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Mounting evidence indicates that low levels of n-3 polyunsaturated fatty acids (PUFAs) play a role in the pathophysiology of a large number of psychiatric disorders. In light of the suboptimal n-3 PUFAs intake due to poor dietary habits among substance abusers and the strong associations between aggression, anxiety and substance use disorders we examined if insurance of adequate intakes of n-3 PUFAs with supplementation would decrease their anger and anxiety scores.
Substance abusers (n=22) were assigned to either 3 g of n-3 PUFAs, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) or soybean oil in identically looking capsules. The trial was double-blind, randomized and lasted 3 months. Anger and anxiety scales were administered at baseline and once a month thereafter. Blood samples were collected at baseline and at the end of the trial.
Patients' dietary intakes of n-3 PUFAs fell below recommended levels. Assignment to n-3 PUFA treatment was accompanied by significant decreases in anger and anxiety scores compared to placebo assignment. These changes were associated with increases in plasma levels of both EPA and DHA but an increase in EPA was more robustly correlated with low end-of-trial anxiety scores and an increase in DHA was more robustly correlated with low end-of-trial anger scores.
These pilot data indicate that ensuring adequate n-3 PUFA intake via supplementation benefits substance abusers by reducing their anger and anxiety levels. The strong correlations between an increase in plasma EPA and lower anxiety scores and between an increase in plasma DHA and lower anger scores suggests a need for the further exploration of the differential responses to these two n-3 PUFAs in different psychiatric conditions.
Evidence has been mounting indicating that deficient intakes of n-3 PUFAs could play a role in the pathophysiology of a wide range of psychiatric disorders that have included (but are not limited to) mood disorders, schizophrenia , attention deficit and hyperactivity disorders, Alzheimer disease and dementias (Alessandri et al., 2004; Freeman et al., 2006; Young et al., 2005). This evidence has been based in part on international comparison studies of associations between seafood consumption and the prevalence of some psychiatric disorders (Hibbeln, 1998; Hibbeln, 2002; Noaghiul and Hibbeln, 2003) and on cross-sectional, case control or cohort studies investigating associations between these disorders and fish or n-3 polyunsaturated fatty acids (PUFAs) intake or tissue PUFAs concentrations (Alessandri et al., 2004; Freeman et al., 2006; Young et al., 2005). These studies are observational and do not constitute absolute proof that fish or n-3 PUFAs affect behavior. Studies involving the administration of supplements could theoretically provide stronger evidence for the beneficial role of n-3 PUFAs in mental health.
Supplements of two n-3 PUFAs, DHA (docosahexaenoic acid, 22:6n-3) and EPA (eicosapentaenoic acid, 20:5n-3), found mainly in fish and other seafood, have been reported to have psychotropic effects (Alessandri et al., 2004; Freeman et al., 2006; Young et al., 2005). They have different mechanisms of action (Sinclair AJ et al., 2007). DHA is concentrated in synaptic neuronal membranes and can alter the fluidity of these membranes that influences the conformation of embedded proteins (Salem et al., 2001). These proteins have important functions as they can act as receptors or transporters and can alter the passage of ions. DHA can thus affect the function of neurotransmitter systems. EPA is not concentrated in neuronal membranes but may affect neuronal function via neuroimmunological and vascular effects (Sinclair AJ et al., 2007). It competes with an n-6 PUFA, AA (arachidonic acid, 20:4n-6), for conversion to potent eicosanoids (Simopoulos, 2002). It can competitively attenuate the formation of n-6 eicosanoids that mediate immune-inflammatory responses that have been linked to the pathophysiology of depressive disorders, bipolar disorders and schizophrenia. EPA could also counteract the vasoconstrictive effects of AA, thereby increasing blood flow in the brain.
Although both EPA and DHA, used singly or in combination, have been reported to have psychotropic properties, their respective roles in specific psychiatric disorders have not yet been firmly established. Optimum doses of these PUFAs have not yet been determined either. Improvements were observed in unipolar depression with a dose of 6.4 g/day of EPA in combination with 3.2 g/day of DHA in one study (Su et al., 2003) and with a lower EPA dose (2 g/day) in another study (Nemets et al., 2002). On the other hand, in a dose finding study, a positive effect was found in unipolar depression with 1 g/day of EPA but doses of 2 and 4 g/day were less effective (Peet and Horrobin, 2002). Studies of bipolar disorders have also yielded discrepant results. Significant benefits were observed with a dose of 6.16 g/day of DHA in combination with 3.36 g/day of EPA in one study and with doses of 1 and 2 g/day of EPA in another study (Frangou et al., 2006; Stoll et al., 1999) whereas other authors reported that a dose of 6 g/day of EPA had no effect in bipolar depression and rapid cycling bipolar disorder (Keck et al, 2006). Heterogeneity of designs and of populations could account for discrepant outcomes. It could be hypothesized for example that n-3 PUFA supplementation could be beneficial only to individuals who are deficient in these PUFAs. Much work remains to be done to determine who might benefit from n-3 supplementation. Additional information is needed about the differential effect of EPA and DHA as well as about the optimum doses and treatment duration of these two important PUFAs.
