabolite 201241002A). Y.  Kageyama is supported by a RIKEN Junior hydrocortisone, which is also called cortisol. The results of the Research Associate fellowship. current study showed that patients with SZ exhibited increased levels of cortisone. These results may reflect the well-estab- lished finding of abnormalities of the hypothalamic-pituitary- Statement of Interest adrenal axis in patients with SZ (Walker et al., 2008). A previous T. Kato has received honoraria for lectures, manuscripts, and/ report has shown that plasma cortisone levels were increased in or consultancy from Kyowa Hakko Kirin Co., Ltd.; Eli Lilly Japan patients with MDD compared with controls (Weber et al., 2000). K.K.; Otsuka Pharmaceutical Co., Ltd.; GlaxoSmithKline K.K.; However, no significant differences were found in this study. Taisho Toyama Pharmaceutical Co., Ltd.; Dainippon Sumitomo The discrepancy of these results might have been a result of dif- Pharma Co., Ltd.; Meiji Seika Pharma Co., Ltd.; Pfizer Japan Inc.; ferences in methodology (LC-TOFMS vs specific radioimmuno- Mochida Pharmaceutical Co., Ltd.; Shionogi & Co., Ltd.; Janssen assay) (Hayashi-Takagi et al., 2014). Pharmaceutical K.K.; Yoshitomiyakuhin; Astellas Pharma Inc.; This study had several limitations. First, effects of diet Wako Pure Chemical Industries, Ltd.; and Takeda Pharmaceutical (Cevallos-cevallos et al., 2009 Y; oung et al., 2017), age (Jové et al., Co., Ltd. within the last 3 years. T. Kato also received a research 2014), circadian variation (Dallmann et al., 2012), and other con- grant from Takeda Pharmaceutical Co., Ltd. These companies founding factors (Lawton et  al., 2008) on the plasma levels of played no role in the study design, data collection and analysis, metabolites cannot be excluded. Because we did not strictly con- decision to publish, or preparation of the manuscript. trol the meal and sample collection time, some of the observed changes might be attributable to these confounding factors. Additional studies involving larger sample size and controlling for References all confounding factors are needed to validate the use of plasma Allen JA, Halverson-Tamboli RA, Rasenick MM (2007) Lipid raft nervonic acid levels as a biomarker of MDD. Second, we obtained microdomains and neurotransmitter signalling. Nat Rev data only for patients with MDD, BD, and SZ, as well as controls. Neurosci 8:128–140. Additional studies of patients with other neuropsychiatric disor - Altamura AC, Buoli M, Caldiroli A, Caron L, Cumerlato Melter ders and demyelinating diseases, such as adrenoleukodystrophy C, Dobrea C, Cigliobianco M, Zanelli Quarantini F (2015) (Moser et al., 1981), are necessary to determine if increased levels Misdiagnosis, duration of untreated illness (DUI) and out- of plasma nervonic acid level are specific to patients with MDD. come in bipolar patients with psychotic symptoms: a natur - Third, the number of participants examined in this study was alistic study. J Affect Disord 182:70–75. too small to draw definitive conclusions. Fourth, a confounding American Psychiatric Association (1994) Diagnostic and statis- effect of metabolic syndrome on plasma nervonic acid cannot be tical manual of mental disorders. 4th ed. 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Annu Rev Clin Psychol 4:189–216. 117:1257–1269. Weber B, Lewicka S, Deuschle M, Colla M, Vecsei P, Heuser I Young RC, Biggs JT, Ziegler VE, Meyer DA (1978) A rating scale (2000) Increased diurnal plasma concentrations of cortisone for mania: reliability, validity and sensitivity. Br J Psychiatry in depressed patients. J Clin Endocrinol Metab 85:1133–1136. 133:429–435. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/207/4356809 by Ed 'DeepDyve' Gillespie user on 16 March 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Journal of Neuropsychopharmacology Oxford University Press
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Abstract

Background: Diagnostic biomarkers of major depressive disorder, bipolar disorder, and schizophrenia are urgently needed, because none are currently available. Methods: We performed a comprehensive metabolome analysis of plasma samples from drug-free patients with major depressive disorder (n = 9), bipolar disorder (n= 6), schizophrenia (n = 17), and matched healthy controls (n = 19) (cohort 1) using liquid chromatography time-of-flight mass spectrometry. A  significant effect of diagnosis was found for 2 metabolites: nervonic acid and cortisone, with nervonic acid being the most significantly altered. The reproducibility of the results and effects of psychotropic medication on nervonic acid were verified in