Similar to other chronic neurodevelopmental disorders, traditional medicine does not offer any cure for autism, and the best evidence-based treatments for this condition involve intensive early behavioral and special educational strategies (1,2). Although psychopharmacological therapies for autism have been studied for >50 years, there are presently no US Food and Drug Administration-approved indications for the treatment of the core symptoms of autism for any agent (3). As a result, complementary and alternative treatments are widely provided to children with autism by parents desperate for effective biomedical interventions for their children (4). Perhaps acting on suspicion or distrust of standard medical practices, a desire not to have their children “drugged” or the desire to seek curative treatment because of the frustration with deficiencies in traditional medical interventions, therapies based on dietary interventions appeal to parents of children with autism as more safe, natural, and holistic approaches to treating their children (5).
At present, there is considerable interest in the possibility that dietary supplementation with the long chain, n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA, 22:6n-3), will decrease the symptoms of autism. DHA is an important structural lipid of brain cell membranes, and it is available in the diet through consumption of fish. It has been estimated that approximately 55% of children with autism take psychotropic medications or dietary supplements (6,7) and that 23% of children with autism are specifically using n-3 or fish oil supplements (8), despite the lack of any evidence to support the safety and/or efficacy of this treatment. Thus, the primary objective of this study was to test the hypothesis that dietary DHA supplementation is a safe and effective treatment for reducing symptoms of autism using a randomized, double-blind, placebo-controlled design.
Children from 3 to 10 years of age with autism were recruited and randomized in a double-blind manner to a placebo-controlled trial of dietary DHA supplementation. The primary outcome measure was a significant positive response on the Clinical Global Impressions-Improvement (CGI-I) scale (9) at the completion of the study. Secondary outcome measures were changes in behavior or development noted on the Aberrant Behavior Checklist (ABC) (10), the Behavior Assessment Scale for Children (BASC) (11), and the Child Development Inventory (CDI) (12) completed at the baseline and at the study completion. The patterns for plasma phospholipid fatty acid were drawn on all of the subjects at study entry and conclusion. This study was approved by the Mayo Clinic institutional review board, and written informed consent was obtained from a parent or guardian before enrollment of any child.
Subjects were recruited through the placement of recruitment flyers across the Mayo Clinic campus at Rochester, MN. These flyers were also sent to local and regional autism support groups to be distributed to members and at meetings, workshops, and conferences. The parents and/or guardians of children with autism who responded as possible study volunteers were first screened by the study coordinator by telephone to determine whether their children were between 3 and 10 years of age and had been diagnosed to have autistic disorder. The subjects were excluded if they had a diagnosis of pervasive developmental disorder—not otherwise specified or Asperger disorder. The subjects were also excluded if they had used a dietary supplement containing DHA within 90 days of study inclusion or had a medical history of a disorder of lipid metabolism.
Consistent with practice guidelines for the diagnosis of autism, which report the “criterion standard” for an autism diagnosis to be the clinical judgment of an experienced clinician (13), children who met these inclusion and exclusion requirements were scheduled for a confirmatory medical diagnostic evaluation with a single developmental-behavioral pediatrician to confirm that each subject met the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria for autistic disorder (14). The Childhood Autism Rating Scale (CARS) (15), a standardized quantitative measure of autism symptomatology, was also administered as part of this confirmatory consultation, and only subjects who scored in the autistic range on the CARS (scores ≥ 30) were included. Thus, only those subjects who both met DSM-IV criteria for autistic disorder on confirmatory medical diagnostic assessment and scored in the autistic range on the CARS were offered enrollment in the study.
All of the participants, families, and study personnel (including those performing baseline and outcome assessments) were blinded to group assignment for the entire study. All of the subjects were randomized in a double-blind fashion to receive either a triglyceride oil capsule containing 200 mg DHA from algal oil with high oleic acid sunflower oil to make 500 mg total oil or a placebo capsule containing 250 mg of corn oil and 250 mg of soybean oil, daily for 6 months. The oils were orange flavored, and the capsules were identical in appearance. All study capsules were provided by Martek Biosciences Corporation (Columbia, MD).
The supplements were dispensed by the Mayo Clinic Investigational Pharmacy according to a randomization scheme stratified by sex and generated using a block approach by the study statistician. The number of capsules dispensed was always somewhat more than needed before the next appointment, and subjects were instructed to return unused capsules at that time. Comparison of the number of capsules returned to the number that should have been returned was used to monitor compliance, which was excellent.
