Bimodal Distribution of Polyunsaturated Fatty Acids in Schizophrenia Suggests Two Endophenotypes of the Disorder

Bimodal Distribution of Polyunsaturated Fatty Acids in Schizophrenia Suggests Two Endophenotypes of the Disorder Håvard Bentsen, Dag K. Solberg, Helge Refsum, Jon Michael Gran, Thomas Bøhmer, Peter A. Torjesen, Ola Halvorsen, and Odd Lingjærde Background: There is conflicting evidence of whether polyunsaturated fatty acids (PUFA) in red blood cells are bimodally distributed in schizophrenia. The purpose of this study was to examine the distribution of PUFA, as well as its links to plausible causal factors. Methods: A 16-week cohort study and a case-control study as part of a randomized controlled trial. Ninety-nine patients with DSM-IV schizophrenia, schizoaffective disorder, or schizophreniform disorder, aged 18 to 39, were consecutively included at admission to psychiatric departments of nine Norwegian hospitals. Fatty acids were measured in 97 of these patients and in 20 healthy control subjects. The primary outcome measure was the bimodality test statistic T, assessed by a ␹2 test of the likelihood of one or two normal distributions of PUFA. Results: At baseline, levels of polyunsaturated fatty acids were highly significantly bimodally distributed among patients. One third of patients constituted a group (low PUFA) who had PUFA levels at one fifth (p ⬍ .001) of those in high PUFA patients and healthy control subjects, which did not differ. Bimodality was mainly accounted for by docosahexaenoic acid and arachidonic acid. Bimodality was confirmed after 16 weeks. ␣-tocopherol was a robust predictor of PUFA at both occasions. Desaturase and elongase indexes differed between PUFA groups. Smoking, gender, antipsychotic medication, and dietary factors did not explain the bimodal distribution. Conclusions: Red blood cell PUFA were bimodally distributed among acutely ill patients with schizophrenia and schizoaffective disorder. Endogenous deficiencies of redox regulation or synthesis of long-chain PUFA in the low PUFA group may explain our findings. Key Words: ␣-tocopherol, desaturases, elongases, fatty acids, oxidative stress, schizophrenia isturbed membrane phospholipids metabolism and a deficient redox regulatory system may account for many abnormalities in schizophrenia. They may also be targets for preventive and therapeutic interventions (1–3). Two British studies have shown a bimodal distribution of red blood cell (RBC) polyunsaturated fatty acids (PUFA) in schizophrenia, whereas the distribution was unimodal in healthy control subjects (4,5). Specifically, docosahexaenoic acid (DHA; C22:6[n-3]) and arachidonic acid (ARA; C20:4[n-6]), the predominant PUFA in the brain, were depleted in one third of the patients. In the first study, the main patient sample (n ⫽ 68) was representative of all individuals with schizophrenia in the catchment areas. The validity of PUFA findings has been questioned because RBCs were stored at only ⫺20°C (6). In the second study, the patient sample was small (n ⫽ 23) and only long-term patients with prominent negative symptoms were included. Red blood cells were stored at ⫺80°C. After these two studies, bimodality has not been reported. Especially, Hibbeln et al. (7) addressed this issue in a sample of 72 patients with schizophrenia or schizoaffective disorder who had completed a clinical trial of ethyl-eicosapentaenoic acid (EPA). They found no bimodal distribution of RBC

D

From the Center for Psychopharmacology (HB, DKS, HR), Diakonhjemmet Hospital; Division of Psychiatry (HB, OL); Nutritional Laboratory (TB), Department of Medicine; and Hormone Laboratory (PAT), Oslo University Hospital; and Department of Biostatistics (JMG), Institute of Basic Medical Sciences, Universty of Oslo, Oslo, Norway; and Department of Psychiatry (OH), Akershus University Hospital, Lørenskog, Norway. Address correspondence to Håvard Bentsen, M.D., Ph.D., Diakonhjemmet Hospital, Center for Psychopharmacology, P.o.b. 85, Vinderen, N-0319 Oslo, Norway; E-mail: [email protected]. Received Aug 18, 2010; revised Jan 31, 2011; accepted Feb 1, 2011.

0006-3223/$36.00 doi:10.1016/j.biopsych.2011.02.011

long-chain polyunsaturated fatty acids (LCPUFA) at baseline. Bimodality could suggest two genetically distinct subgroups of schizophrenia. Because of this important implication and the conflicting findings, we tested the hypothesis that RBC PUFA are bimodally distributed in schizophrenia and related psychoses during an acute phase of the disorder. This study was a part of a randomized placebocontrolled trial of ethyl-EPA and/or vitamins E and C. The relation of PUFA to clinical and biochemical characteristics and treatment effects are reported separately.