The 2001−2002 National Health and Nutrition Examination Survey (NHANES) data indicate that dietary intakes of n-3 PUFAs declines among men as binge drinking frequency increases (Kim et al., 2007). In addition loss of tissue PUFAs due to alcohol use has been documented in several reviews (Lands et al., 1998; Salem and Olsson, 1997). We had hypothesized that a deficiency in n-3 PUFAs would not be limited to alcoholics but would also be found in polysubstance abusers and that they would respond positively to the administration of n-3 PUFAs supplements. A group of patients was studied and found to have a suboptimal dietary intake of long chain (LC) n-3 PUFAs. Significant decreases in anger and anxiety scores following a 3-month treatment with a daily dose of 2.25 g of EPA, 500 mg of DHA and 250 mg of other n-3 PUFAs were observed in these patients (Buydens-Branchey and Branchey, 2006; Buydens-Branchey and Branchey, in press). Plasma samples had been collected in these patients prior to the start and at the end of the therapeutic trial but fatty acids (FAs) levels had not been determined in these samples and no association could thus be established between changes in anger and anxiety scores and biochemical changes. Samples were subsequently assayed and the entire FA series determined. The assessment of associations between biochemical and psychological changes is the topic of the present report. The following questions were addressed: 1) Were lower anger and anxiety scores at the end of the trial associated with more important increases in plasma n-3 PUFAs? 2) Did increases in EPA and DHA levels have a differential effect on anger and anxiety?
Patients initially selected for participation in this study were 24 men admitted to the New York Harbor Healthcare System (Brooklyn site) substance abuse outpatient clinics. All consecutively admitted patients who met the study admission criteria were considered for enrollment after they had completed the first phase of their rehabilitation treatment that lasted 3 months. They underwent a physical examination and were administered a battery of laboratory tests. They were considered for inclusion if they did not have a major physical illness (e.g. cardiovascular, pulmonary, gastrointestinal, renal, neuromuscular, immunodeficiency or endocrine disorder or liver function tests greater than one SD above maximum normal values). Patients were interviewed by a psychiatrist. They were screened for concomitant Axis I disorder using the Structured Clinical Interview for DSM IV (SCID) (First et al., 1997). They were also administered the Addiction Severity Index questionnaire (ASI) (McLellan et al., 1992). Patients were considered for inclusion if they did not present evidence of schizophrenia, major depressive disorder, bipolar disorder, mental retardation or dementia. If subjects were judged to be appropriate for enrollment, they were offered to participate and, if they accepted, a written informed consent was obtained after complete description of the study. The study was approved by the institution IRB. The plasma samples of two patients were lost. Data presented below are those of the 22 patients whose psychological and biochemical assessments were available.
The following assessments covering the year and the month preceding the study entry were obtained: nature of the substances used, amount of each substance used and frequency of use.
A diet questionnaire covering the month preceding the study entry was obtained. This questionnaire was an adaptation of the National Institute of Health Diet History Questionnaire, downloaded from the National Cancer Institute web site (National Cancer Institute, 2002). Based on the results of this questionnaire, each patient's intake of Kcalories, n-6 and n-3 PUFAs were calculated, using the USDA National Nutrient Database Reference, Release 17 downloaded from its website (U.S. Department of Agriculture, 2004).
A modified version of the Profiles of Mood States (POMS) (McNair et al., 1971) was administered. The POMS is a self-report questionnaire which requires subjects to rate the intensity of 65 mood items on a 5-point scale. Ratings are combined into six scores assessing vigor, depression, anxiety, anger, confusion and fatigue. It was modified for use in this study because we had observed that patients attending our clinics asked to make 325 choices when given the complete POMS quickly lost interest in the task and gave unreliable answers. In order to limit the total number of choices, a modified version of the POMS was administered. In this modified version, patients were offered two choices for each item: present or absent. This made the questionnaire more readily acceptable. Changes in anger and anxiety scores are reported below.