The choice of DHA dose involved both biochemical and practical considerations. The dose selected (200 mg/day) has been shown to significantly increase erythrocyte and plasma phospholipid DHA levels (16). A higher dose was considered but administration of even 1 study capsule per day is often problematic in this population.
Our placebo capsules were designed to be identical in appearance to the study capsules. They needed to contain oil, similar to the DHA-containing capsules, but to serve as an appropriate control they could not contain longer-chain ω-3 fatty acids. Corn and soybean oil were used in the placebo capsules because they have a similar texture and are composed of fatty acids, but not longer-chain ω-3 fatty acids. This allowed us to determine the specific effects of the long-chain ω-3 fatty acid, DHA, compared to non–ω-3 fatty acids. Soybean oil contains some α-linolenic acid (∼5%–14% of total fatty acids), which is an 18-carbon ω-3 fatty acid that is a precursor of DHA; however, the contribution of this in terms of affecting DHA status would be negligible. Corn oil contains virtually no α-linolenic acid (∼1% of total fatty acids). Longer-chain ω-3 fatty acids have been generally accepted as essential for neurological membrane functioning. The corn and soybean oil in the placebo capsules did not contain any of these neurologically critical longer-chain ω-3 fatty acids, and there have never been any reports of corn or soybean oils improving symptoms of children with autism.
The study consisted of a baseline confirmatory diagnostic medical evaluation, along with follow-up medical evaluations after 3 and 6 months of supplementation with a single developmental-behavioral pediatrician. Blood samples were obtained via venipuncture from the children at the baseline and after 6 months. The parent or guardian completed a demographic questionnaire at the baseline visit. Behavioral, developmental, and adverse effect questionnaires were completed by the parent/guardian and/or investigator at each visit as outlined below.
Plasma Phospholipid Fatty Acid Patterns
Blood samples (5 mL) were obtained at the baseline and after 6 months of supplementation to determine plasma phospholipid fatty acid patterns. All analyses were performed by the Mayo Clinic Biochemical Genetics Laboratory using an established assay (17). In brief, a 2-step, acid-base hydrolysis was followed by hexane extraction and derivatization with pentafluorobenzyl bromide. Separation and detection were accomplished by capillary gas chromatography electron-capture negative ion-mass spectrometry. Quantitation was based on analysis in the selected ion-monitoring mode by using 13 stable isotope-labeled internal standards. In this study, the baseline plasma phospholipid levels in children with autistic disorder were compared to pediatric reference ranges established at the Mayo Clinic to detect nutritional deficiencies and metabolic disorders involving C8-C26 fatty acids (17).
Behavioral and Developmental Outcome Measures
The parents and/or guardians of all of the children enrolled in this study completed specific questionnaires at the baseline and at 3 and 6 months after supplementation. The behavioral and developmental outcome measures selected for this study were based on consensus recommendations in the medical literature (3), and prior published studies investigating effects of alternative medicine (18) and psychopharmacological (19) interventions in children with autism. The primary aim in selecting these measures was to find measures that have been shown to be most sensitive to behavioral changes as a result of short-term biomedical interventions. All of the outcome measures selected for this study are valid and reliable measures of the childhood behavior and development. Prior studies have indicated that global measures of intelligence, adaptive behavior, language, and autistic symptomatology, which have been designed for diagnostic purposes, are not sensitive to assessing effects of biomedical therapeutic interventions (3). Regardless of the objective of the study involving children with autism, the CGI-I scale is considered to be the single measure that should be used universally in all of the clinical trials of children with autism (3). Thus, changes in the core symptoms of autism (social interaction, communication, repetitive/stereotyped behavior) were assessed by CGI-I scores derived from both parents and investigator ratings after 3 and 6 months of supplementation. The CGI-I score scale consists of a 7-point Likert scoring system with scores ranging from 1 (very much improved) to 7 (very much worse). Significant positive responses were defined as either a 1 (very much improved) or a 2 (much improved).
Several secondary outcome measures of the behavior and development were included in this study to provide data on a wide range of developmental and behavioral outcome incident to DHA supplementation. The ABC (10) was completed by parents at the baseline and after 6 months of supplementation. The ABC is a 58-item parent-completed rating scale that was developed to measure the effects of pharmacological interventions in children with developmental disabilities, and it is a recommended outcome measure in clinical trials of children with autism (3,18). The ABC consists of 5 subscales: irritability, lethargy, stereotypy, hyperactivity, and inappropriate speech. The CDI (12) was completed by parents and the BASC (11) was completed by both parents and teachers at the baseline and after 6 months of supplementation. The CDI consists of 9 subscales and is a 300-item “yes-no” questionnaire that offers a valid, reliable, and easily administered assessment of a child's overall general development, and their development in social, self-help, fine motor, gross motor, and expressive and receptive language development. The BASC includes both parent- and teacher-rating scales (18 and 20 subscales, respectively) that provide reliable and valid measures of a broad sample of behaviors, including attention span, hyperactivity, social skills, adaptability, functional communication, and atypical behaviors (11).