Methods and Materials Patients For the clinical trial, we included patients with schizophrenia, schizoaffective disorder, or schizophreniform disorder, established by the Structured Clinical Interview for DSM-IV. They were aged 18 to 39 years, had been admitted to a psychiatric department within the previous 21 days, were prescribed antipsychotics, had no substance dependence (DSM-IV), and gave informed consent. The study protocol was approved by the Regional Committee of Medical Research Ethics. Patients were recruited from the psychiatric emergency departments of nine hospitals in Norway from September 2001 to December 2003. They were randomized to receive capsules of ethyl-eicosapentaenoate 2 g a day or placebo oil and tablets of ␣-tocopherol 364 mg plus ascorbic acid 1000 mg a day or placebo. Blood was sampled after 8 hours of fasting before and after the 16-week trial. For analysis of fatty acids, ethylenediaminetetraacetic acid blood was kept cool until centrifugation within 1 hour. Washed red blood cells were stored at ⫺70°C within 1 hour after centrifugation and sent within 3 months in dry ice to Mylnefield Research Services Ltd, Dundee, United Kingdom. There lipids were extracted, converted into fatty acid methyl esters, and analyzed by gas chromatography, yielding fatty acid profiles. Twenty-eight species of fatty acids from C14:0 to C24:1 were reported as micrograms per gram of BIOL PSYCHIATRY 2011;70:97–105 © 2011 Society of Biological Psychiatry

98 BIOL PSYCHIATRY 2011;70:97–105 RBC. The sum of ␻3 fatty acids was C18:3(n-3) ⫹ C18:4(n-3) ⫹ C20:3(n-3) ⫹ C20:5(n-3) ⫹ C22:5(n-3) ⫹ C22:6(n-3). The sum of ␻6 fatty acids was C18:2(n-6) ⫹ C18:3(n-6) ⫹ C20:2(n-6) ⫹ C20:3(n-6) ⫹ C20:4(n-6) ⫹ C22:4(n-6) ⫹ C22:5(n-6). The sum of ␻3 and ␻6 fatty acids was named polyunsaturated fatty acids, the key variable of the present study. The sum of ␻3 and ␻6 PUFA with 20 or 22 carbon atoms was named long-chain polyunsaturated fatty acids. Serum ␣-tocopherol was analyzed at the Nutrition Laboratory, Oslo University Hospital, Aker (OUHA), Oslo, Norway, with kits from Bio-Rad Lab., GmBH (Munich, Germany), high-performance liquid chromatography, and ultraviolet light detection. The adjusted term (␣-tocopherol)/([triglycerides] ⫹ [cholesterol]) was used in statistical analyses. Serum total antioxidant status (TAS) was analyzed at the Nutrition Laboratory, OUHA, with kits from Randox Laboratories Ltd., (Antrim, United Kingdom). Total serum malondialdehyde (MDA) was analyzed with the colorimetric thiobarbituric acid reactive substances test. A coefficient of variation of 6.5% was achieved. Analyses were done by Vitas AS, Oslo, Norway. For analysis of free F2-isoprostane (8-epi-PGF2␣ or iPF2␣-III), ethylenediaminetetraacetic acid blood was kept cool until centrifugation within 1 hour. Plasma was stored at ⫺70°C within 1 hour after centrifugation. After purification on F2-isoprostane affinity column, the plasma levels were quantified with competitive enzyme-linked immunoassay followed by ultraviolet light detection at 405 to 420 nm (8-Isoprostane Enzyme Immunoassay Kit, Cat# 516351; Cayman Chemical Comapny, Ann Arbor, Michigan). Analyses were done by the Hormone Laboratory, OUHA. Healthy Control Subjects Healthy control subjects were 18 to 39 years old and had no mental disorder according to the Mini International Neuropsychiatric Interview 5.0.0, DSM-IV. They were employees of a hospital and a kindergarten. They were examined for RBC fatty acids in June and July and re-examined in October and November. If available (n ⫽ 17), average values across these two sessions were used for statistical analyses to attenuate the effect of seasonal fluctuations. Otherwise, single measures were used (n ⫽ 3). Statistical Methods Bimodality of PUFA was tested using a likelihood ratio test (8). A two-component normal mixture model p N(␮1, ␴12) ⫹ (1⫺ p) N(␮2, ␴22) was fitted, where p is the proportion in the first component. The parameters were estimated using an expectation-maximization procedure (9). The likelihood function Lb for the bimodal model was compared with the likelihood Lu for the single normal distribution model N(␮, ␴2). Doehlert et al. (8) found that the distribution of T ⫽ 2 ln (Lb/Lu) for normally distributed data in its tail resembled a ␹2 distribution with df ⫽ 4. Simulations with our data support this result. We generated 999 samples from a one-component normal distribution using the sample mean and variance (10). The p values for all PUFA were the same or smaller than those obtained using the ␹2 distribution with df ⫽ 4. We therefore kept the latter ones. We used a strict definition of statistical significance (␣ ⫽ .005), corresponding to T ⫽ 14.9. The above-mentioned methods were implemented in the statistical software package R, version 2.11.0 (http://cran.r-project.org/mirrors.html) (11). All other tests were done with the PASW Statistics 18 program (SPSS, Inc., Chicago, Illinois). Parametric or nonparametric tests were chosen depending on the distribution of the variables. The ␣-level was set at .05 (twosided). Adjustment for multiple testing was inappropriate for the analwww.sobp.org/journal

H. Bentsen et al. ysis of our key hypothesis but was done for the confirmatory bimodality analyses of C22:6(n-3), C20:4(n-6), sum of ␻3 PUFA, sum of ␻6 PUFA, and LCPUFA. Other analyses were considered as exploratory and therefore did not require adjustment (12).

