Psychiatry Research 227 (2015) 353–359

Contents lists available at ScienceDirect

Psychiatry Research journal homepage: www.elsevier.com/locate/psychres

Fatty acid composition of the postmortem prefrontal cortex of patients with schizophrenia, bipolar disorder, and major depressive disorder Kei Hamazaki a,b,n, Motoko Maekawa a, Tomoko Toyota a, Brian Dean c,d, Tomohito Hamazaki a, Takeo Yoshikawa a a

Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Saitama 351-0198, Japan Department of Public Health, Faculty of Medicine, University of Toyama, 2630 Sugitani, Toyama City, Toyama 930-0194, Japan c The Molecular Psychiatry Laboratory, The Florey Institute of Neuroscience and Mental Health, Howard Florey Laboratories, The University of Melbourne, Parkville, Victoria, Australia d The Department of Psychiatry, The University of Melbourne, Victoria 3010, Australia b

art ic l e i nf o

a b s t r a c t

Article history: Received 11 August Received in revised 12 December 2014 Accepted 2 January Available online 23

Postmortem brain studies have shown abnormal levels of n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid, in the frontal cortex (particularly the orbitofrontal cortex) of patients with depression, schizophrenia, or bipolar disorder. However, the results from regions in the frontal cortex other than the orbitofrontal cortex are inconsistent. In this study we investigated whether patients with schizophrenia, bipolar disorder, or major depressive disorder have abnormalities in PUFA levels in the prefrontal cortex [Brodmann area (BA) 8]. In postmortem studies, fatty acids in the phospholipids of the prefrontal cortex (BA8) were evaluated by thin layer chromatography and gas chromatography. Specimens were evaluated for patients with schizophrenia (n ¼ 15), bipolar disorder (n ¼15), or major depressive disorder (n ¼15) and compared with unaffected controls (n ¼15). In contrast to previous studies, we found no significant differences in the levels of PUFAs or other fatty acids in the prefrontal cortex (BA8) between patients and controls. Subanalysis by sex also showed no significant differences. No significant differences were found in any individual fatty acids between suicide and nonsuicide cases. These psychiatric disorders might be characterized by very specific fatty acid compositions in certain areas of the brain, and BA8 might not be involved in abnormalities of PUFA metabolism. & 2015 Elsevier Ireland Ltd. All rights reserved.

2014 form 2015 January 2015

Keywords: Brodmann area 8 Polyunsaturated fatty acids Postmortem brain Prefrontal cortex

1. Introduction The findings of some large-scale observational studies (Colangelo et al., 2009; Lucas et al., 2011; Sanchez-Villegas et al., 2007), but not all (Hakkarainen et al., 2004; Miyake et al., 2006; Murakami et al., 2008), suggest that a higher dietary intake of n-3 polyunsaturated fatty acids (PUFAs) may lead to a decreased risk of depressive disorders. A meta-analysis of 14 case-control studies revealed that levels of n-3 PUFAs in peripheral tissue were significantly decreased in individuals with depression (Lin et al., 2010), and a meta-analysis of 5 pooled datasets of clinical trials in patients with bipolar depressive symptoms showed that n-3 PUFAs had clinical benefits but did not attenuate bipolar mania (Sarris et al., 2012).

Abbreviations: AA, arachidonic acid; BA8, Brodmann area 8; CVD, cardiovascular disease; DPA, docosapentaenoic acid, DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; PL, phospholipid; PMI, postmortem interval; PUFAs, polyunsaturated fatty acids n Corresponding author at: Department of Public Health, Faculty of Medicine, University of Toyama, 2630 Sugitani, Toyama City, Toyama 9300194, Japan. E-mail address: [email protected] (K. Hamazaki). http://dx.doi.org/10.1016/j.psychres.2015.01.004 0165-1781/& 2015 Elsevier Ireland Ltd. All rights reserved.

Two meta-analyses of clinical trials of n-3 PUFAs for the treatment of patients with schizophrenia showed inconclusive results (Freeman et al., 2006; Joy et al., 2006). However, a recent meta-analysis of 14 studies focusing on the PUFAs in erythrocyte membranes of patients with schizophrenia found that all major PUFAs, including arachidonic acid (AA, 20:4n-6), docosahexaenoic acid (DHA, 22:6n-3), and docosapentaenoic acid (DPA, 22:5n-3), were decreased in medication-naive patients and patients taking typical antipsychotics (van der Kemp et al., 2012), although a contemporaneous meta-analysis of trials using only purified eicosapentaenoic acid (EPA, 20:5n-3) or EPA-enriched oils involving schizophrenia patients showed no beneficial effects (FusarPoli and Berger, 2012). Furthermore, a trial conducted with adolescents and young adults at ultra-high risk of psychosis revealed that n-3 PUFAs not only reduced the rate of progression to first-episode psychotic disorders, but also improved positive, negative, and general symptoms (Amminger et al., 2010). The brain is known to consist of relatively high levels of longchain PUFAs such as DHA and AA. The postmortem frontal cortex is the most studied area in relation to n-3 PUFAs and psychiatric disorders. McNamara et al. (2007b) examined the postmortem

