Journal of Autism and Developmental Disorders (2022) 52:5342–5355

https://doi.org/10.1007/s10803-021-05396-9

ORIGINAL PAPER

Randomized Controlled Trial of Omega‑3 and ‑6 Fatty Acid

Supplementation to Reduce Inflammatory Markers in Children
with Autism Spectrum Disorder
Sarah A. Keim1,2,3 · Abigail Jude1 · Katie Smith1 · Aiman Q. Khan1 · Daniel L. Coury1,2 · Joseph Rausch1,2 ·
Shivika Udaipuria1 · Megan Norris1,2,4 · Lindsay R. Bartram1,2 · Anita R. Narayanan1,2 · Lynette K. Rogers1,2

Accepted: 3 December 2021 / Published online: 11 January 2022

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
This double-blind, randomized controlled trial, tested fatty acid (FA) supplementation in children (ages 2- < 6 years) recently
diagnosed with Autism Spectrum Disorder (ASD). Participants received daily oral FA supplement containing omega-3 and
omega-6 FA, or a placebo for 90 days based on participant weight. Erythrocyte FAs and the cytokines, IL-1β, IL-2, IFNγ,
were measured in plasma obtained from serial blood collections. Treatment increased omega-3 and omega-6 FA levels
(1.40 mol% for EPA and 1.62 mol% for DHA) and reduced IL-2 levels compared to placebo (− 0.17 pg/mL, 95% CI − 0.31,
− 0.02, d = − 0.62). Omega 3–6 treatment was tolerable and adherence was greater than 70%. Future research will assess
the effects of Omega 3–6 treatment on ASD symptoms. Registered on 06/08/2018 with ClinicalTrials.gov: NCT03550209.

Keywords Autism spectrum disorder · IL-2 · Inflammation · Omega-3 fatty acids · Omega-6 fatty acids · Young child

Introduction 52–95% of families commonly try complementary therapies

including omega fatty acid (FA) dietary supplements, based
Although at least 1 in 54 U.S. children has Autism Spectrum largely on anecdotal evidence and small trials (Whitehouse,
Disorder (ASD), no approved medications exist to treat the 2013).
core symptoms (Maenner et al., 2020). Atypical antipsychot- FA supplements are among the most popular complemen-
ics are commonly prescribed for irritability with ASD, but tary health interventions utilized by children with ASD. To
side effects are significant, common, and of particular con- date, the results of efficacy trials testing FA supplementation
cern for young children (McPheeters et al., 2011). Behav- for ASD are mixed for reasons including small sample size,
ioral interventions remain the most effective approaches for variation in the formulations tested, and design shortcom-
addressing atypical behaviors and social deficits of ASD, but ings including lack of blinding or placebo control. Prior trials
they are intensive, costly, and inaccessible for some fami- using FA therapies have almost exclusively tested a combina-
lies (Dawson et al., 2010; Grosse et al., 2021; Reichow and tion of omega-3 (n − 3) docosahexaenoic acid (DHA) and
Wolery, 2009). Because of the dearth of effective treatments, eicosapentaenoic acid (EPA) derived from fish oil. However,
the addition of alternative anti-inflammatory FAs such as
omega-6 (n − 6) gamma-linoleic acid (GLA), may enhance
* Sarah A. Keim the anti-inflammatory effects of supplementation and may also
[email protected]
improve behavioral outcomes. In fact, a recent meta-analysis
1
Nationwide Children’s Hospital, 700 Children’s Dr, reported larger effect sizes and more consistent improve-
Columbus, OH 43205, USA ment in Attention Deficit-Hyperactivity Disorder (ADHD)
2
Department of Pediatrics, College of Medicine, The Ohio for DHA + EPA + GLA than for DHA + EPA alone (standard
State University, Columbus, OH, USA difference in means -0.31 favoring DHA + EPA + GLA (CI
3
Division of Epidemiology, College of Public Health, The − 0.46, -0.6)) (Puri and Martins 2014). The Preemie Tots trial
Ohio State University, Columbus, OH, USA recently reported improvements in ASD symptoms based on
4
Department of Psychology, College of Arts and Sciences, the Brief Infant Toddler Social and Emotional Assessment
The Ohio State University, Columbus, OH, USA (BITSEA; (Briggs-Gowan et al., 2004) ASD scale, gesture