Results Flow of Participants Ninety-nine patients satisfied the eligibility requirements and constituted the intention-to-treat sample. Red blood cell fatty acids were measured in 97 of these patients at baseline and in 77 patients after 16 weeks. Twenty healthy control subjects were examined, 17 of these twice. Baseline Patient Characteristics Key characteristics of the patient sample and details regarding psychotropic medication are shown in Table 1 and Table S1 in Supplement 1, respectively. For 3 months before hospitalization, 48 patients were not prescribed any antipsychotic medication. After admission to the hospital, all patients were prescribed antipsychotics. Twenty patients had used fish oil supplements during the last 3 months (16 patients used ⬍ .2 g/day). Seventeen patients had used vitamin E supplements, all ⱕ 30 mg/day. The Distribution of Fatty Acids in Patients Baseline. The Kolmogorov-Smirnov tests indicated one normal distribution for saturated and monounsaturated fatty acids (Figure S1 in Supplement 1). In contrast, all major fatty acids with at least three double bonds, and only these, were bimodally distributed. Both histograms and statistical tests showed bimodality for the C20 to C22 fatty acids dihomo-gamma-linolenic acid (DGLA) C20:3(n-6) (T ⫽ 20.2, p ⫽ 5*10⫺4), ARA C20:4(n-6) (T ⫽ 46.1, p ⫽ 2*10⫺9), EPA C20:5(n-3) (T ⫽ 18.5, p ⫽ 10⫺3), and DHA C22:6(n-3) (T ⫽ 49.6, p ⫽ 4*10⫺10). Gamma-linolenic acid (GLA) C18:3(n-6) was statistically bimodally distributed (p ⫽ 3*10⫺11), whereas bimodality was less evident in its histogram. The statistical evidence for bimodality of ␣-linolenic acid C18:3(n-3) was weaker (p ⫽ 8*10⫺3), though the histogram showed bimodality. The sum of ␻3 and the sum of ␻6 PUFA were bimodally distributed, with parameters T ⫽ 47.4, p ⫽ 10⫺9 and T ⫽ 24.0, p ⫽ 8*10⫺5, respectively (Figure 1A,B). All confirmatory analyses yielded significant findings after Bonferroni correction. The histogram of RBC PUFA is compatible with bimodality (Figure 1C). The bimodality test yielded T ⫽ 33.7, df ⫽ 4, p ⫽ 9*10⫺7. Thus, the distribution represented a mixture of two distributions assumed to be normal. The low PUFA distribution had a probability p1 ⫽ .30, a mean ␮1 ⫽ 101.7 ␮g/g, and a standard deviation ␴1 ⫽ 50.5 ␮g/g. The high PUFA distribution had the parameters p2 ⫽ .70, ␮2 ⫽ 440.9 ␮g/g, and ␴2 ⫽ 121.6 ␮g/g. We assume that the trough, i.e., the point with the lowest density of observations, belonged to the interval between (␮1 ⫹ 2␴1) and (␮2 ⫺ 2␴2), i.e., between 202.7 and 197.8 ␮g/g, with a mean 200 ␮g/g. Thirty percent of the patients had a value at or below this cutoff score (low PUFA patients), whereas 70% had a value above the threshold (high PUFA patients). The histogram (Figure 1D) and parameters of RBC LCPUFA (T ⫽ 46.4, p ⫽ 2*10⫺9) are compatible with bimodality. The low and high LCPUFA distributions had the parameters p1 ⫽ .36, ␮1 ⫽ 55.1 ␮g/g, ␴1 ⫽ 40.8 ␮g/g and p2 ⫽ .64, ␮2 ⫽ 307.2 ␮g/g, ␴2 ⫽ 72.8 ␮g/g, respectively. Thirty-six percent of the patients had a value at or below the trough 149 ␮g/g (low LCPUFA patients), whereas 64% had a value above this threshold (high LCPUFA patients). All low PUFA patients were also low LCPUFA patients, whereas 6% of subjects were high PUFA but low LCPUFA patients. Low PUFA may thus be viewed as a subgroup in the low LCPUFA group with a more extensive reduction of PUFA. In the schizophrenia subgroup (n ⫽

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H. Bentsen et al. Table 1. Sample Characteristics at Baseline Patients Characteristics

ITT Sample (n ⫽ 99 or b–h)

Low PUFA (n ⫽ 30 or c–h)

High PUFA (n ⫽ 67 or c–h)

Healthy Control Subjects (n ⫽ 20)

Age, Mean (SD), Years Male, n (%) Education, % [(ⱕ 1):2:3]a Smoker, n (%) DSM-IV Diagnosis, n (SZ:SA:SF) Duration of Illness, Median (25–75 percent), Years First Hospitalization, n (%) RBC PUFA, Median (Interquartile Range)b ␻-3 PUFA ␻-6 PUFA ␻-6/␻-3 Ratio EPA DHA ARA ARA/EPA ARA/DHA Mead Acid (⌬6 Desaturase Index GLA/LA) ⫻ 104 ⌬5 Desaturase Index ARA/DGLA Elongase Index DGLA/GLA Lipid-Adjusted Serum-␣-Tocopherolc S-Uric Acidd S-Albumine Total Antioxidant Capacityf F2-Isoprostaneg Malondialdehyde (TBARS)h

27.4 (6.1)i 61 (64) 29:52:19k 63 (64)i 71:20:8 4 (1–9) 31 (31) 384 (343)k 81 (88)k 296 (246)k 4.0 (2.0)j 8.1 (10.0)j 45.0 (55.5)k 114.0 (121.9)j 13.0 (8.2) 2.8 (1.2)j .8 (1.1)k 107 (100)k 6.5 (2.9)j 11.5 (11.4)k 4.7 (3.4) 313 (137) 42 (7) 1.3 (.2) 29 (20) .42 (.21)