cohort 2, an independent sample set of medicated patients [major depressive disorder (n= 45), bipolar disorder (n= 71), schizophrenia (n = 115)], and controls (n = 90) using gas chromatography time-of-flight mass spectrometry. Results: The increased levels of nervonic acid in patients with major depressive disorder compared with controls and patients with bipolar disorder in cohort 1 were replicated in the independent sample set (cohort 2). In cohort 2, plasma nervonic acid levels were also increased in the patients with major depressive disorder compared with the patients with schizophrenia. Significance Statement Box Sample In cohort 2, nervonic acid levels were increased in the depressive state in patients with major depressive disorder compared with the levels in the remission state in patients with major depressive disorder and the depressive state in patients with bipolar disorder. Conclusion: These results suggested that plasma nervonic acid is a good candidate biomarker for the depressive state of major depressive disorder. Keywords: biomarker, bipolar disorder, major depressive disorder, metabolomics, nervonic acid Received: June 19, 2017; Revised: September 21, 2017; Accepted: October 2, 2017 © The Author(s) 2017. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 207 provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/207/4356809 by Ed 'DeepDyve' Gillespie user on 16 March 2018 208 | International Journal of Neuropsychopharmacology, 2018 Significance Statement Plasma nervonic acid levels were increased in patients with major depressive disorder (MDD) compared with those in patients with bipolar disorder and healthy controls. Nervonic acid levels were higher in the depressive state in the patients with MDD compared with the remission state in the patients with MDD and the depressive state of patients with bipolar disorder, suggesting state-dependent alterations. Therefore, plasma nervonic acid is a good candidate diagnostic biomarker for MDD. Introduction Although a proper diagnosis and medical treatment are import- City University Hospital and Hannan Hospital, where controls ant for improving the prognosis of patients with mental illness were recruited from among the healthy spouses of the patients. (Post et al., 2010; Gaebel and Zielasek, 2015), diagnoses are diffi- The control samples in the NCNP Biobank were collected by cult to make, especially when major depressive disorder (MDD) advertising in local free magazines and on websites. needs to be distinguished from the depressive state of bipolar The sex ratios and ages did not significantly differ among the disorder (BD). In addition, patients with schizophrenia (SZ) can groups in cohort 1 (Table 1 ). Among the groups in cohort 2, the also show depressive symptoms. Because treatment approaches sex ratios did not significantly differ, while age did (Table ). 2 The differ among patients with MDD, BD, and SZ, diagnostic bio- age of participants in the MDD and control groups was higher markers that distinguish MDD, BD, and SZ are required to avoid than that of SZ (P = .000046 and .000004, respectively). ANCOVA misdiagnosis (Altamura et al., 2015) and obtain a better progno- was used for the analysis of age. sis (Drancourt et al., 2013). Although candidate biomarkers have All participants provided written informed consent after the been studied in various ways (Singh and Rose, 2009), the results entire study was explained to them. This study was conducted in of previous studies lack reproducibility, sensitivity, and selectiv- accordance with the Declaration of Helsinki and approved by the ity (Kunugi et al., 2015; Buoli et al., 2016). following: the Ethics Committee of NCNP, the Ethics Committee The plasma levels of hydrophobic molecules, such as ster - of Hannan Hospital, the Research Ethics Committee of Osaka oids and unsaturated fatty acids, have been studied as candi- City University, and Wako First Research Ethics Committee of date biomarkers of MDD (Halbreich et al., 1985Lin et  ; al., 2010), RIKEN. BD (Chiu et al., 2003; Marx et al., 2006; Sublette et al., 2007), and/ or SZ (Peet et al., 2004; Reddy et al., 2004; Freeman et al., 2006; Diagnosis and Assessment of Symptoms Marx et al., 2006; Amminger et al., 2015). However, these candi- Trained psychiatrists or psychologists conducted structured date molecules were examined in patients taking medication, interviews of the participants using a Japanese version of and the potential effects of medication cannot therefore be the Mini-International Neuropsychiatric Interview (M.I.N.I.) excluded. (Sheehan et  al., 1998), and the results were used to make the In the