354

K. Hamazaki et al. / Psychiatry Research 227 (2015) 353–359

orbitofrontal cortex [Brodmann area (BA) 10] from patients with schizophrenia (n ¼21) and age-matched controls (n¼ 26) and found that DHA was the only PUFA that was significantly reduced by 20% in patients with schizophrenia compared with controls. They also investigated the fatty acid composition of the same regions from patients with bipolar disorder (McNamara et al., 2008a) and major depressive disorder (MDD) (McNamara et al., 2007a), finding that levels of DHA were significantly reduced by 24% and 22%, respectively, in patients compared with controls. We previously measured PUFA levels in the postmortem medial temporal lobe including the hippocampus (Hamazaki et al., 2010), amygdala (Hamazaki et al., 2012), and entorhinal cortex (Hamazaki et al., 2013) from patients with psychiatric disorders; however, we found no significant differences between the groups except for small changes in n-6 PUFAs. Two postmortem brain (BA10) studies in patients with schizophrenia have recently been reported, but neither showed any differences in DHA or AA levels (Tatebayashi et al., 2012, Taha et al., 2013). The frontal eye field (BA8) is part of the prefrontal cortex and known to be responsible for eye tracking dysfunction which is the most widely replicated behavioral deficit in schizophrenia [for review, refer to Levy et al., (2010)]. Two meta-analyses revealed large effect sizes for global measures of eye movement dysfunction and for some specific measures not only in patients with schizophrenia (O’Driscoll and Callahan, 2008) but also in first-degree biological relatives (Calkins et al., 2008). In view of these findings, we hypothesized that there might be differences in PUFA levels in BA8. To date, there have been no reports of fatty acid profiles in the frontal eye field (BA8) in individuals with psychiatric disorders. Therefore, in this study we investigated the fatty acid composition in BA8 in this patient population and compared the findings with those from unaffected controls.

especially interested in DHA, we decided to measure fatty acids in total phospholipids that play an important role in maintaining the structural and functional integrity of membranes. For an internal standard, 1, 2-diheptadecanoyl-sn-glycero3phosphocholine (Avanti Polar Lipids, Inc., Alabaster, AL) was added. Total phospholipid fractions were separated by silica gel thin-layer chromatography using petroleum ether:diethyl ether:acetic acid (80:30:1, vol/vol/vol) as the developing solvent (Noda and Ikegami, 1966). For detection, 0.005% primuline (in acetone:H20; 4:1, vol/vol) was used under ultraviolet light. Total phospholipid fractions were transmethylated with HCl-methanol. The fatty acid composition was analyzed by gas chromatography (GC-2014 Shimadzu Corporation, Kyoto, Japan) using a DB-225 capillary column (length 30 m; internal diameter 0.25 mm; film 0.25 μm; J&M Scientific, Folsom, CA). The entire system was controlled using the gas chromatography software GC-solution version 2.3 (Shimadzu Corporation). Fatty acids were expressed as percentage area of total fatty acids and absolute amount (μg/g brain wet weight). We were not able to detect EPA in 12 of 60 samples. When detectable, the highest composition of EPA was 0.12% and the lowest was 0.02%. Because the coefficient of variation (intra-assay) for EPA was 50% and we could not confirm whether these small amounts (0.02–0.12%) were from brain or from other areas such as vascular endothelial cells or blood, we decided not to use EPA for further analysis.

2.3. Statistical analysis Characteristics of the postmortem prefrontal cortex samples are expressed as means 7S.D. Differences between groups were examined using the chi-squared test for categorical variables and one-way ANOVA for continuous variables. For significant results with one-way ANOVA, Bonferroni post-hoc tests were performed. We used the Kruskal–Wallis test (Table 2) and ANOVA (Supplementary Tables 1 and 2) to compare individual fatty acids between the four groups. Chlorpromazine equivalents were used to calculate doses of antipsychotic drugs. Further comparisons of individual fatty acids adjusted for age, sex, and PMI between the four groups were made by analysis of covariance (ANCOVA). Moreover, we added doses of antipsychotic as a covariate. Spearman's rank correlation test was used to calculate correlation coefficients between each fatty acid level, and chlorpromazine equivalents, age, PMI, and pH. Fisher's exact test was used to compare the prevalence of suicide between groups. In the case of single comparisons between suicide and non-suicide cases, and between men and women, the Mann–Whitney U-test was performed; p o 0.05 was considered significant. Data were analyzed using the statistical software SPSS, version 19.0 (IBM Japan, Tokyo, Japan).

2. Methods 2.1. Postmortem prefrontal cortex samples Brain tissue samples were obtained from the Victorian Brain Bank Network (VBBN) at the Florey Institute for Neuroscience and Mental Health. The collection of tissue was approved by the Ethics Committee of the Victorian Institute of Forensic Medicine, and the supply of tissue for the study was approved by the Tissue Access Committee of the VBBN following approval of the study by the Ethics Committee of RIKEN Brain Science Institute and the University of Toyama. Samples were from patients with schizophrenia, bipolar disorder, or MDD (n¼ 15 for each psychiatric disorder) and non-pathological conditions to serve as controls (n¼ 15). Following ethical approval, left central nervous system (CNS) hemispheres were collected, sliced, and rapidly frozen to  80 1C as described previously (Dean et al., 1999). For this study, tissues were provided as frozen blocks of gray matter of the superior frontal gyrus (BA8) from the left hemisphere. All cadavers were stored at 4 1C within 5 h of death. During case history review, age at death, gender, postmortem interval (PMI), and brain pH were determined [according to Kingsbury et al. (1995)]. With regards to PMI, where death was witnessed, PMI was from the time of death to autopsy; if death had not been witnessed, PMI was taken as the mid-point between the time the person was last observed alive and the time found dead (maximum of 5 h). For all cases, psychiatric diagnoses were performed according to DSM-IV criteria following a review of clinical records using the Diagnostic Instrument for Brain Studies, a structured instrument allowing a consensus psychiatric diagnosis to be made after death (Hill et al., 1996; Roberts et al., 1998). For all non-psychiatric cases, an extensive review of case histories, along with questioning of families and treating clinicians, was undertaken to exclude any history of psychiatric illness. During this review a neuropsychopharmacological profile was also obtained. The demographic characteristics of this study are shown in Table 1. 2.2. Tissue preparation and lipid extraction Frozen sections of prefrontal cortex tissue were homogenized in ice-cold saline, and aliquots were used for lipid analysis. Total lipids were extracted according to the method of Bligh and Dyer (1959). DHA content is reported to be about 100 to 1000 times higher in phospholipids than in triglycerides, cholesterol esters, or unesterified fatty acids in human brain (Taha et al., 2013). Because we were