29.9 (6.5)l 18 (60) 27:50:23 19 (63) 23:7:0 5 (2–10) 10 (33)n 93 (70)n 11 (11)n 84 (64)n 5.8 (2.4)n 1.5 (1.4)n 4.8 (5.3)n 17.0 (17.9)n 10.8 (8.3)n 3.5 (1.8)n 0n 194 (100)n 4.4 (2.4)n 3.9 (3.8)n 3.9 (1.2) 297 (122) 42 (5) 1.3 (.3) 32 (22) .39 (.18)

26.3 (5.4)p 43 (64) 30:52:18q 42 (63)o 47:12:8 3 (1–7) 20 (30) 460 (148) 104 (43) 338 (101)o 3.6 (1.2) 10.6 (7.2) 59.5 (30.4)o 141.6 (58.6) 13.3 (8.2) 2.5 (1.0) 1.0 (.4)o 86 (100)o 7.1 (2.6) 15.1 (10.3)p 6.0 (3.6) 321 (139) 42 (7) 1.3 (.2) 28 (18) .45 (.22)

31.1 (5.3) 11 (55) 15:40:45 7 (35)

479 (49) 111 (26) 378 (58) 3.3 (1.0) 11.7 (6.0) 65.8 (15.7) 154.9 (25.0) 13.4 (6.7) 2.3 (.6) 1.2 (.1) 65 (⬍ 100) 7.7 (1.7) 19.2 (5.7)

Empty cells mean that the specific assessments were not performed in healthy control subjects. Sample sizes of patients differed according to whether the specific assessment was performed. Groups were compared with ␹2 or Fisher’s exact tests, Kendall’s tau-B, or two-sample Mann-Whitney tests of rank differences, depending on types of data. ARA, arachidonic acid; DGLA, dihomo-gamma-linolenic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; GLA, gamma-linolenic acid; ITT, intent-to-treat; LA, linolenic acid; PUFA, polyunsaturated fatty acids; RBC, red blood cell; S, serum; SA, schizoaffective disorder; SF, schizophreniform disorder; SZ, schizophrenia; TBARS, thiobarbituric acid reactive substances. a Highest completed education: ⱕ 1 ⫽ primary school completed or noncompleted, 2 ⫽ secondary school, 3 ⫽ college or university. b RBC PUFA ⫽ sum of ␻-3 and ␻-6 polyunsaturated fatty acids in red blood cells, ␮g/g RBC. For all fatty acids derived variables: patients n ⫽ 97, healthy control subjects n ⫽ 20. c Adjusted serum-␣-tocopherol ⫽ serum ␣-tocopherol/(triglycerides ⫹ cholesterol), (␮mol/L)/(mmol/L), n ⫽ 91, low PUFA n ⫽ 28, high PUFA n ⫽ 61. d ␮mol/L, n ⫽ 72, low PUFA n ⫽ 16, high PUFA n ⫽ 54. e g/L, n ⫽ 92, low PUFA n ⫽ 27, high PUFA n ⫽ 63. f mmol/L, n ⫽ 97, low PUFA n ⫽ 29, high PUFA n ⫽ 66. g pg/mL, n ⫽ 97, low PUFA n ⫽ 29, high PUFA n ⫽ 66. h ␮mol/L, n ⫽ 95, low PUFA n ⫽ 28, high PUFA n ⫽ 66. b– h Median (interquartile range). Patients versus healthy control subjects: i.01 ⱕ p ⬍ .05, j.001 ⱕ kp ⬍ .01, p ⬍ .001. Only differences without parentheses are significant after Bonferroni correction: p’ ⬍ .05, p’ ⫽ 3p. Low versus high PUFA patients: l.01 ⱕ p ⬍ .05, m.001 ⱕ p ⬍ .01, np ⬍ .001. All marked differences are significant also after Bonferroni correction: p’ ⬍ .05, p’ ⫽ 3p. High PUFA patients versus healthy control subjects: o.01 ⱕ p ⬍ .05, p.001 ⱕ p ⬍ .01, qp ⬍ .001. Only differences without parentheses are significant after Bonferroni correction: p’ ⬍ .05, p’ ⫽ 3p.

70), both PUFA and LCPUFA were bimodally distributed (T ⫽ 32.7, p ⫽ 10⫺6 and T ⫽ 39.1, p ⫽ 7*10⫺8, respectively). In the schizoaffective subgroup (n ⫽ 19), there was no significant bimodal distribution of PUFA (T ⫽ 8.5, p ⫽ .08), whereas LCPUFA was bimodally distributed (T ⫽ 17.8, p ⫽ .001). In the schizophreniform subgroup (n ⫽ 8), PUFA and LCPUFA were unimodally distributed. 16-Week Follow-Up. We examined the stability of PUFA and LCPUFA by the Wilcoxon signed-rank test among completers in the placebo EPA group (n ⫽ 37). Neither PUFA nor LCPUFA changed significantly over 16 weeks. Seven of the 13 baseline low PUFA subjects and 7 of the 16 baseline LCPUFA subjects moved to the high PUFA or LCPUFA groups, respectively, at follow-up. Three of the 24 high PUFA