present study, we performed a comprehensive metab- diagnoses according to the criteria of the DSM-IV (American olome analysis using liquid chromatography time-of-flight mass Psychiatric Association, 1994). In cohort 2, we divided the spectrometry (LC-TOFMS) to investigate the use of nonpolar or patients with MDD into remitted and depressive state based medium-polar metabolites as biomarkers in plasma samples of on M.I.N.I. We also divided the patients with BD into manic, drug-free patients with MDD, BD, and SZ. The reproducibility of remitted, and depressive states based on M.I.N.I. Patients with the results and the effects of psychotropic medication on ner - any comorbid axis I disorders, histories of central nervous sys- vonic acid, which was the most significantly altered metabolite, tem diseases, substance abuse/dependence, or severe head were verified in an independent sample set of mostly medicated trauma were excluded. Depressive symptoms were assessed patients. Here, we report that the levels of nervonic acid were using the 21-item Hamilton Depression Rating Scale (HAMD- increased in patients with MDD in 2 independent sample sets. 21) (Hamilton, 1960). The Positive and Negative Syndrome Scale (PANSS) (Kay and Fiszbein, 1987) was used to measure the symp- Materials and Methods tom severity of the patients with SZ. The Young Mania Rating Scale (YMRS) (Young et  al., 1978) was utilized to measure the Participants severity of the manic episodes in patients with BD. Cohort 1 of the samples used for LC-TOFMS [MDD (n= 9), BD (n = 6), SZ (n = 17)] and matched healthy controls (n = 19) w as Plasma Sampling derived from the National Center of Neurology and Psychiatry Blood samples were collected between 9:00 am and 4:00 pm (NCNP) Biobank (project no. NCNPBB-0002). The patients in with or without overnight fasting. Information on meals was cohort 1 had not taken any antipsychotic or antidepressant not available and not considered in the statistical analyses. medications for at least 2 weeks. To measure the absolute con- Blood samples were drawn from a peripheral vein, collected centrations of nervonic acid in cohort 1, gas chromatography in ethylenediaminetetraacetic acid-2Na-containing vacuum TOFMS (GC-TOFMS) was performed, but only 30 samples [MDD blood collection tubes (VENOJECT II, Terumo Corporation), (n = 6), BD (n = 4), SZ (n = 13)] and controls (n= 7) of the 51 sam- and immediately placed on ice. Within 30 minutes of the ples of cohort 1 from the NCNP Biobank were available. A second blood collection, plasma samples were isolated via centrifu- cohort of samples [MDD (n= 45), BD (n = 71), SZ (n = 115)] and con- gation at 2500× g at 4°C for 10 minutes and stored at –80°C trols (n= 90) was obtained from the NCNP Biobank (project no. until use. NCNPBB-0002-1). Cohort 2 also contained samples from Osaka Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/207/4356809 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Kageyama et al. | 209 Table 1. Demographic and Clinical Data for the Participants in Cohort 1 SZ MDD BD Control Statistics Number (male/female) 17 (8/9) 9 (3/6) 6 (1/5) 19 (10/9) P = .41 Age, years (mean ± SD) 33.6 ± 15.7 39.1 ± 10.2 41.8 ± 13.3 36.1 ± 12.9 P = .57 c b Duration of illness, years (mean ± SD) 9.1 ± 13.7 4.8 ± 2.9 7.8 ± 6.3 N.A. P = .81 HAMD-21, score (mean ± SD) N.A. 11.2 ± 8.5 14.4 ± 5.9 N.A. N.A PANSS positive, score (mean ± SD) 21.4 ± 5.4 N.A. N.A. N.A. N.A. PANSS negative, score (mean ± SD) 22.4 ± 6.4 N.A. N.A. N.A. N.A. PANSS general pathology, score (mean ± SD) 47.3 ± 11.4 N.A. N.A. N.A. N.A. YMRS, score (mean ± SD) N.A. N.A. 6.3 ± 4.0 N.A. N.A. Abbreviations: BD, bipolar disorder; HAMD-21, 21-item Hamilton Depression Rating Scale; MDD, major depressive disorder; N.A., not applicable; PANSS, Positive and Negative Symptom Scale; SZ, schizophrenia; YMRS, Young Mania Rating Scale. Chi-squared test. ANOVA. Missing data for 5 individuals in the MDD patient group and 1 individual in the BD patient group. Missing data for 4 individuals in the SZ patient group. Table 2. Demographic and Clinical Data for the Participants in Cohort 2 SZ MDD BD Control Statistics Number (male/female) 115 (59/56) 45 (19/26) 71 (37/34) 90 (46/54) P = .72 Age, years (mean ± SD) 39.0 ± 13.6 50.4 ± 16.0 45.2 ± 13.3 49.2 ± 15.0 P = .000046 Number of drug-free patients (male/female) 7 (5/2) 4 (3/1) 0 N.A. N.A. Number of patients with depressed state N.A. 34 25 N.A. N.A. Number of patients with remitted state N.A. 11 34 N.A. N.A. Number of patients with manic state N.A. N.A. 12 N.A. N.A. HAMD-21, score (mean ± SD) N.A. 17.0 ± 8.6 9.2 ± 8.1 N.A. N.A PANSS positive, score (mean ± SD) 17.7 ± 5.6 N.A. N.A. N.A. N.A. PANSS negative, score (mean ± SD) 18.7 ± 6.2 N.A. N.A. N.A. N.A. PANSS general pathology, score (mean ± SD) 36.0 ± 8.7 N.A. N.A. N.A. N.A. YMRS, score (mean ± SD) N.A. N.A. 5.7 ± 7.4 N.A. N.A. Abbreviations: BD, bipolar disorder; HAMD-21, 21-item Hamilton Depression Rating Scale; MDD, major depressive disorder; N.A., not applicable; PANSS, Positive and Negative Symptom Scale; SZ, schizophrenia; YMRS, Young Mania Rating Scale. Chi-squared test. ANOVA. at 800× g for 2 minutes at room temperature. Next, 920 µL of LC-TOFMS Analysis supernatant (upper organic phase) was dispensed in a conical Plasma samples (500 µL) were added to 1200 µL of 1% formic acid/ glass insert for autosampler vials, dried in vacuo, and added to a acetonitrile containing an internal standard solution (Solution mixture of 40 µL of pyridine, 40 µL of N-(tert-butyldimethylsilyl)- ID: H3304-1002, Human Metabolome Technologies, Inc.) at 0°C. N-methyl trifluoroacetamide, and 30  µL of acetonitrile to obtain The solution was thoroughly mixed and centrifuged at 2300 × g tert-butyldimethylsilyl (TBDMS) esters from the free fatty acids. and 4°C for 5 minutes. The supernatant was filtered through The sample solutions were placed in sealed autosampler vials, Hybrid SPE-Phospholipid (product no.  55261-U; Sigma-Aldrich vortexed, and maintained at 60°C for at least 60 minutes before Corporation). The filtrate was desiccated and then dissolved the analysis. Absolute concentrations were quantified using a with 80 µL of isopropanol and Milli-Q (1:1, v/v) for the LC-TOFMS series of standards of known concentrations of nervonic acid analysis. The detection limit was determined at a signal-to- (Sigma-Aldrich, Japan K.K.) mixed with the internal stand- noise ratio>3. The candidate peaks were assigned a specific ard. All other regents were obtained from Wako Pure Chemical metabolite identity based on their m/z (±10 ppm) and retention Industries, Ltd. time (±0.5 minutes) determined by TOFMS. The absolute concen- We used a JMS-T100GCV time-of-flight mass spectrometer trations were not determined, and the relative peak areas were (JEOL Ltd) equipped with a 7890A GC (Agilent Technologies, used in the comparisons. The metabolome measurements were Inc.) and 7693 autosampler (Agilent Technologies, Inc.). Each conducted at Human Metabolome Technologies, Inc. TBDMS-derivatized sample solution was injected (2 µL) in the splitless injection mode up to a temperature of 320°C. We used a Zebron ZB-1 MS column (30 m × 0.32  mm id; film thickness GC-TOFMS Analysis 0.25 µm, Phenomenex, Inc.). The carrier gas was helium, and it We measured the levels of nervonic acid using GC-TOFMS was applied at a constant flow-rate of 1.5 mL/min. The GC oven according to a protocol described in a previous report temperature was maintained at 120°C for 4 minutes, ramped up (Quehenberger et al., 2011) with slight modifications. Oleic acid linearly from 120°C to 360°C at 30°C/min, and then maintained d9 (2.5 ng) (Avanti Polar Lipids, Inc) was added to each plasma at 360°C for 0.5 minutes. The outlet of the column was directly sample (100  µL) as an internal standard. The plasma sample was connected to a 70-eV electron ionization source of the mass added to a mixture of 400 µL methanol, 25 µL 1N HCl, and 1000 µL spectrometer, with a resolving power >6000 (typical full width isooctane. The solution was thoroughly mixed and centrifuged at half-maximum mass resolution, 9000). The typical mass Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/207/4356809 by Ed 'DeepDyve' Gillespie user on 16 March 2018 210 | International Journal of Neuropsychopharmacology, 2018 accuracies were <5  ppm after the application of single-point (χ = 13.41, P = .0038). A nominally significant increase was found internal mass drift compensation using the column bleed peak in cortisone, a precursor of cortisol, in patients with SZ com- (m/z, 281.0517). Peak areas of high-resolution accurate-mass pared with controls (P = .048) (Figure 1a). None of these differ - extracted ion chromatograms for the characteristic fragment ences were statistically significant after correcting for multiple ions at [M -  57] from TBDMS-derivatized analyte and internal comparisons. For nervonic acid, a Kruskal-Wallis test showed standards were used for quantitation. Examples of chromato- nominally significant effect of diagnosis. However, over one-half grams of nervonic acid (m/z, 423.3658± 0.005; retention time, of the nervonic acid measures were below the detection limit of 11.50 minutes) and oleic acid d9 (m/z, 348.32842 ± 0.005; reten- LC-TOFMS. To determine the groups that