The trial lasted 3 months and was double blind. Patients received either capsules containing n-3 PUFAs or placebo capsules. Assignment to the active substance group or to the placebo group was done according to a table of random numbers. Both types of capsules were purchased from “Nordic Naturals” (94 Hangar Way, Watsonville, CA 95076). The PUFA capsules contained 450 mg of EPA, 100 mg of DHA and 50 mg of other n-3 PUFAs, consisting mainly of ALA (alpha-linolenic acid, 18:3 n-3) and of DPA n-3 (docosapentaenoic acid, 22:5n-3). The placebo capsules contained soybean oil. All capsules contained vitamin E as an antioxidant and lemon oil to mask their taste. Patients were advised to take 5 capsules each day. Those taking the active substances were thus given a daily amount of 2.25 g of EPA, 500 mg of DHA and 250 mg of other n-3 PUFAs.
One, 2 and 3 months following the start of the study, dietary questionnaire and modified POMS covering the one-month period elapsed between two interviews were administered to all patients.
Blood samples were drawn twice under fasting conditions, the first time prior to the beginning of the trial and the second time, at the end of the trial. Patients were instructed to abstain from taking the study capsules on the morning of the second blood drawing. Plasma was immediately separated by centrifugation and kept at −80°C until assayed. Fatty acids, measurements were performed at the National Institute of Alcohol Abuse and Alcoholism (NIAAA) Laboratory of Membrane Biophysics and Biochemistry according to the method of Masood et al. (2005). They included total SFAs (saturated fatty acids), total MUFAs (monounsaturated fatty acids), total n-6 PUFAs, LA (linoleic acid, 18:2n-6), AA, DPA n-6 (docosapentaenoic acid, 22:5n-6), total n-3 PUFAs, ALA, EPA, DPA n-3 and DHA.
The statistical software SPSS for Windows (version 11.5) was used to perform the statistical analyses described below.
Comparisons of the two patient groups' demographic and baseline dietary data (including fish and PUFAs intakes) were made with two-tailed Student's t-tests, chi square tests with Yates' continuity correction or Fisher's exact tests, as appropriate.
Comparisons of the changes over time in the anger and anxiety scores of the two patient groups were done with repeated measures ANCOVAs with baseline values as covariates. The significance level was set by default at .05 by the software program. Because of the small number of study participants and the resulting low statistical power of the analyses, effect sizes (f) for the differences between the two groups were also calculated. Small, medium and large effect sizes correspond to f values of .10, .25 and .50, respectively (Cohen, 1988).
Statistical comparisons of baseline and end of trial FA levels were done separately for each patient group, using Wilcoxon matched-pairs signed-ranks tests. For each FA, the percent change from baseline to end-of-trial value was calculated.
The levels of association between the end-of-trial anger and anxiety scores and the percent changes in FAs and their significance were assessed in the two patient groups, using the Spearman's rank correlation coefficient.
There were no significant differences between patients assigned to the two treatment groups in age, marital status, educational level, employment status or types and amounts of drugs used for a period of one year prior to the start of the study. Eight patients (four in the PUFA group and four in the placebo group) were maintained on doses of methadone ranging from 70 to 120 mg. These doses remained stable throughout the study. Five patients in the placebo group and 3 in the PUFA group were treated with antidepressants. Doses of antidepressants remained stable throughout the study.
Baseline dietary data are shown in table 1. The mean daily fish intake of the 22 study participants was 37.9±17.8 g. Their mean daily total n-6 PUFA intake was 15.8±9.2 g and their mean daily total n-3 PUFA intake was 1.48±0.90 g. Patients consumed thus 11 times more n-6 than n-3 PUFAs. There were no significant differences between the PUFA and control groups in mean Body Mass Index, mean daily Kcalories, mean daily amounts of fish, mean daily amounts of total, short chain or long chain n-6 PUFAs and of total, short chain or long chain n-3 PUFAs consumed during the month preceding the start of the study. The fish most frequently consumed were catfish, whiting, flounder or canned tuna. Calculations of differences between the daily intake of 500 mg of LC n-3 PUFAs, recommended for cardiovascular health by ISSFAL, the International Society for the Study of Fatty Acids and Lipids (2004) and patients' actual daily intake did not reveal differences between the groups but showed that both groups fell short of the ISSFAL recommended daily intake. The n-3 PUFA group patients consumed on average 29.8% and the placebo group 33.1% of this recommended intake. Patients' n-3 PUFAs intake fell even shorter of the daily amount of 3.5 g for a 2000 Kcal diet recommended by Hibbeln et al. (2006) to protect the US population against both cardiovascular and major psychiatric diseases. The n-3 PUFA and placebo group patients consumed only 4.2% and 4.7%, respectively of this recommendation. There were no significant changes in dietary PUFA intake over time in any of the two groups, as assessed by repeated measures ANOVAs.