subjects and 2 of the 21 high LCPUFA subjects changed affiliation. The stability seemed stronger for the LCPUFA than the PUFA grouping (Kendall tau-b, .51, p ⫽ .003, vs. .37, p ⫽ .04). At follow-up, histograms and bimodality tests for EPA-placebo patients (n ⫽ 39) indicated a bimodal distribution of PUFA: T ⫽ 21.5, p ⫽ .0002.Forpatientswithschizophrenia,the distribution was bimodal (n ⫽ 30, T ⫽ 21.7, p ⫽ .0002). The corresponding parameters for LCPUFA were T ⫽ 32.7, p ⫽ 10⫺6 and T ⫽ 30.4, p ⫽ 4*10⫺6, respectively. Correlates of Polyunsaturated Fatty Acids Omega-6/Omega-3 PUFA Ratios. Mann-Whitney tests showed much higher ␻6/␻3 PUFA (Z ⫽ ⫺6.29, p ⬍ .001) and ARA/DHA (Z ⫽ www.sobp.org/journal

100 BIOL PSYCHIATRY 2011;70:97–105

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Figure 2. The relation between ␻3 and ␻6 polyunsaturated fatty acids in patients and healthy control subjects is best described as a quadratic curve: y ⫽ 12.7 ⫹ 5.6x ⫺ .02x2, where x ⫽ ␻3 and y ⫽ ␻6. Adjusted R2 ⫽ .83, p ⬍ .001. dy/dx ⫽ 5.6 ⫺ .04x. dy/dx ⫽ 0 when x ⫽ 140. Then y ⫽ 404.7 and y/x ⫽ 2.9. PUFA, polyunsaturated fatty acids; RBC, red blood cell.

⫺4.64, p ⬍ .001) in the low than in the high PUFA group (Table 1). Thus, though both ␻3 and ␻6 PUFA were reduced in the low PUFA groups, ␻3, specifically DHA, was much more reduced than ␻6 PUFA. The analogous was true for ␻6/␻3 LCPUFA and ARA/DHA in relation to LCPUFA groups. The relationship between ␻3 and ␻6 PUFA was best described by a quadratic curve (Figure 2). The curve’s slope decreased as ␻3 PUFA increased. The ␻3 and ␻6 PUFA were positively linked among patients (Spearman’s rho ⫽ .86, p ⬍ .001, n ⫽ 97). In contrast, they were negatively linked in healthy control subjects (rho ⫽ ⫺.59, p ⫽ .006, n ⫽ 20). Mead Acid. Mead acid (C20:3[n-9]) is made by ⌬6-desaturase from oleic acid when both C18:3(n-3) and C18:2(n-6) are lacking. Thus, elevated blood concentration of this fatty acid indicates essential fatty acid deficiency (13). No mead acid was detected among low PUFA patients (Table 1). Desaturase and Elongase Indexes. Product-to-precursor ratios, called desaturase indexes, are conventionally used to estimate desaturase activity. The most specific indexes are ARA/DGLA for ⌬5-desaturase and GLA/linolenic acid for ⌬6-desaturase (14,15). The key elongase enzyme for the conversion of C18 to C20 PUFA in humans is the elongation of very long chain fatty acids 5 (ELOVL5 or HELO1); thus, we defined the elongase index as DGLA/GLA. Gammalinolenic acid/linolenic acid was higher and ARA/DGLA and DGLA/GLA were lower among low PUFA patients (Table 1). All differences were highly significant, especially for elongase (Mann-Whitney test, Z ⫽ ⫺7.2, p ⬍ .001).

Figure 1. (A–D) Distribution of fatty acids at baseline. Each panel displays how red blood cell concentrations, in micrograms per gram of red blood cells, of different groups of polyunsaturated fatty acids were distributed in the sample of 97 intent-to-treat patients. RBC, red blood cell.

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Plausible Determinants of PUFA Bimodality We examined the following variables as plausible determinants of PUFA and LCPUFA groups (low, high): age, sex, completed education, schizoaffective disorder, schizophreniform disorder, antipsychotic medication before hospitalization, fish oil supplement, vitamin E supplement, vitamin C supplement, smoker, and lipid adjusted ␣-tocopherol. The optimal multivariate models are shown in Tables 2 and 3. ␣-tocopherol correlated strongly with PUFA (Figure 3), both in

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H. Bentsen et al. Table 2. Optimal Predictor Model for High Versus Low PUFA Groups at Baseline Characteristics Age, Years Antipsychotic Drugs, Defined Daily Doses Adjusted Serum-␣-Tocopherol, (␮mol/L)/ (mmol/L) Constant

Adjusted Odds Ratio (95% CI)

p Value

.92 (.84–1.01) 1.88 (.94–3.75)

.06 .07

1.66 (1.20–2.28) .73

.002

n ⫽ 89. Goodness-of-fit: Hosmer-Lemeshow test ␹2 ⫽ 5.3, df ⫽ 8, p ⫽ .72. Percentage correctly classified: 75. Difference between ⫺2 log likelihood of models with ␣-tocopherol with and without age and antipsychotic defined daily doses is ␹2 ⫽ 6.4, df ⫽ 2, p ⫽ .01. CI, confidence interval; PUFA, polyunsaturated fatty acids.