3. Results As shown in Table 1, participants' age at death, sex, and PMI did not differ significantly between the groups. Brain pH, which increases during the postmortem hydrolysis process, did differ significantly between the groups, but post-hoc analysis showed no significant differences in pH between any psychiatric disorder and the control group. Suicide was the cause of death in 20 individuals (control n¼ 0, schizophrenia n ¼3, bipolar disorder n ¼4, and MDD n ¼13), and the remaining 39 died from other causes (data were missing on cause of death in 1 patient with MDD). Despite missing information on cause of death for 1 patient with MDD, the suicide rate was notably high in the MDD group (p o0.0001). By contrast, the most common cause of death among controls was cardiovascular disease (CVD); there were 14, 2, 6, and 0 CVD deaths among control subjects and patients with schizophrenia, bipolar disorder, and MDD, respectively (po 0.0001). Fatty acid levels in the phospholipid fraction of BA8 in controls and patients with schizophrenia, bipolar disorder, and MDD are shown in Table 2 [composition, median (25th percentile, 75th percentile)] and Supplementary Table 1 (composition, mean7S.D.) and 2 (absolute concentrations, mean7S.D.). No differences were observed in any individual fatty acids or the n-6:n-3 ratios. Subanalysis by sex also showed no significant differences (data not shown). Comparison between suicide and non-suicide cases showed no significant differences in any individual fatty acids; however, significant decreases were seen in total saturated fatty acids in suicide cases (  1.4%, p o0.05).

K. Hamazaki et al. / Psychiatry Research 227 (2015) 353–359

355

Table 1 Characteristics of patients and control subjects.

Age at death (years) Sex (male/female) Postmortem interval (PMI, hours) Brain tissue pH No. of suicidesa

Control, n ¼15

Schizophrenia, n¼ 15

Bipolar disorder, n¼15

Major depressive disorder, n¼15

p Value

577 13 8/7 43 718 6.34 7 0.23 0

587 14 8/7 437 13 6.18 70.27 3

587 14 8/7 367 15 6.26 70.27 4

57 712 8/7 427 17 6.52 7 0.19 13

0.99 1.00 0.61 0.002 o 0.0001

p Value: 2  4 chi-squared test for categorical variables and one-way ANOVA for continuous variables. a

Information on cause of death was missing for 1 patient with major depressive disorder.

Table 2 Fatty acid composition of phospholipids in the postmortem frontal cortex (BA8) of patients with schizophrenia, bipolar disorder, or major depressive disorder, and of unaffected controls. Fatty acids (area %)

Controls

Schizophrenia

Bipolar disorder

Major depression

Median

0.25, 0.75

Median

0.25, 0.75

Median

0.25, 0.75

Median

0.25,0.75

p Value

0.41 22.20 24.59 0.22 0.12 0.23 47.66

(0.39, 0.43) (21.49, 22.79) (24.11, 24.71) (0.21, 0.25) (0.09, 0.18) (0.14, 0.35) (46.96, 48.31)

0.42 21.59 24.25 0.22 0.14 0.30 46.87

(0.39, 0.44) (20.94, 22.07) (23.88, 24.73) (0.20, 0.24) (0.12, 0.16) (0.18, 0.33) (46.52, 47.31)

0.41 21.88 24.50 0.23 0.12 0.21 47.46

(0.39, 0.45) (21.33, 22.29) (24.05, 24.87) (0.22, 0.24) (0.10, 0.14) (0.16, 0.28) (46.71, 47.70)

0.40 21.32 24.36 0.23 0.12 0.29 46.52

(0.36, 0.41) (20.08, 21.74) (23.91, 24.56) (0.22, 0.24) (0.10, 0.14) (0.20, 0.53) (45.29, 47.20)

0.17 0.16 0.78 0.65 0.52 0.45 0.11

Monounsaturated fatty acids 16:1 n-7 Palmitoleic acid 18:1 n-9 Oleic acid 18:1 n-7 Vaccenic acid 20:1 n-9 Gondoic acid 22:1 n-9 Erucic acid 24:1 n-9 Nervonic acid Total monounsaturated fatty acids

0.47 15.15 3.56 0.58 0.04 0.74 20.88

(0.43, 0.58) (14.48, 16.33) (3.44, 3.84) (0.42, 0.67) (0.03, 0.08) (0.55, 1.13) (19.70, 22.23)

0.57 16.14 3.86 0.70 0.06 1.12 22.36

(0.54, 0.61) (15.55, 16.56) (3.69, 4.15) (0.62, 0.78) (0.05, 0.08) (0.86, 1.38) (21.53, 23.25)

0.48 15.90 3.75 0.62 0.05 0.99 21.83

(0.42, 0.53) (15.09, 16.91) (3.55, 3.95) (0.52, 0.89) (0.01, 0.08) (0.57, 1.26) (20.50, 23.48)