showed significantly tion time, 10.00 minutes) are shown in supplementary Figure 1. different levels of nervonic acid, we reanalyzed the samples for nervonic acid using GC-TOFMS, which is more sensitive than LC-TOFMS, to obtain more precise data. We measured the abso- Statistical Analysis lute concentrations of plasma nervonic acid in the 30 available The data are presented as mean± SD. The means were compared samples [MDD (n = 6), BD (n = 4), SZ (n = 13)] and controls (n= 7) using Welch t test, 1-way ANOVA followed by posthoc Tukey’s of the 51 subjects of cohort 1. We were not able to measure the test, or Kruskal-Wallis test. Any missing values for the relative nervonic acid in the remaining 21 subjects, because sufficient area were replaced with machine epsilon (≃ 0). In the full Institute sample volumes were not available. We detected plasma ner - of Electrical and Electronics Engineers system, the spacing was vonic acid in all 30 samples. The inter-assay CV for nervonic acid - 52 2 × 10 . ANCOVA that controlled for age and sex was used to (n = 12) was 9.2%. Patients with MDD had significantly increased evaluate the effects of age and sex on the nervonic acid levels. levels of nervonic acid (0.62± 0.20 µM) compared with controls Categorical variables were compared using chi-squared test. The (0.29 ± 0.053 µM, P = .005) and patients with BD (0.35± 0.17 µM, threshold for statistical significance was set at <P .05 for all anal - P = .049) (Figure 1b). To evaluate the effects of potential con- yses, and multiple comparisons were corrected for using the founding factors on the plasma levels of nervonic acid, we false discovery rate. Statistical power and effect size were calcu- applied an ANCOVA with a dependent variable of the metabolite lated using G-Power version 3.1 (Faul et al., 2007). The statistical levels, an independent variable of diagnosis, and the covariates analyses were conducted using R version 3.2.5 (https://www . of age and sex. The effects of diagnosis on the nervonic acid lev- r-project.org/) and IBM Statistical Package for Social Sciences els remained significant (P = .009). The nervonic acid levels were Statistics 23, Japanese version (IBM Japan). not significantly correlated with age (P = .082) or sex (P = .42). Results Increased Levels of Nervonic Acid Level in Patients with MDD Metabolome Analysis in Cohort 1 We focused on nervonic acid, because it was the most signifi- The LC-TOFMS metabolome analysis detected 126 candidate cantly altered metabolite (P = .005, f = 0.78). We confirmed the peaks in the cohort 1 plasma samples (supplementary Table 1). reproducibility of the results in the medicated condition and the We used an ANOVA or Kruskal-Wallis test to identify the peaks effects of psychotropic medication on nervonic acid by measur - that significantly differed among the 4 sample groups (MDD, ing the levels of nervonic acid using GC-TOFMS in an independ- BD, SZ, and controls). A nominally significant effect of diagno- ent sample set. We performed a power analysis to determine the sis on the relative peak area was found for 2 peaks, which were sample size required to replicate the initial nervonic acid find- assigned to cortisone (F = 2.851, P = .047, f = 0.42) and nervonic acid ing with reasonable statistical power at an alpha value of 0.05, Cortisone a Nervonic acid 0.00004 2.5 P = 0.048 P = 0.005 2.0 0.00003 P = 0.049 1.5 0.00002 1.0 0.00001 0.5 0.0 0.00000 Figure  1. Plasma level of metabolites with significant differences in the cohort 1 samples. The bars indicate the mean of each group. The error bars represent the SDs. Comparison of the plasma levels of metabolites among the patients with schizophrenia (SZ), major depressive disorder (MDD), and bipolar disorder (BD), and healthy controls (1-way ANOVA with posthoc Tukey’s test). (a) Cortisone was measured using liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) in 51 samples. The Y-axis shows the relative concentrations. (b) The levels of nervonic acid were measured using gas chromatography time-of-flight mass spectrometry (GC-TOFMS) in the 30 available samples of cohort 1. The y-axis shows the absolute concentrations. The data on the relative concentrations of nervonic acid are listed in supplementary Table 1. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/207/4356809 by Ed 'DeepDyve' Gillespie user on 16 March 2018 SZ MDD BD control SZ MDD BD control Relative area μM Kageyama et al. | 211 (1  – β) of 0.80, and an effect size of f= 0.2. Because the sample M.I.N.I. results: depressed (dMDD) patients (n = 34) and remitted conditions differed between the sets (cohort 1, drug-free; cohort (rMDD) patients (n= 11) (supplementary Table  2). The levels of 2, mostly medicated), we estimated that the effect size would be nervonic acid were higher in the dMDD patients (0.65 ± 0.45 µM) relatively small. The total number of samples required to attain compared with the rMDD patients (0.40± 0.17 µM; effect size of sufficient statistical power to detect a difference was = n 280. We Cohen’s d = 0.75; P = .0091) (Figure 3a). measured 321 samples of mostly medicated patients or con- The patients with BD were divided into the following 3 trols (MDD, n = 45; BD, n = 71; SZ, n = 115; control, n = 90) (Table 2). groups based on the M.I.N.I.  results: depressed (dBD) patients Similar to the results obtained for cohort 1, patients with MDD in (n = 25), remitted (rBD) patients (n= 34), and manic (mBD) cohort 2 had significantly increased plasma nervonic acid levels patients (n= 12) (supplementary Table  3). No significant state- (0.59 ± 0.41 µM) compared with controls (0.40 ± 0.29 µM; P = .0022), dependent difference were found in the patients with BD (dBD, patients with BD (0.39± 0.25 µM; P = .0013), and patients with SZ 0.44 ± 0.30  µM; rBD, 0.32 ± 0.17 µM; mBD, 0.46 ± 0.31  µM; f = 0.57, (0.44 ± 0.24 µM; P = .021) (Figure 2). The effect size was f = 0.26. The P = .09) (Figure  3b). We compared the plasma levels of nervonic sensitivity and specificity of the plasma nervonic acid levels acid in the dMDD and dBD patients (supplementary Table 4) and that discriminated between patients with MDD and controls found that the dMDD patients had increased plasma nervonic were 67.1% and 62.2%, respectively. To evaluate the effects of the acid levels compared with those in the dBD patients (d = 0.54, confounding factors on the plasma levels of nervonic acid, we P = .038) (Figure 3c). The plasma nervonic acid levels did not sig- applied an ANCOVA with a dependent variable of the metabolite nificantly differ between the rMDD and rBD patients (d = 0.47, levels, an independent variable of diagnosis, and the covariates P = .20) (Figure 3d; supplementary Table 5). of age and sex. The effects of diagnosis on the nervonic acid lev- We then investigated the relationships among the symp- els remained significant (P = .0016), although the levels of ner - toms of the patients (PANSS positive, negative, or general path- vonic acid were affected by age (P = .010). ology scores; YMRS scores; and HAM-D21 scores), duration of illness, and nervonic acid levels with correlation analyses using Spearman’s rank correlation coefficients. There were no signifi- Relationship between Nervonic Acid Levels and the cant correlations between nervonic acid levels and the clinical Clinical Assessments scores. However, the nervonic acid concentrations positively To determine whether the levels of nervonic acid changed in correlated with the duration of illness in the patients with MDD a state-dependent manner in patients with MDD, we divided (ρ = 0.55, P = .0011). the patients with MDD into the following 2 groups based on the Discussion P = 0.0022 To the best of our knowledge, this is the first study to examine plasma nervonic acid as a potential biomarker of MDD. Because P = 0.0013 the profile of plasma metabolites might be potentially affected 2.5 by antipsychotic (Cai et  al., 2012) and antidepressant drugs P = 0.021 (Webhofer et  al., 2011), we investigated candidate metabolites in a drug-free sample set of patients, who were not currently on medication, to avoid detection of metabolites that are affected by medication. Subsequently, we confirmed that the levels of 2.0 plasma nervonic acid were altered in an independent sample set of patients who were mostly medicated. Nervonic acid [(Z)-Tetracos-15-enoic acid or 24:1, n-9 by the International Union of Pure and Applied Chemistry nomencla- 1.5 ture] is a long chain of monounsaturated omega-9 fatty acids that are particularly abundant in the white matter of the brain. Nervonic acid is an essential molecule for the growth and main- tenance of the brain and peripheral nervous tissue enriched in 1.0 sphingomyelin (Martínez and Mougan, 2002) and related to psy- chiatric disorders (Amminger et al., 2012). Because sphingomye- lin is a key constituent of myelin, it is abundant in the white matter of the brain. Our results showed that plasma nervonic 0.5 acid levels were increased in patients with MDD, in whom the integrity of the white matter has been reported as impaired (Nobuhara et al., 2006; Li et al., 2007). Thus, increased levels of plasma nervonic acid might reflect white matter dysfunction in patients with MDD. 0.0 White matter dysfunction has also been reported in patients with