Adverse Effect Assessment
The parents and/or guardians were instructed to contact the study coordinator at any time throughout the study with concerns regarding possible adverse effects. To monitor for safety of the supplementation, the Treatment Emergent Symptoms Scale (TESS) (20) was completed via parent interview after 3 and 6 months of supplementation. The TESS is a standardized rating scale that measures the occurrence or nonoccurrence of 24 potential adverse effects of medication in 6 categories (gastrointestinal, urinary, respiratory, skin, neurological, and psychological). Each adverse effect is scored on a 3-point Likert scale, with 2 indicating severe, 1 mild, and 0 absent adverse effects.
The data were summarized using standard descriptive statistics. The baseline demographic and clinical characteristics were compared between the 2 treatment groups, using the Fisher exact test for categorical variables, the 2-sample t test for age, and the Wilcoxon rank sum test for parental education and family income categories. The questionnaire responses obtained during the follow-up visits at 3 and 6 months were compared between the 2 treatment groups based on the participating subjects. The proportions with a “significant positive response” on each of the CGI-I scales were compared between the 2 groups using the Fisher exact test. In addition, the full 7-point CGI-I ratings were compared between the 2 groups using the Wilcoxon rank sum test. The CDI, ABC, and BASC scores at 6 months of treatment were compared between the 2 groups based on fitting separate analysis of covariance models to adjust for the corresponding baseline scores. All calculated P values were 2-sided and P < 0.05 were considered statistically significant. All analyses were performed using the SAS version 9.1 software package (SAS Institute, Cary, NC).
In adherence with the intent-to-treat principle, an additional analysis was performed for the primary outcome variable, the overall CGI-I scale at 6 months. In this analysis, all of the subjects were analyzed according to their randomized treatment assignment, regardless of their level of compliance, and subjects who dropped out before the end of the study were classified as not having a significant positive response.
The primary outcome variable for this study was defined as a significant positive response on the 7-point CGI-I scale. A significant positive response was defined as either a 1 (very much improved) or 2 (much improved) on at least 1 of the subcomponents of this scale. Using a power of 0.8, a significance level of 0.05, and a 2-sided χ2 test, the study was designed to enroll 32 subjects in each group to have sufficient statistical power to detect a 25% difference in the proportion of subjects, with a positive CGI-I response in the DHA-supplemented group relative to the placebo group.
The CONSORT (21,22) flow diagram for this study is shown in Figure 1. A total of 48 children with autism (40 [83%] male, mean age [standard deviation] 6.1 [2.0] years) were enrolled; 24 received DHA and 24 placebo (Fig. 2). There were no differences between the groups in demographic factors (age, sex, race, parental education, family income) or baseline total plasma phospholipid or n-3 fatty acid levels (Table 1). A group of 34 children completed the study (19 in DHA group, 15 in placebo group).
Plasma Total Fatty Acids
Table 2 shows the total plasma n-3 fatty acid levels in the 48 children with autistic disorder at the baseline. Only 3 (6%) children were found to have low DHA levels and 1 (2%) had a low eicosapentaenoic acid (20:5n-3) level, but none had low α-linolenic acid (ALA; 18:3n-3), docosapentaenoic acid (DPA; 22:5n-3), or total n-3 fatty acid levels. In addition, 10 children (21%) were found to have high DHA levels, 11 (23%) high ALA levels, 4 (8%) high eicosapentaenoic acid levels, and 7 (15%) high total n-3 fatty acid levels. The mean values for each of these n-3 fatty acids were within the normal reference ranges. Figure 2 shows the mean total plasma n-3 fatty acid levels after 6 months of treatment. All of the children in the DHA group evidenced a significant increase in their plasma phospholipid DHA levels from the baseline to study conclusion, confirming their compliance with taking the DHA supplements. The DHA group showed a median 431% increase in total plasma DHA levels after 6 months of supplementation.