0.53 16.35 3.76 0.75 0.07 1.12 22.46

(0.45, 0.57) (15.43, 17.51) (3.54, 3.93) (0.56, 0.88) (0.05, 0.08) (0.79, 1.76) (20.90, 24.41)

0.08 0.41 0.20 0.22 0.50 0.19 0.23

n-3 Polyunsaturated fatty acids 22:5 n-3 Docosapentaenoic acid 22:6 n-3 Docosahexaenoic acid Total n-3 polyunsaturated fatty acids

0.41 15.90 16.17

(0.24, 0.45) (14.27, 16.27) (14.66, 16.67)

0.35 14.97 15.28

(0.31, 0.44) (13.98, 15.63) (14.29, 16.06)

0.37 14.92 15.34

(0.35, 0.41) (14.15, 15.25) (14.46, 15.59)

0.38 15.04 15.39

(0.33, 0.44) (13.82, 15.55) (14.14, 15.97)

0.87 0.61 0.57

n-6 Polyunsaturated fatty acids 18:2 n-6 Linoleic acid 20:2 n-6 Eicosadienoic acid 20:3 n-6 Dihomo-γ-linolenic acid 20:4 n-6 Arachidonic acid 22:4 n-6 Docosatetraenoic acid Total n-6 polyunsaturated fatty acids

0.70 0.08 0.84 8.94 4.96 15.47

(0.64, 0.87) (0.05, 0.10) (0.79, 0.97) (8.51, 9.29) (4.65, 5.02) (15.01, 16.19)

0.71 0.08 0.86 8.57 4.73 15.05

(0.64, 0.88) (0.06, 0.10) (0.78, 0.96) (8.22, 8.77) (4.61, 5.31) (14.64, 15.63)

0.68 0.07 0.86 8.81 5.22 15.69

(0.54, 0.75) (0.06, 0.08) (0.80, 0.89) (8.43, 9.25) (4.85, 5.39) (14.95, 16.08)

0.66 0.09 0.86 8.79 5.29 15.35

(0.62, 0.79) (0.07, 0.10) (0.78, 1.02) (8.31, 8.90) (4.81, 5.53) (14.79, 16.11)

0.75 0.51 0.99 0.22 0.31 0.31

(0.96, 1.09)

1.04

(0.99, 1.08)

(0.96, 1.12)

0.85

Saturated 14:0 16:0 18:0 20:0 22:0 24:0

fatty acids Myristic acid Palmitic acid Stearic acid Arachidic acid Behenic acid Lignoceric acid Total saturated fatty acids

n-6/n-3

0.98

(0.92, 1.12)

1.00

1.04

p value: Kruskal–Wallis test

Comparison by sex showed that AA (p o0.05) was higher in men (n ¼32) than in women (n¼ 28) and that levels of linoleic acid (18:2n-6) (p o0.01), dihomo-γ-linolenic acid (20:3n-6) (p o0.05), erucic acid (22:1n-9) (po 0.05), and lignoceric acid (24:0) (p o0.05) were lower in men. Age was positively associated with 3 fatty acids—vaccenic acid (18:1n-7), arachidic acid (20:0), and nervonic acid (24:1n-9)—and total monounsaturated fatty acids, and inversely associated with 2 fatty acids—stearic acid (18:0) and AA—and total n-6 PUFAs (Fig. 1). We further explored which condition (schizophrenia, bipolar disorder, or MDD) contributed to these significant associations. In MDD, a negative association was found between age and arachidonic acid (20:4n-6) and positive associations were observed between age and both nervonic acid (24:1n-9) and monounsaturated fatty acids (data not shown). Among patients with schizophrenia, only n-6 PUFAs were negatively associated with age (data not shown). No associations were found in patients with bipolar disorder (data not shown). PMI and pH were not

associated with any fatty acids (data not shown). The chlorpromazine equivalent dose was positively associated with palmitoleic acid (16:1n-7) and inversely associated with docosapentaenoic acid (22:5n-3) (Fig. 2). Further comparison of individual fatty acids between the four groups adjusted for age sex, and PMI (ANCOVA) showed no significant differences in any individual fatty acids. Furthermore, addition of doses of antipsychotic as a covariate did not result in any significant differences in any individual fatty acids.

4. Discussion Although some postmortem studies have investigated fatty acid levels in the prefrontal cortex of patients with psychiatric disorders, to the best of our knowledge this is the first study to examine fatty acid levels in BA8. Our aim was to investigate whether BA8 from patients with psychiatric disorders showed

356

K. Hamazaki et al. / Psychiatry Research 227 (2015) 353–359

2 0 20

8

40

60

80

Spearman’ srho =0.277

p =0.032 0.3 0.2 0.1

4 2 0

20

40

60

80

100

Spearman’ srho=0. 275 (%) p =0.033

50

S pearman’ srho =0.287

6

0

100

p =0.026

40 30 20 10 0

0

20

40

60

80

0

100

20

40

60

80

100

12 A A(20:4n-6)

30 Stearic acid (18:0)

0.4

0.0 0

Nervonic acid (24:1n -9)

Arachidic acid (20: 0)

4

Total monounsaturated fatty acids

V accenic acid (18: 1n-7)

S pearman’ srho=0. 320

p =0.013

6

20 10

Spearman’ srho =-0. 353 p =0.006

10 8 6 Spearman’ srho =-0. 261

4

p =0.044

2 0

0 0

20

40

60

80

0

100

20

40

60

80

100

age

total n-6 P U FA s

20 15 10 Spearman’ srho =-0. 378 p =0.033

5 0 0

20

40

60

80

100

age

0.8 0.6 0.4 0.2

Spearman’ s rho=0.482 p =0.020

0.0 0

500

1000

1500

2000

Chlorpromazine equivalent dose

Docosapentaenoic acid (22: 5n-3)

Palmitoleic aci d (16:1n-7)

Fig. 1. Correlations between age (n¼ 60) and levels of vaccenic acid (18:1n-7) (% total fatty acids) (A), arachidic acid (20:0) (B), nervonic acid (24:1n-9) (C), total monounsaturated fatty acids (D), stearic acid (18:0) (E), arachidonic acid (20:4n-6) (F), and total n-6 polyunsaturated fatty acids (G). Spearman's rank correlation coefficients and p values are presented.