BD and SZ (Sussmann et al., 2009; Skudlarski et al., 2013). Why were the levels of plasma nervonic acid increased only in the patients with MDD? One possibility is that the cause of white matter dysfunction might be different in the patients with Figure  2. Absolute plasma levels of nervonic acid in the cohort 2 samples. MDD compared with the patients with BD and SZ (Johnston- The bars indicate the mean of each group. The error bars represent the SDs. Wilson et  al., 2000). Whereas sphingomyelin is abundant in Comparison of the absolute plasma levels of nervonic acid among the patients myelin-forming oligodendrocytes, it also exists in lipid rafts with schizophrenia (SZ), major depressive disorder (MDD), bipolar disorder (BD), and healthy controls (1-way ANOVA with posthoc Tukey’s test). of neuronal cells, which influence the potency and efficacy of Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/207/4356809 by Ed 'DeepDyve' Gillespie user on 16 March 2018 SZ MDD BD control Nervonicacid(μM) 212 | International Journal of Neuropsychopharmacology, 2018 a b P = 0.0091 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 P = 0.038 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 Figure 3. State-dependent changes in plasma levels of nervonic acid in patients with MDD and BD in cohort 2. The bars indicate the mean for each group. The error bars represent the SDs. (a) Comparison between patients in the depressive state of major depressive disorder (dMDD) and the remitted state of major depressive dis- order (rMDD) (Welch t test). (b) Comparison among patients in the depressive state of bipolar disorder (dBD), the remitted state of bipolar disorder (rBD), and the manic state of bipolar disorder (mBD) (1-way ANOVA with posthoc Tukey’s test). (c) Comparison between patients with dMDD and dBD (Welch t test). (d) Comparison between patients with rMDD and rBD (Welch t test). neurotransmitter receptors and transporters. The effects of types of samples (plasma vs erythrocyte membrane) (Hayashi- lipid rafts on neurotransmitter signaling have been implicated Takagi et  al., 2014). In addition, previous studies (Assies et  al., in neurological and psychiatric diseases (Allen et  al., 2007). 2010; Lin et  al., 2010) have shown that omega-3 and omega-6 Sphingomyelin has also been reported to be associated with polyunsaturated fatty acids are altered in patients with MDD the regulation of neurogenesis. An association of decreased compared with controls. We detected omega-3 and omega-6 neurogenesis in the hippocampus and the dysregulation of polyunsaturated fatty acids (supplemental Table 1), but no sig- sphingomyelin metabolic pathway has been suggested to play nificant differences were found between patients with MDD and a role in depression (Gulbins et  al., 2015), and antidepressants controls. Because nervonic acid does not cross the blood-brain have been postulated to enhance neurogenesis by inhibiting the barrier (Coupland et al., 2003), it is not clear whether there is a acid sphingomyelinase/ceramide system, which is part of the relationship between peripheral nervonic acid levels and its lev- sphingomyelin metabolic pathway (Gulbins et  al., 2013). Thus, els in the brain. More studies, including studies of postmortem increased levels of plasma nervonic acid might reflect dysregu- brains or cerebrospinal fluid, are necessary to fully elucidate the lation of oligodendrocytes, sphingomyelin-rich lipid rafts, and/ relationship between MDD and nervonic acid. or the sphingomyelin metabolic pathway in patients with MDD. A previous study (Kim et al., 2016) has reported a correlation A previous report has shown that the nervonic acid levels between erythrocyte nervonic acid and positive symptoms on that were measured in erythrocyte membranes from patients PANSS in ultra-high-risk subjects. However, plasma nervonic with recurrent MDD were decreased (Assies et  al., 2010). This acid levels and PANSS scores were not correlated in the results finding might be due to differences in MDD subtype (recurrent of the present study, and the difference between the previous vs not recurrent), methodology (LC-TOFMS vs GC-TOFMS), or the findings and our findings might have been due to the differences Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/207/4356809 by Ed 'DeepDyve' Gillespie user on 16 March 2018 dMDD rMDD dBD rBD dMDD mBD dBD rMDD rBD Nervonic acid (μM) Nervonic acid (μM) Nervonic acid (μM) Nervonic acid (μM) Kageyama et al. | 213 in the subjects (SZ vs ultra-high-risk subjects) or types of sam- Acknowledgments ples (plasma vs erythrocyte membrane). We thank Drs Taku Doi and Mariko Yamada