Core Symptoms of Autism
Table 3 shows the number and percentage of children with a significant positive response from parent and investigator CGI-I ratings after 3 and 6 months of treatment. We found no statistically significant differences in the percentage with a positive response between the groups in either parental or investigator CGI-I ratings after 3 or 6 months of treatment; however, when the ratings were analyzed using the full 7-point scale, the DHA-supplemented group received slightly worse investigator ratings of repetitive/stereotypic behaviors at 6 months compared to the placebo group (P = 0.02, as per the Wilcoxon rank sum test; percentage scored as 3, 4, and 5 are 0%, 83%, and 17% in the DHA group and 33%, 60%, and 7% in the placebo group, respectively).
In addition, the overall CGI-I rating scale at 6 months was analyzed based on considering all of the 48 randomized subjects. In this analysis, subjects who dropped out before the end of the study were classified as not having a significant positive response. Among the 24 patients randomized to receive DHA, 5 (21%) subjects had a significant positive response based on the parent ratings for the overall CGI-I rating scale at 6 months, compared to 2 (8%) subjects randomized to receive placebo (95% exact confidence interval for difference in proportions −17.6 to 41.0). Based on the investigator ratings at 6 months, none of the subjects had a significant positive response, compared to just 1 (4%) subject randomized to receive placebo (95% exact confidence interval for difference in proportions −33.4 to 25.6).
The CDI, ABC, and BASC scores at 6 months of treatment were compared between the 2 treatment groups, after accounting for the baseline scores. Among the 52 subscales that were evaluated, only 2 statistically significant differences were found (Table 4). On average, parents (but not teachers) provided a more favorable average change in their rating of social skills on the BASC for the children in the placebo group compared to those in the DHA group (mean change, 3.0 vs −0.2). In addition, teachers (but not parents) provided a more favorable average change in their rating of functional communication on the BASC for the children in the DHA group compared to those in the placebo group (mean change, 1.5 vs −4.5); however, after adjusting for the multiple comparisons using a Bonferroni correction, none of these comparisons reached statistical significance with a P < 0.01.
Adverse Effect Assessment
The parents or the guardians of children in the placebo group did not report any severe level of adverse effects on the TESS. One parent of a child in the DHA group reported a severe level of headaches and 1 a severe level of restlessness at 3 months but not at 6 months, and 1 parent of a child in the DHA group reported a severe level of agitation at 6 months (but not at 3 months). Overall, there was no significant difference between groups in treatment-emergent adverse effects.
Autism is a neurodevelopmental disability characterized by clinically significant impairments in social interaction and communication, in association with atypical repetitive, stereotypic, and ritualistic behaviors (14). The prevalence of autism appears to be increasing, likely in large part the result of increased awareness and improved identification and the availability of federally mandated early intervention services (23). In a study, we found the incidence of autism to be 2.9/1000 in our geographic region (Olmsted County, MN) (23). In 2012, the Autism and Developmental Disabilities Monitoring Network of the Centers for Disease Control and Prevention reported the prevalence of autism to be 1 in every 88 children (24). The prevalence of autism is greater in the pediatric population than that of diabetes, congenital heart disease, cystic fibrosis, inflammatory bowel disease, and chronic renal disease combined, making autism a major public health concern (25).
The epidemiological data suggest that the population with lower dietary n-3 fatty acid consumption and, hence, lower plasma and, presumably, brain contents of n-3 fatty acids, including DHA, has higher rates of psychiatric disorders, including depression and bipolar disorder (26,27). In addition, children with attention-deficit/hyperactivity disorder (ADHD) (28) and adults with schizophrenia (29) have been shown to have lower plasma phospholipid DHA contents than controls. DHA is the predominant long-chain polyunsaturated fatty acid (LC-PUFA) present in the structural lipids of brain cell membranes, especially those at the synaptic terminals (30–33). DHA and other long-chain polyunsaturated fatty acid may influence synaptic function directly through effects on membrane structure, and indirectly through the generation of eicosanoids/docosanoids (prostaglandins, leukotrienes, thromboxanes, resolvins, neuroprotectins) or through immune system/cytokine interactions (34,35). Thus, there has been considerable interest in the role of DHA in the etiology and treatment of a variety of psychiatric and neurodevelopmental disorders, including depression (36), schizophrenia (37), aggression (38), bipolar disorder (39), Alzheimer disease (40–42), dyslexia (43), ADHD (44–46), and developmental coordination disorder (43,47).