schizophrenia (rho ⫽ .46, p ⬍ .001, n ⫽ 66) and schizoaffective disorder (rho ⫽ .74, p ⫽ .002, n ⫽ 15). ␣-tocopherol was equally linked to ␻3 (rho ⫽ .45) and ␻6 PUFA (rho ⫽ .47) (both p ⬍ .001, n ⫽ 89). Also at follow-up, ␣-tocopherol was the strongest determinant of PUFA and LCPUFA. Low ␣-tocopherol was a necessary but not sufficient condition for low PUFA (Figure S2 in Supplement 1). Lipid-adjusted ␣-tocopherol values were categorized according to healthy reference values (16). Respectively to low PUFA and LCPUFA groups, 22% and 25% had a value below the reference interval and 4% and 3% had a value above it. In the high PUFA or LCPUFA groups, respectively, 44% and 48% of the patients had a value above the reference interval and 12% and 9% had a value below it. Antipsychotic Medication Before Admission and at Baseline. Types of antipsychotic drugs were defined by being first or second generation and by propensity for metabolic side effects and antioxidant effect (17,18). Users of quetiapine/olanzapine/clozapine had 27% lower levels of F2-isoprostane than users of other antipsychotics (p ⫽ .02), suggesting a higher antioxidant capacity. However, lack of or type of antipsychotic drugs did not relate to PUFA groups (Table 2). The mean antipsychotic daily defined dose at baseline was lower in the low PUFA (1.3 vs. 1.7, T ⫽ ⫺2.23, p ⫽ .03) and LCPUFA groups (1.3 vs. 1.7, T ⫽ ⫺2.72, p ⫽ .008). Diagnosis. Twenty-three of 70 patients with schizophrenia, 7 of 19 patients with schizoaffective disorder, and none of 8 patients with schizophreniform disorder belonged to the low PUFA group (Fisher’s exact test, p ⫽ .13). The LCPUFA groups were not significantly linked to diagnoses. Biomarkers of Redox Regulation. Among biomarkers of redox regulation, only ␣-tocopherol was related significantly to PUFA or LCPUFA groups (see above). Uric acid, albumin, TAS, F2-isoprostane, and MDA had no significant links to PUFA (Table 1). At both Table 3. Optimal Predictor Model for High Versus Low LCPUFA Groups at Baseline Characteristics Age, Years Schizoaffective Disorder (No, Yes) Antipsychotic Drugs, Defined Daily Doses Adjusted Serum-␣-Tocopherol (␮mol/L)/ (mmol/L) Constant

Adjusted Odds Ratio (95% CI)

p Value

.91 (.82–.996) .25 (.05–1 .20) 2.25 (1.09–4.64)

.04 .08 .03

1.95 (1.36–2.80) .37

⬍.001

n ⫽ 89. Goodness-of-fit: Hosmer-Lemeshow test ␹2 ⫽ 4.9, df ⫽ 8, p ⫽ .77. Percentage correctly classified: 79. CI, confidence interval; LCPUFA, long-chain polyunsaturated fatty acids.

Figure 3. Polyunsaturated fatty acids by serum-␣-tocopherol adjusted for serum-triglycerides ⫹ serum-cholesterol at baseline in 97 intent-to-treat patients. cholest., cholesterol; PUFA, polyunsaturated fatty acids; RBC, red blood cell; triglyc., triglycerides.

sessions, weaker antioxidant capacity was related to smoking (lower serum [s]-uric acid, p ⫽ .004 and .03; lower TAS, p ⫽ .004 and .02), female gender (lower TAS, p ⬍ .01; s-uric acid and s-albumin, both p ⱕ .001), and increasing age (lower s-albumin, p ⫽ .02). Plausible Determinants of ␣-Tocopherol. Adjusted serum-␣tocopherol had a unimodal positively skewed distribution. A baseline multiple linear regression model with age, sex, completed education, antipsychotic medication before hospitalization, smoker, vitamin E supplement, vitamin C supplement, and EPA supplement explained only 11% of the variance of serum-␣-tocopherol (n ⫽ 88, adjusted R2 ⫽ .01, p ⫽ .34, markedly skewed distribution of residuals). Being younger (p ⫽ .03) and female (p ⫽ .04) predicted higher ␣-tocopherol. Comparison of PUFA Levels Between Patients and Healthy Control Subjects The distribution of PUFA values among patients (n ⫽ 97) and healthy control subjects (n ⫽ 20) is displayed in Table 1. MannWhitney test of PUFA yielded Z ⫽ ⫺3.57, p ⬍ .001. The link was independent of the season for patient assessment. All healthy control subjects belonged to the high PUFA group versus 67 out of 97 patients (Fisher’s exact test, p ⫽ .003). A multiple linear regression analysis was performed with PUFA as a dependent variable and patient-control subject status, sex, age, completed education, and smoking as independent variables. Assumptions of normality and homoscedasticity of residuals were fulfilled. The model explained 19% of the variance of PUFA [adjusted R2 ⫽ .15, F ⫽ 5.06, df ⫽ (5,111), p ⬍ .001]. Age (adjusted B ⫽ ⫺9.6 [95% confidence interval ⫺15.3 to ⫺3.9], p ⫽ .001) and patient status (adjusted B ⫽ ⫺177.3 [95% confidence interval ⫺273.7 to ⫺80.1], p ⬍ .001) were negatively linked to PUFA. No other variables in the model had p ⬍ .20. Ethnicity was not included in the model: about 95% in both groups were Caucasian (as in the general Norwegian population). Mostly, fatty acid variables did not differ between high PUFA subjects and healthy control subjects but both differed from low PUFA subjects (Table 1). Similar results were obtained for LCPUFA groups. At follow-up, patients in the EPA-placebo group still had a lower PUFA level than healthy control subjects (Z ⫽ ⫺2.25, p ⫽ .03). www.sobp.org/journal