0.6

0.4

0.2

Spearman’ s rho=-0. 448

p =0.032 0.0

0

500

1000

1500

2000

Chlorpromazine equivalent dose

Fig. 2. Correlations between chlorpromazine equivalent dose (n¼23) and palmitoleic acid (16:1n-7) (A) and docosapentaenoic acid (22:5n-3) (B). Spearman's rank correlation coefficients and p values are presented.

different PUFA levels, especially DHA, compared to those without psychiatric disorders, as reported previously (Horrobin et al., 1991; McNamara et al., 2007b). However, we did not find any marked

alterations in PUFAs. Although there was no statistical difference or trend in nervonic acid levels, they were higher by 51% in patients with schizophrenia than in control subjects. It is

K. Hamazaki et al. / Psychiatry Research 227 (2015) 353–359

interesting that this difference was in the opposite direction in a previous study by Amminger et al. (2012). Their re-analysis (observational study) of the control subjects who participated in the original clinical trial showed that a lower erythrocyte nervonic acid level predicted higher risk of transition to psychosis in young patients (Amminger et al., 2012). This discrepancy can presumably be explained by the disease stage (early vs. late stage) or brain region (peripheral tissue vs. brain for fatty acid analysis). At present, not much is known about this fatty acid in relation to the pathology of psychosis, and studies are therefore required. Antipsychotic medication has been shown to exert effects on PUFA metabolism in animal studies (Cheon et al., 2011; Modi et al., 2013), an observational study (Khan et al., 2002), and a clinical study (Evans et al., 2003). In the latter it was demonstrated that 6 months of antipsychotic treatment in first-episode psychotic patients normalized peripheral DHA levels. In the present study, patients who were taking chlorpromazine had a lower level of eicosadienoic acid (20:2n-6) than those who were not. Furthermore, chlorpromazine equivalents positively correlated with palmitoleic acid and inversely correlated with DPA levels (Fig. 2). The correlation between chlorpromazine equivalents and DPA is somewhat inconsistent with a previous clinical study showing that antipsychotic treatment normalized DHA levels (Evans et al., 2003). Future research is warranted to evaluate the association between antipsychotic medications and levels of other fatty acids. We previously found that low levels of peripheral n-3 PUFAs were associated with a high risk of suicide attempt in China (Huan et al., 2004). The risk of suicide completion was also inversely associated with peripheral n-3 PUFAs in US military personnel (Lewis et al., 2011). Of interest, suicide among women in Japan, where fish is regularly consumed, was found to be associated with very low levels of fish consumption (Poudel-Tandukar et al., 2011). In the present study, no significant differences in PUFA levels were found between the suicide (n ¼20) and non-suicide (n ¼39) groups, which is consistent with previous postmortem brain studies (Lalovic et al., 2007; McNamara et al., 2009). Aging is known to be inversely correlated with DHA and AA levels in the orbitofrontal cortex, and declines in their levels were compensated for by increased monounsaturated fatty acid concentrations (McNamara et al., 2008b). Although in the present study we found no association between age and DHA, vaccenic acid, AA, and total n-6 PUFAs showed similar inverse trends with age. Furthermore, our findings of a negative association between age and arachidonic acid (20:4n-6) in MDD were consistent with the results of McNamara et al. (2007a) from their analysis of a combined group of MDD patients and controls. Although they did not describe nervonic acid (24:1n-9) in the orbitofrontal cortex, the same series of n-9 PUFAs, namely oleic acid (18:1n-9), were found to be positively associated with age. Nervonic acid (24:1n-9) is an elongase product of oleic acid (18:1n-9) which is converted from stearic acid (18:0) by stearoyl-CoA desaturase (SCD). McNamara et al. (2008b) reported that increasing age was associated with transient elevations in elongase mRNA expression and SCD activity, which might be the same mechanism as that in the case of MDD in the present study. With regard to the negative association between age and total n-6 PUFAs found in patients with schizophrenia in the present study, McNamara et al. (2007b) also reported a negative association between patients with schizophrenia who had been taking typical antipsychotic and arachidonic acid (20:4n-6), the major n-6 PUFA in the brain (total n-6 PUFAs were not calculated). Levels of both n-6 PUFAs and AA are known to decline with age, which is presumably due to reduced biosynthesis from dietary precursors (McNamara et al., 2008b). With regard to sex differences, McNamara et al. previously found in the orbitofrontal cortex (BA 10) that female patients with