for their help during We calculated the effect sizes to evaluate if plasma nervonic sample collection. We are grateful to the members of our labora- acid levels would be useful as a diagnostic biomarker. The effect tory for their valuable discussions and technical assistance and size of the nervonic acid level comparisons between the dMDD to the patients and controls who participated in this study. and rMDD patients (d = 0.75, Figure  3a) and dMDD and dBD patients (d = 0.54, Figure  3c) was medium, while the effect size was small in the comparisons among MDD, BD, SZ, and controls Author Contributions (f = 0.26, Figure 2). These results suggested that nervonic acid lev- T. Kasahara, T. Kato, and Y. Kageyama conceived and designed els can be used as a biomarker to discriminate the depressive the study. Y. Kageyama, T. Nakamura, T. Kasahara, and T. Kato state of patients with MDD from the euthymic state of patients acquired and analyzed the data. K. Hattori, S. Yoshida, Y. Goto , with MDD or the depressive state of patients with BD. On the K. Inoue, M. Tani, Y. Deguchi, K. Kuroda, and Y. Kageyama other hand, nervonic acid levels would not be useful to discrim- collected the human samples and drafted the manuscript. inate euthymic patients with MDD from controls or patients Y. Kageyama, T. Kasahara, and T. Kato wrote the paper. with other mental disorders (SZ or BD). Additional longitu- dinal measurements of plasma nervonic acid levels along with detailed information on clinical symptoms in the same individ- Funding ual are required to determine whether nervonic acid levels show This work was supported by the Japan Agency for Medical state or trait-dependent changes. Research and Development (T. Kato: 16815678), MEXT/JSPS Cortisone is a 21-carbon steroid that is one of the main KAKENHI grants (T. Kato: 24249063 and 17H01573), and a grant- hormones released by the adrenal gland in response to stress in-aid from the Japanese Ministry of Health and Labor (T. Kato: (Tacker et  al., 1978). It is converted to the active metabolite 201241002A). Y.  Kageyama is supported by a RIKEN Junior hydrocortisone, which is also called cortisol. The results of the Research Associate fellowship. current study showed that patients with SZ exhibited increased levels of cortisone. These results may reflect the well-estab- lished finding of abnormalities of the hypothalamic-pituitary- Statement of Interest adrenal axis in patients with SZ (Walker et al., 2008). A previous T. Kato has received honoraria for lectures, manuscripts, and/ report has shown that plasma cortisone levels were increased in or consultancy from Kyowa Hakko Kirin Co., Ltd.; Eli Lilly Japan patients with MDD compared with controls (Weber et al., 2000). K.K.; Otsuka Pharmaceutical Co., Ltd.; GlaxoSmithKline K.K.; However, no significant differences were found in this study. Taisho Toyama Pharmaceutical Co., Ltd.; Dainippon Sumitomo The discrepancy of these results might have been a result of dif- Pharma Co., Ltd.; Meiji Seika Pharma Co., Ltd.; Pfizer Japan Inc.; ferences in methodology (LC-TOFMS vs specific radioimmuno- Mochida Pharmaceutical Co., Ltd.; Shionogi & Co., Ltd.; Janssen assay) (Hayashi-Takagi et al., 2014). Pharmaceutical K.K.; Yoshitomiyakuhin; Astellas Pharma Inc.; This study had several limitations. First, effects of diet Wako Pure Chemical Industries, Ltd.; and Takeda Pharmaceutical (Cevallos-cevallos et al., 2009 Y; oung et al., 2017), age (Jové et al., Co., Ltd. within the last 3 years. T. Kato also received a research 2014), circadian variation (Dallmann et al., 2012), and other con- grant from Takeda Pharmaceutical Co., Ltd. These companies founding factors (Lawton et  al., 2008) on the plasma levels of played no role in the study design, data collection and analysis, metabolites cannot be excluded. Because we did not strictly con- decision to publish, or preparation of the manuscript. trol the meal and sample collection time, some of the observed changes might be attributable to these confounding factors. Additional studies involving larger sample size and controlling for References all confounding factors are needed to validate the use of plasma Allen JA, Halverson-Tamboli RA, Rasenick MM (2007) Lipid raft nervonic acid levels as a biomarker of MDD. Second, we obtained microdomains and neurotransmitter signalling. Nat Rev data only for patients with MDD, BD, and SZ, as well as controls. Neurosci 8:128–140. 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Journal

International Journal of NeuropsychopharmacologyOxford University Press

Published: Mar 1, 2018

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