Children with autism tend to be extremely picky eaters as a result of their ritualistic tendencies (48), and this may result in a lack of appropriate nutrients, including DHA intake. Based on neuroanatomical and functional MRI data, it appears that the symptoms of autism may be mediated by the medial-temporal lobe (hippocampus and amygdala) and the cerebellum (49,50). Interestingly, animal studies have shown that the hippocampus and cerebellum are the most vulnerable brain structures to effects of malnutrition (51,52). Permanent alterations are often seen in these structures, whereas other brain areas tend to recover. If this is also true in humans, it can be hypothesized that the picky eating of children with autism may result in a lack of appropriate nutrient intake, including DHA. As DHA is a critical component of brain cell membranes, a lack of dietary DHA, and hence, low levels of DHA in the plasma and brain, would be expected to affect the cognitive processes mediated by these most nutritionally sensitive brain areas and result in autistic symptomatology. Two of 3 prior studies have reported lower levels of ω-3 fatty acids in children with autism compared to controls (53–55). We, however, did not find children with autism to have consistently low plasma DHA or other n-3 fatty acid levels, as 94% of our subjects had normal or high DHA levels compared to the reference range determined by our laboratory.
In addition to any long-term effects owing to altering the structural membranes of neurons, DHA supplementation may act in a more short-term pharmacological manner. A direct relation between plasma phospholipid DHA content and metabolism of serotonin and dopamine within the central nervous system has been reported (56,57). In addition, limbic areas of the medial-temporal lobe (amygdala and hippocampus), critical for emotional expression and social behavior, are richly innervated with serotonergic neurons, and while serotonergic innervation of the cerebellum is less prominent, serotonin projections are also critical to cerebellar function (58); however, although we found that dietary DHA supplementation was safe (few treatment-emergent adverse effects reported), despite a median increase in total plasma DHA levels of 431%, we did not find that DHA supplementation improved the core symptoms of autism based on either parental or investigator ratings. We also found that DHA supplementation did not generally affect the development or behavior of children with autism based on both parental and teacher ratings. Teachers (but not parents) reported an improved functional communication for children in the DHA group compared to those in the placebo group; however, after adjusting for multiple comparisons, this improvement no longer reached statistical significance.
Our findings are potentially limited by our small sample size. Although the study was designed to enroll 32 patients per group, we were only able to enroll 24 subjects per group and retain 15 and 19 subjects per group at 6 months. Thus, our study had 80% power to detect a difference between group means of 1 standard deviation between the DHA-supplemented and placebo groups after 6 months. Therefore, our study could not determine whether DHA supplementation may produce more subtle developmental or behavioral improvements in children with autism; however, although this study may not detect smaller differences, improvements of a magnitude less than that designed to be detected in this study may not be clinically meaningful for children with autism. It is also possible that supplementation with a higher dose of DHA for a longer time period or that supplementation with a combination of n-3 fatty acids may produce different results. Numerous prior studies have, however, found DHA supplementation for as little as 2 to 4 months to result in clinically significant improvements in the neurobehavioral symptoms of ADHD (46,47), bipolar disorder (38), and developmental coordination disorder (46), and thus our 6-month study duration should have been long enough to capture similar neurobehavioral changes in children with autism.
Another limitation of our study is that although we found a median increase in total plasma DHA levels of 431% in our DHA supplemented group, it is unknown whether plasma DHA levels reflect levels in neuronal membranes in children, as results from the required postmortem studies have not been reported. In 1 study, the correlation coefficient between brain cortex DHA and erythrocyte DHA in infants who underwent postmortem examinations because of sudden death was 0.33 (59). It is, however, unclear to what degree these results would apply to our study population, and extrapolation from results of animal studies is problematic.
Despite concerns about nutritional deficiency derivative of their picky eating, we did not find children with autism to be deficient in DHA or other n-3 fatty acids at the baseline. In addition, although dietary DHA supplementation is a widely popular treatment for children with autism, we did not find this intervention to improve the core symptoms of autism or a broad range of associated developmental and behavioral difficulties in children with autism, although children who received the supplementation experienced a 431% median increase in their plasma total fatty acid DHA levels; however, although none of our comparisons reached statistical significance after adjusting for multiple comparisons, our finding of a favorable change in functional communication reported by teachers in children with autism who received DHA supplementation may be confirmed by further investigation in a larger randomized, placebo-controlled clinical trial.
We thank our study coordinator Candice Klein and all of the children and families who volunteered to participate in this study. We also thank Martek Biosciences Corporation (Columbia, MD) for providing the DHA supplements and placebos used in this study.
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