Figure 1 illustrates the changes in the anger scores during the 3-month administration of n-3 PUFAs or placebo capsules. The figure shows a decline in anger scores in the n-3 PUFA group but not in the placebo group. A comparison of the two groups by repeated measures ANCOVA (with baseline values as covariates) revealed a significant difference (F1,21=4.857, P=.040) and a large effect size (f=0.506).
Figure 2 illustrates the changes in the anxiety scores during the 3-month trial. The figure shows a decline in anxiety scores in the n-3 PUFA group but not in the placebo group. A comparison of the two groups by repeated measures ANCOVA (with baseline values as covariates) revealed a significant difference (F1,19=6.115, P=.023) and a large effect size (f=0.567).
Baseline and end-of-trial FA values, expressed in μg/ml and as % of total fatty acids (% TFA), are shown in table 2 for patients given the placebo capsules and in table 3 for patients treated with PUFA capsules. Changes from baseline to end-of-trial values, expressed as percentages of baseline values, are also shown in the two tables. Comparisons of baseline and end-of-trial FA values done with the Wilcoxon matched-pairs signed-ranks test did not reveal significant changes in the patients treated with the placebo. On the other hand, these comparisons revealed significant decreases at the end of the supplementation period in AA, DPA n-6, and total n-6 LC PUFAs, expressed as % TFA in patients given PUFAs. In these patients, there were significant increases in EPA, DPA n-3, DHA, and total n-3 LC PUFAs, expressed in μg/ml and in EPA, DPA n-3, and total n-3 LC PUFAs, expressed as % TFA. The increase in DHA expressed as % TFA was almost significant. Changes in the AA/EPA and in the DPA n-6/DHA ratios were also significant, reflecting increases in EPA and DHA.
Correlations between the end-of-trial anger and anxiety scores and the percent changes in FAs from the first to the second measurement were not significant in the placebo group but some of these correlations were significant in the n-3 PUFA group. The negative correlations (see table 4) indicate that lower end-of-trial anger and anxiety scores were correlated with more important changes from baseline in LC PUFAs of the n-3 series. As shown in the table, a low anxiety score was robustly associated with an increase in EPA but not significantly associated with an increase in DHA whereas a low anger score was robustly associated with increases in DPA n-3 and DHA.
In this study, the daily administration of 2.250 g of EPA and 500 mg of DHA for 3 months was accompanied by significant decreases in anger and anxiety scores in a group of substance abusers. These changes were associated with increases in plasma levels of EPA and DHA but an increase in EPA was more robustly correlated with low end-of-trial anxiety scores and an increase in DHA was more robustly correlated with low end-of-trial anger scores.
The present data provide further support to emerging evidence of a link between n-3 PUFAs and hostility. Rates of homicides are greater in countries with lower seafood intake (Hibbeln, 2001). This is consistent with a study showing that a reduced plasma level of DHA predicted greater hostility in violent male subjects with antisocial personality (Virkkunen et al., 1987) and with other data showing that young adults whose dietary intake of DHA and fish was high had a lower likelihood of high hostility (Iribarren et al., 2004). Few studies have investigated the effects of n-3 supplements on hostility or aggression. In one study, DHA supplementation with oil capsules prevented an increase in aggression at times of stress among Japanese students (Hamazaki et al., 1996) but had no effect in non-stressful situations (Hamazaki et al., 1998). Similar findings by the same authors were reported in an elderly cohort of white-collar workers but not in rural villagers (Hamazaki et al., 2002). In a study of women with untreated borderline personality disorder, ethyl ester EPA was found to diminish aggression and hostility measures as well as the severity of depressive symptoms (Zanarini and Frankenburg, 2003). Prisoners treated with supplements of vitamins, minerals and n-6 and n-3 PUFAs had fewer disciplinary incidents than those who had received a placebo (Gesch et al., 2002).