357

MDD exhibited greater DHA deficiency (  32%) than male patients ( 16%) (McNamara et al., 2007a) and that female patients with schizophrenia had reduced DHA deficits (  2%) compared to male patients (  27%) (McNamara et al., 2007b); there were similar DHA deficits in female and male patients with bipolar disorder ( 26% and  22%, respectively) (McNamara et al., 2008a). In the present study, we found no significant differences in a subanalysis by sex. Although the mechanism underlying these sex differences is still unclear, fatty acid composition is known to show a sex difference (Childs et al., 2008). This area of research is still at an early stage and therefore further investigation is needed. Several limitations of this postmortem brain study should be acknowledged. Firstly, no information was available on dietary intake of fatty acids; consequently, it is impossible to investigate the relationship between the prior intake of fatty acids and brain fatty acid level. Secondly, no information was available regarding smoking which is known to affect PUFA metabolism (Hibbeln et al., 2003). Thirdly, because we analyzed only a small part of the BA8 region, and brain lipids are known to be distributed in a heterogeneous pattern even within small brain regions (Matsumoto et al., 2011), our analysis might show a weakened relationship between each fatty acid and schizophrenia. Fourthly, selection bias might have occurred. The control group consisted of a large number of subjects who died from CVD and whose n-3 fatty acid levels were deemed to be low (Mozaffarian and Wu, 2011). However, DHA (22:6n-3) and AA (20:4n-6) levels were not significantly different between those who died from CVD (n ¼22) and those who died from other causes (n ¼38) in the control group (data not shown). In conclusion, we found no marked alteration in the levels of any fatty acid in the prefrontal cortex (BA8) of patients with psychiatric disorders. These results suggest that there might be differential fatty acid metabolism in the prefrontal cortex of these patients. Further studies are needed to elucidate the neuropathology of schizophrenia in relation to fatty acid metabolism.

Role of funding source This work was supported by Grant-in-Aid for Scientific Research (C) (25461726), Japan. BD is a National Health and Medical Research Council Senior Research Fellow (APP1002240). This work was supported in part by the Victorian Government's Operational Infrastructure Support. The funding source had no role in the study design; the collection, analysis, and interpretation of data; writing the report; or the decision to submit the paper for publication.

Conflict of interest statement KH has received research support from an Intramural Research Grant for Neurological and Psychiatric Disorders from the National Center of Neurology and Psychiatry, the Japan Society for the Promotion of Science, the Tamura Foundation for Promotion of Science and Technology, and the Ichiro Kanehara Foundation for Promotion of Medical Sciences and Medical Care; consultant fees from Polyene Project, Inc. and scholarship donations from Otsuka Pharmaceutical Co., Ltd.; and has been a paid speaker for DHA & EPA Association. TH has received research support from the Japan Society for the Promotion of Science, Open Research Center for Lipid Nutrition (Kinjo Gakuin University), and Nippon Suisan Kaisha, Ltd.; consultancy fees from Polyene Project, Inc. and Otsuka Pharmaceutical Co., Ltd.; lecture fees from Otsuka Pharmaceutical Co., Ltd.; and travel expenses from Aker BioMarine.

358

K. Hamazaki et al. / Psychiatry Research 227 (2015) 353–359

Acknowledgments We are grateful to Ms. Shizuko Takebe (University of Toyama) for her technical assistance and Ms. Mika Kigawa (University of Toyama) for her statistical assistance. We thank Drs. Ken-ichi Moto and Toshihide Kobayashi (RIKEN Lipid Biology Laboratory) for their technical advice. The Victorian Brain Bank Network is supported by the Florey Institute for Neuroscience and Mental Health, the Alfred Hospital, the Victorian Forensic Institute of Medicine, and the University of Melbourne, and funded by Australia's National Health and Medical Research Council, Helen Macpherson Smith Trust, and Parkinson's Victoria and Perpetual Philanthropic Services.

Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.psychres.2015.01.004.