102 BIOL PSYCHIATRY 2011;70:97–105 Discussion Bimodal Distribution of PUFA Our finding of bimodality of PUFA is in agreement with two previous studies (4,5). In all three studies, RBC arachidonic acid and docosahexaenoic acid had an unequivocally bimodal distribution and linolenic acid was unimodally distributed. In the American study, no bimodality was found (7); however, their sample consisted of stable outpatients who had completed a trial. This might have effectively excluded a group of low PUFA patients who had a higher dropout rate in our study. Furthermore, we assume that bimodality is more pronounced during oxidative stress. Biomarkers of lipid peroxidation maximize at the peak of relapse and decline during remission, fitting with our finding of a weakened bimodality at follow-up (19,20). However, bimodality has also was been found in a mostly stabilized community sample (4). Plausible Predictors of Bimodality of PUFA Polyunsaturated fatty acid levels may be low because of increased consumption or reduced synthesis of PUFA. Bimodality implies at least one binary causal factor. This factor might be environmental, such as exposure or nonexposure to a toxic agent or a nutritional supplement, or genetic, such as gender or the presence of a polymorphism. Smoking. We found some evidence that smoking was related to weaker antioxidant capacity, but not to lower PUFA. In most epidemiological and clinical studies, there has been no effect of smoking on redox regulatory biomarkers or PUFA (7,21–27). Antipsychotic Drugs. The proportions of antipsychotic users did not differ between PUFA groups, contrary to expectations (22,28,29). As expected, we found lower F2-isoprostane among users of quetiapine/olanzapine/clozapine (2,17,25,29,30). It is unlikely that dosage, being unimodally distributed, caused bimodality of PUFA. Also, the minor differences of dosage should not entail major differences of PUFA in only 3 weeks (28,29,31). Gender. Gender did not predict PUFA, in keeping with some studies (24,32) but not others (7,33). Serum biomarkers of redox regulation, except ␣-tocopherol, indicated higher oxidative stress in women, as in some other studies (21,34). Age. Contrasting with most previous studies of psychotic patients, higher age predicted low PUFA and lower antioxidant capacity (7,22,24,35). However, because age was normally distributed, it cannot explain the bimodality of PUFA. Diet. The fatty acid compositions in red blood cells and plasma reflect intake of lipids in the previous couple of months and weeks, respectively (31). Nutritional intake of total PUFA in a Norwegian population explained only 4% of the variance in serum PUFA (36). Intake of fish oil and body ␻3-LCPUFAs are robustly linked in normal subjects (37). Body ARA is much less influenced by diet, and the content of ARA is tightly regulated (37,38). The links between diet and RBC PUFA are markedly weaker in schizophrenic than in normal subjects (7,39,40). Red blood cell PUFA reflects diet, incorporation, and metabolism in membrane phospholipids, whereas plasma PUFA mainly reflects diet. Intake and plasma levels of PUFA do not differ consistently between patients and healthy subjects (40,41). In contrast, LCPUFA in erythrocytes has repeatedly been found reduced in patients (4,5,22, 24,40,42,43). In schizophrenia, LCPUFA was unimodally distributed in plasma but bimodally distributed in RBC (4). These previous data imply that deficient diet does not explain low levels of RBC PUFA in schizophrenia (1). Data from our study regarding mead acid, nutritional supplements, and geographic center, as well as ethnic homogeneity and www.sobp.org/journal