There is a limited amount of information about the effects of n-3 PUFAs in anxiety. The present data are among the first suggesting the possible existence of a relationship between EPA and anxiety in humans. Yehuda et al. (2005) who investigated the effects of the administration of a mixture of n-3 and n-6 PUFAs on test anxiety in college students observed an improvement in variables associated with this type of anxiety (i.e., appetite, mood, concentration, fatigue, academic organization, and sleep). They also observed a decrease in elevated cortisol levels. Green et al. (2006) found decreased levels of most n-3 PUFAs in the red blood cell membranes of patients with social anxiety disorder. Furthermore, their data suggest the existence of a relationship between the degree of n-3 PUFA deficiency and the severity of the anxiety disorder. Fux et al. (2004), on the other hand found EPA to be ineffective in a preliminary study of 11 patients with obsessive-compulsive disorder treated with selective serotonin reuptake inhibitors. Preclinical studies have shown that the administration of EPA decrease anxiety-like behaviors in rodents. The inflammatory-sickness response, stress and anxiety-like behaviors and stimulation of corticosterone secretion induced in rats by interleukin-1beta, the most potent proinflammatory cytokine, were found to be significantly reversed by the administration of EPA (Song et al., 2004).
In light of the discrepant results yielded by different studies using comparable amounts of n-3 PUFAs to treat mood disorders, one could wonder whether participants in some of these studies could have been unresponsive because they were not deficient in n-3 PUFAs. This was not the case in the present study as lower anger and anxiety scores at the end of the trial were associated with more important changes from baseline in EPA and DHA. Unlike most therapeutic agents, FAs are available from dietary sources but the upper limits of plasma concentrations of EPA and DHA beyond which additional increments would become ineffective have not yet been determined. Within the FA ranges observed in this study, no ceiling effect was observed and the magnitude of EPA and DHA changes appeared sufficient to induce psychological effects.
Little is presently known about the nature of the specific mechanisms underlying the efficacy of EPA and DHA in different psychiatric disorders. N-3 PUFAs have extraordinarily complex and diverse effects ranging from modifications of the biophysical properties of neuronal membranes that lead to alterations in receptor activity and signal transduction to the production of potent eicosanoids that control immune-inflammatory responses and blood flow as well as the regulation of gene expression. EPA and DHA are generally used in the treatment of one specific, more or less well delineated psychiatric condition. In the present study, changes in two psychological dimensions were assessed simultaneously and an attempt was made to determine the relative contribution of EPA and DHA to these changes. Increases in EPA (but not in DHA) were found to be robustly correlated with end-of-trial low anxiety scores whereas increases in DHA were more robustly correlated with low anger scores. It would be tempting to speculate that the modes of action of LC n-3 PUFAs could be different for anger and anxiety. The stress and anxiety-like behaviors and the stimulation of corticosterone induced in rats by interleukin-1beta were found to be significantly reversed by the administration of EPA (Song et al., 2004). On the other hand, modifications of serotonin (5-HT) neurotransmission have been implicated in violence and increasing DHA consumption may increase brain 5-HT neurotransmission as indicated by a study showing that higher concentrations of plasma DHA predicted higher levels of cerebrospinal fluid 5-HIAA (5-Hydroxyindole Acetic Acid) in healthy controls and late onset alcoholics (Hibbeln et al., 1998). It can be pointed out, however, that a small number of subjects took part in the present study. The correlation between EPA increases and anger that was not significant could have become significant if a larger number of subjects had been included, raising the possibility that DHA and EPA could act in synergy on the modulation of hostility.
The present data need to be confirmed in larger samples. Studies are needed to determine the effectiveness of EPA and DHA administered separately and in combination in different psychiatric conditions. The most effective doses of these two PUFAs need to be determined. These doses might depend on the pre-trial plasma levels of n-3 PUFAs as it is possible that non-deficient individuals might be non-responders. Ideally, n-3 PUFAs should be measured prior to the start of therapeutic interventions. Doses of EPA and DHA will also depend on the pre-trial levels of PUFAs of the n-6 series as they compete with PUFAs of the n-3 series for conversion to biologically active derivatives. Recommendations might thus vary as a function of the country of origin of individuals who are candidates for treatment. The duration of treatment needed to restore PUFAs to healthy levels and whether supplementation can be replaced by sound dietary habits should also be investigated.
This pilot study, performed in substance abusers whose dietary intake of LC n-3 PUFAs was suboptimal, showed that a 3-month EPA and DHA supplementation was accompanied by significant decreases in anger and anxiety scores. Strong correlations were found between an increase in plasma EPA and end-of-trial anxiety scores and between an increase in plasma DHA and lower end-of-trial anger scores. The differential responses to these two n-3 PUFAs should be further explored in different psychiatric conditions.
This work was supported by grant R01-DA15360 from the National Institute on Drug Abuse.
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