References Amminger, G.P., Schafer, M.R., Klier, C.M., Slavik, J.M., Holzer, I., Holub, M., Goldstone, S., Whitford, T.J., McGorry, P.D., Berk, M., 2012. Decreased nervonic acid levels in erythrocyte membranes predict psychosis in help-seeking ultra-highrisk individuals. Molecular Psychiatry 17, 1150–1152. Amminger, G.P., Schafer, M.R., Papageorgiou, K., Klier, C.M., Cotton, S.M., Harrigan, S.M., Mackinnon, A., McGorry, P.D., Berger, G.E., 2010. Long-chain omega-3 fatty acids for indicated prevention of psychotic disorders: a randomized, placebo-controlled trial. Archives of General Psychiatry 67, 146–154. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37, 911–917. Calkins, M.E., Iacono, W.G., Ones, D.S., 2008. Eye movement dysfunction in firstdegree relatives of patients with schizophrenia: a meta-analytic evaluation of candidate endophenotypes. Brain and Cognition 68, 436–461. Cheon, Y., Park, J.Y., Modi, H.R., Kim, H.W., Lee, H.J., Chang, L., Rao, J.S., Rapoport, S.I., 2011. Chronic olanzapine treatment decreases arachidonic acid turnover and prostaglandin E concentration in rat brain. Journal of Neurochemistry 119, 364–376. Childs, C.E., Romeu-Nadal, M., Burdge, G.C., Calder, P.C., 2008. Gender differences in the n-3 fatty acid content of tissues. Proceedings of the Nutrition Society 67, 19–27. Colangelo, L.A., He, K., Whooley, M.A., Daviglus, M.L., Liu, K., 2009. Higher dietary intake of long-chain omega-3 polyunsaturated fatty acids is inversely associated with depressive symptoms in women. Nutrition 25, 1011–1019. Dean, B., Pavey, G., Chai, S.W., Mendelsohn, F.A.O., 1999. The localisation and quantification of molecular changes in the human brain using in situ radioligand binding and autoradiography. In: Dean, B., Hyde, T.M., Kleinman, J. (Eds.), The Use of CNS Autopsy Tissue in Psychiatric Research: A Practical Guide. Gordon & Breach Science Publishers, Sydney, pp. 67–83. Evans, D.R., Parikh, V.V., Khan, M.M., Coussons, C., Buckley, P.F., Mahadik, S.P., 2003. Red blood cell membrane essential fatty acid metabolism in early psychotic patients following antipsychotic drug treatment. Prostaglandins, Leukotrienes, and Essential Fatty Acids 69, 393–399. Freeman, M.P., Hibbeln, J.R., Wisner, K.L., Davis, J.M., Mischoulon, D., Peet, M., Keck Jr., P.E., Marangell, L.B., Richardson, A.J., Lake, J., Stoll, A.L., 2006. Omega-3 fatty acids: evidence basis for treatment and future research in psychiatry. Journal of Clinical Psychiatry 67, 1954–1967. Fusar-Poli, P., Berger, G., 2012. Eicosapentaenoic acid interventions in schizophrenia: meta-analysis of randomized, placebo-controlled studies. Journal of Clinical Psychopharmacology 32, 179–185. Hakkarainen, R., Partonen, T., Haukka, J., Virtamo, J., Albanes, D., Lonnqvist, J., 2004. Is low dietary intake of omega-3 fatty acids associated with depression? American Journal of Psychiatry 161, 567–569. Hamazaki, K., Choi, K.H., Kim, H.Y., 2010. Phospholipid profile in the postmortem hippocampus of patients with schizophrenia and bipolar disorder: no changes in docosahexaenoic acid species. Journal of Psychiatric Research 44, 688–693. Hamazaki, K., Hamazaki, T., Inadera, H., 2012. Fatty acid composition in the postmortem amygdala of patients with schizophrenia, bipolar disorder, and major depressive disorder. Journal of Psychiatric Research 46, 1024–1028. Hamazaki, K., Hamazaki, T., Inadera, H., 2013. Abnormalities in the fatty acid composition of the postmortem entorhinal cortex of patients with schizophrenia, bipolar disorder, and major depressive disorder. Psychiatry Research 210, 346–350. Hibbeln, J.R., Makino, K.K., Martin, C.E., Dickerson, F., Boronow, J., Fenton, W.S., 2003. Smoking, gender, and dietary influences on erythrocyte essential fatty acid composition among patients with schizophrenia or schizoaffective disorder. Biological Psychiatry 53, 431–441.