H. Bentsen et al. no substance dependency indicate that diet does not explain bimodality of PUFA (13,37). ␣-Tocopherol. The strongest and most robust predictor of PUFA groups was serum ␣-tocopherol, a principal protector of polyunsaturated fatty acids (44). We found that the more fatty acids were unsaturated, the more their distribution was bimodal. This agrees with the hypothesis that lack of ␣-tocopherol and resulting unhampered breakdown contributed to low PUFA (44). However, ␣-tocopherol was unimodally distributed and low levels were a necessary but not sufficient condition of low PUFA. Figure 1 suggests that an additional necessary bimodal factor caused low PUFA and low ␣-tocopherol and/or that low PUFA caused low ␣-tocopherol (discussed below). We accounted for only 11% of the variance of ␣-tocopherol. A bimodal deficiency in a redox regulator could explain the depletion and would most likely be genetically determined (3,45,46). The distributions of ␣-tocopherol both in the low and high PUFA groups differed distinctly from that in a healthy reference population (16). Abnormally high levels of ␣-tocopherol in the high PUFA group could best be accounted for by CYP3A4 deficiency or inhibition because of inflammation (44,47,48). Lipid Peroxidation. Against expectations, plasma F2-isoprostane and MDA did not differ between PUFA groups. The F2-isoprostane is recognized as the “gold standard” biomarker of lipid peroxidation in vivo (49 –51). The only published study in schizophrenia, using an immunoassay kit (Oxis International, Inc.), revealed a fourfold increase of urine F2-isoprostane in acutely psychotic patients compared with healthy control subjects (52). In contrast, the median plasma F2-isoprostane among patients in our study was 29 pg/mL compared with a median of 22 and a range of 7 to 34 in eight similar studies of healthy control subjects. We did not find expected links to PUFA, other biomarkers of oxidative stress, or clinical phenomena (21,53). This might indicate that our isoprostane measurement lacked sensitivity (50,51,54,55). Expected links between F2isoprostane and diseases characterized by oxidative stress or biomarkers of redox regulation have not always been found (21.53– 55). Measuring isoprostane involves major methodological challenges, including sampling, storing, extraction, purification, and chemical analysis (50,51). A range of endogenous and exogenous factors may influence isoprostane formation in vivo (53). Thus, negative findings for isoprostane do not exclude lipid peroxidation. Total plasma MDA analyzed by the thiobarbituric acid test has been widely used, also in schizophrenia research (56). This test lacks specificity (51). Often MDA and isoprostane have weak links, and MDA has been less related to clinical phenomena than isoprostane (21,52,55). Assuming normal lipid peroxidation, the primary abnormality could be reduced synthesis of PUFA, yielding less substrate for lipid peroxidation (57). Also, less PUFA could entail increased consumption of ␣-tocopherol, because EPA and DHA may have antioxidant effects (58 – 60). Desaturation and Elongation. Key enzymes for the synthesis of human PUFA are ⌬5-desaturase, ⌬6-desaturase, and elongases (61,62). Fatty acid desaturase haplotypes and plasma or erythrocyte ARA concentration are strongly associated (63– 65). No significant links have been found with DHA, except for a study on breast milk (66). In this study, the prevalent minor single nucleotide polymorphism rs174575 allele was linked to lower concentrations of ARA, EPA, and DHA. Fatty acid desaturase genotypes are associated with metabolic and neurological conditions (67). In schizophrenia, data regarding desaturases are scant and conflicting (43,68 –70). In our study, only the low PUFA group differed consistently from healthy control sub-

H. Bentsen et al. jects for desaturase indexes. The ⌬5-desaturase index was lower and the ⌬6-desaturase index was higher, indicating an increased risk of metabolic morbidity and total mortality (71–73). Despite low levels, ARA and DHA seemed to be unable to upregulate the chain of desaturases and elongases sufficiently and in a coordinated manner (61) (Discussion text in Supplement 1). The key elongase for conversion from C18 to C20 in humans is ELOVL5 (62). In a small postmortem study, prefrontal cortical ELOVL5 (or HELO1) expression did not differ between patients with schizophrenia and healthy control subjects (70). In our study, the elongase index was the synthesis index that best distinguished low from high PUFA groups. The ELOVL5 maps to chromosome 6p, which is a candidate region for schizophrenia (74). It seems warranted to test for nucleotide changes at this gene (Discussion text in Supplement 1). Strengths and Limitations The validity of our main findings is strengthened by being replications of those from two previous studies. Also, bimodality of PUFA linked to lower ␣-tocopherol was confirmed by repeated measurement. The moderator effect on PUFA of the phase of disorders is avoided by including acutely ill patients only. One limitation is that no patients were drug-naive. However, lack of medication was unrelated to PUFA. Diet was not assessed. However, diet has not explained plasma PUFA in the Norwegian population. Also, dietary variation would not yield bimodality of PUFA. The control group was small and poorly matched to patients. However, differences were highly significant, also when adjusted for relevant factors such as smoking and education. Conclusions The concentration of RBC polyunsaturated fatty acids was bimodally distributed during an acute stage of schizophrenia and schizoaffective disorders. Both PUFA groups were prevalent and may define two distinct endophenotypes of the disorders. Endogenous deficiencies of redox regulation or synthesis of long-chain PUFA in the low PUFA group may explain our findings. The planning, collection of data, and management of this clinical trial was financially supported by The Stanley Medical Research Institute, United States (#01T-106); Laxdale, Ltd., United Kingdom; and Aker University Hospital, Norway. The analysis of data and preparation of manuscript was supported by Diakonhjemmet Hospital, Norway. We received minor grants from the University of Oslo, Josef and Haldis Andresen’s legacy, and H Lundbeck A/S. The Norwegian EPA/Antioxidants Study Group consisted of 16 investigators, all doctors, at nine hospitals in Southern Norway (major contributors: Dag K. Solberg, Håvard Bentsen, Grete Møller-Stray, Torbjørg Jensen, André Blaauw, Kåre Osnes, Gunnar Johannessen, and Ola Halvorsen). The project monitor was Heidi Bjørge. The Steering Committee consisted of Dr. David F. Horrobin (deceased April 2003), replaced by Dr. Harald Murck, United Kingdom; Professor Odd Lingjærde; and Dr. Håvard Bentsen (chairman). Professor Odd Aalen and Professor Inge Helland gave statistical advice. The authors reported no biomedical financial interests or potential conflicts of interest. Supplementary material cited in this article is available online. 1. Horrobin DF, Glen AIM, Vaddadi K (1994): The membrane hypothesis of schizophrenia. Schizophr Res 13:195–207. 2. Mahadik SP, Yao JK (2006): Phospholipids in schizophrenia. In: Lieberman JA, Stroup TS, Perkins DO, editors. Textbook of Schizophrenia. Washington, DC: American Psychiatric Publishing, 117–135.

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