Hill, C., Keks, N., Roberts, S., Opeskin, K., Dean, B., MacKinnon, A., Copolov, D., 1996. Problem of diagnosis in postmortem brain studies of schizophrenia. American Journal of Psychiatry 153, 533–537. Horrobin, D.F., Manku, M.S., Hillman, H., Iain, A., Glen, M., 1991. Fatty acid levels in the brains of schizophrenics and normal controls. Biological Psychiatry 30, 795–805. Huan, M., Hamazaki, K., Sun, Y., Itomura, M., Liu, H., Kang, W., Watanabe, S., Terasawa, K., Hamazaki, T., 2004. Suicide attempt and n-3 fatty acid levels in red blood cells: a case control study in China. Biological Psychiatry 56, 490–496. Joy, C.B., Mumby-Croft, R., Joy, L.A., 2006. Polyunsaturated fatty acid supplementation for schizophrenia. Cochrane Database of Systematic Reviews. Khan, M.M., Evans, D.R., Gunna, V., Scheffer, R.E., Parikh, V.V., Mahadik, S.P., 2002. Reduced erythrocyte membrane essential fatty acids and increased lipid peroxides in schizophrenia at the never-medicated first-episode of psychosis and after years of treatment with antipsychotics. Schizophrenia Research 58, 1–10. Kingsbury, A.E., Foster, O.J., Nisbet, A.P., Cairns, N., Bray, L., Eve, D.J., Lees, A.J., Marsden, C.D., 1995. Tissue pH as an indicator of mRNA preservation in human post-mortem brain. Brain Research. Molecular Brain Research 28, 311–318. Lalovic, A., Levy, E., Canetti, L., Sequeira, A., Montoudis, A., Turecki, G., 2007. Fatty acid composition in postmortem brains of people who completed suicide. Journal of Psychiatry & Neuroscience 32, 363–370. Levy, D.L., Sereno, A.B., Gooding, D.C., O’Driscoll, G.A., 2010. Eye tracking dysfunction in schizophrenia: characterization and pathophysiology. Current topics in Behavioral Neurosciences 4, 311–347. Lewis, M.D., Hibbeln, J.R., Johnson, J.E., Lin, Y.H., Hyun, D.Y., Loewke, J.D., 2011. Suicide deaths of active-duty US military and omega-3 fatty-acid status: a casecontrol comparison. Journal of Clinical Psychiatry 72, 1585–1590. Lin, P.-Y., Huang, S.-Y., Su, K.-P., 2010. A meta-analytic review of polyunsaturated fatty acid compositions in patients with depression. Biological Psychiatry 68, 140–147. Lucas, M., Mirzaei, F., O’Reilly, E.J., Pan, A., Willett, W.C., Kawachi, I., Koenen, K., Ascherio, A., 2011. Dietary intake of n-3 and n-6 fatty acids and the risk of clinical depression in women: a 10-y prospective follow-up study. American Journal of Clinical Nutrition 93, 1337–1343. Matsumoto, J., Sugiura, Y., Yuki, D., Hayasaka, T., Goto-Inoue, N., Zaima, N., Kunii, Y., Wada, A., Yang, Q., Nishiura, K., Akatsu, H., Hori, A., Hashizume, Y., Yamamoto, T., Ikemoto, K., Setou, M., Niwa, S., 2011. Abnormal phospholipids distribution in the prefrontal cortex from a patient with schizophrenia revealed by matrix-assisted laser desorption/ionization imaging mass spectrometry. Analytical and Bioanalytical Chemistry 400, 1933–1943. McNamara, R.K., Hahn, C.G., Jandacek, R., Rider, T., Tso, P., Stanford, K.E., Richtand, N.M., 2007a. Selective deficits in the omega-3 fatty acid docosahexaenoic acid in the postmortem orbitofrontal cortex of patients with major depressive disorder. Biological Psychiatry 62, 17–24. McNamara, R.K., Jandacek, R., Rider, T., Tso, P., Dwivedi, Y., Roberts, R.C., Conley, R.R., Pandey, G.N., 2009. Fatty acid composition of the postmortem prefrontal cortex of adolescent male and female suicide victims. Prostaglandins, Leukotrienes, and Essential Fatty Acids 80, 19–26. McNamara, R.K., Jandacek, R., Rider, T., Tso, P., Hahn, C.G., Richtand, N.M., Stanford, K.E., 2007b. Abnormalities in the fatty acid composition of the postmortem orbitofrontal cortex of schizophrenic patients: gender differences and partial normalization with antipsychotic medications. Schizophrenia Research 91, 37–50. McNamara, R.K., Jandacek, R., Rider, T., Tso, P., Stanford, K.E., Hahn, C.G., Richtand, N.M., 2008a. Deficits in docosahexaenoic acid and associated elevations in the metabolism of arachidonic acid and saturated fatty acids in the postmortem orbitofrontal cortex of patients with bipolar disorder. Psychiatry Research 160, 285–299. McNamara, R.K., Liu, Y., Jandacek, R., Rider, T., Tso, P., 2008b. The aging human orbitofrontal cortex: decreasing polyunsaturated fatty acid composition and associated increases in lipogenic gene expression and stearoyl-CoA desaturase activity. Prostaglandins, Leukotrienes, and Essential Fatty Acids 78, 293–304. Miyake, Y., Sasaki, S., Yokoyama, T., Tanaka, K., Ohya, Y., Fukushima, W., Saito, K., Ohfuji, S., Kiyohara, C., Hirota, Y., 2006. Risk of postpartum depression in relation to dietary fish and fat intake in Japan: the Osaka Maternal and Child Health Study. Psychological Medicine 36, 1727–1735. Modi, H.R., Taha, A.Y., Kim, H.W., Chang, L., Rapoport, S.I., Cheon, Y., 2013. Chronic clozapine reduces rat brain arachidonic acid metabolism by reducing plasma arachidonic acid availability. Journal of Neurochemistry 124, 376–387. Mozaffarian, D., Wu, J.H., 2011. Omega-3 fatty acids and cardiovascular disease: effects on risk factors, molecular pathways, and clinical events. Journal of the American College of Cardiology 58, 2047–2067. Murakami, K., Mizoue, T., Sasaki, S., Ohta, M., Sato, M., Matsushita, Y., Mishima, N., 2008. Dietary intake of folate, other B vitamins, and omega-3 polyunsaturated fatty acids in relation to depressive symptoms in Japanese adults. Nutrition 24, 140–147. Noda, M., Ikegami, R., 1966. Chromatographic separation of the lipids in rice bran lipoprotein. Agricultural and Biological Chemistry 30, 330–337. O’Driscoll, G.A., Callahan, B.L., 2008. Smooth pursuit in schizophrenia: a metaanalytic review of research since 1993. Brain and Cognition 68, 359–370. Poudel-Tandukar, K., Nanri, A., Iwasaki, M., Mizoue, T., Matsushita, Y., Takahashi, Y., Noda, M., Inoue, M., Tsugane, S., 2011. Long chain n-3 fatty acids intake, fish consumption and suicide in a cohort of Japanese men and women—the Japan Public Health Center-based (JPHC) prospective study. Journal of Affective Disorders 129, 282–288.

K. Hamazaki et al. / Psychiatry Research 227 (2015) 353–359

Roberts, S.B., Hill, C.A., Dean, B., Keks, N.A., Opeskin, K., Copolov, D.L., 1998. Confirmation of the diagnosis of schizophrenia after death using DSM-IV: a Victorian experience. Australian and New Zealand Journal of Psychiatry 32, 73–76. Sanchez-Villegas, A., Henriquez, P., Figueiras, A., Ortuno, F., Lahortiga, F., MartinezGonzalez, M.A., 2007. Long chain omega-3 fatty acids intake, fish consumption and mental disorders in the SUN cohort study. European Journal of Nutrition 46, 337–346. Sarris, J., Mischoulon, D., Schweitzer, I., 2012. Omega-3 for bipolar disorder: metaanalyses of use in mania and bipolar depression. Journal of Clinical Psychiatry 73, 81–86.

359

Taha, A.Y., Cheon, Y., Ma, K., Rapoport, S.I., Rao, J.S., 2013. Altered fatty acid concentrations in prefrontal cortex of schizophrenic patients. Journal of Psychiatric Research 47, 636–643. Tatebayashi, Y., Nihonmatsu-Kikuchi, N., Hayashi, Y., Yu, X., Soma, M., Ikeda, K., 2012. Abnormal fatty acid composition in the frontopolar cortex of patients with affective disorders. Translational Psychiatry 2, e204. van der Kemp, W.J., Klomp, D.W., Kahn, R.S., Luijten, P.R., Hulshoff Pol, H.E., 2012. A meta-analysis of the polyunsaturated fatty acid composition of erythrocyte membranes in schizophrenia. Schizophrenia Research 141, 153–161.