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Journal of Autism and Developmental Disorders (2022) 52:5342–5355 5343

use in communication, internalizing behaviors, and interper- and approved by the Institutional Review Board (IRB #17-
sonal relationship adaptive behaviors among preterm toddlers 00517) at the single study site, Nationwide Children’s Hos-
with early ASD symptoms randomized to DHA + EPA + GLA pital (NCH), Columbus, OH, USA. Each child’s parent or
versus placebo (Boone et al., 2021; Keim et al., 2018; Shep- guardian provided written informed consent. A study doctor
pard et al., 2017). Only one trial focused on ASD has tested (D.L.C.) and independent monitoring committee reviewed
a combination of DHA and EPA with evening primrose oil, a adverse events and other safety-related data. Clinicians who
source of GLA and arachidonic acid, but it was not placebo- diagnose and care for children with ASD were involved in
controlled (Ooi et al., 2015). In addition to improvements in the study design and development of study procedures.
attention problems, parent-reported outcomes in that study
indicated medium effect sized improvements related to social Participants, Sample Size, and Power
interaction and communication, similar to our prior reports.
Another major reason for the lack of clarity as to the effec- Children recently diagnosed with ASD at the Nationwide
tiveness of FA supplements is the very limited extent to which Children’s Hospital Child Development Center (Columbus,
studies have evaluated potential biological signatures of inter- OH, USA), a large interdisciplinary assessment and treat-
est to shed light on the pathways through which FAs may exert ment center for ASD, were assessed for eligibility using the
their effect. Inflammation is one potential mechanism by which EPIC electronic medical record system. DSM-5 criteria were
FA may affect autism-related behaviors. Inflammation is well applied in the diagnosis process. Inclusion criteria included
characterized in youth with ASD – increases in inflammatory age 2- < 6 years, receipt of an ASD diagnosis within the
cytokines and chemokines both in the circulation and cerebral prior 6 months with a score in the “autism” (severe) range
spinal fluid have been reported, and ASD symptom severity on the Autism Diagnostic Observation Schedule-2nd edition
has been positively associated with levels of several cytokines (ADOS-2; (Lord et al., 2012)), and English as the primary
(Masi et al., 2017; Theoharides et al., 2016). Only two of the language.We focused on preschool aged children because
prior trials testing FA supplementation examined inflamma- of extensive evidence indicating that ASD therapies are
tory markers in relation to changes in behavior, despite the maximally beneficial if they begin as early as possible after
evidence from observational studies tying altered inflamma- diagnosis (Granpeesheh et al., 2009). This is because neuro-
tory profiles to ASD, and particular long-chain FAs to anti- plasticity decreases significantly as children age, and also the
inflammatory mechanisms (Mankad et al., 2015). Mankad rate of DHA accretion in the brain displays a corresponding
et al. (2015) suggested worsened externalizing behaviors decline (Martinez, 1994). The sample was limited to chil-
with increasing levels of IL-10 and IL-1β, but no clear path dren in the severe range on the ADOS-2 to reduce heteroge-
connecting supplementation to changes in cytokines and to neity and focus on children potentially more likely to show
subsequent behavior. Similarly, Mazahery et al. (2020) tested benefit in this relatively small cohort.
interactions between baseline IL-1β levels and treatment Exclusion criteria included 1) they consumed FA supple-
assignment but did not measure changes in inflammation due ments in the past 6 months, consumed fatty fish more than
to treatment. The mechanisms by which FAs might improve 3 times per week, or were still breast or formula feeding, as
ASD symptoms is not well understood and led us to hypoth- these would potentially provide large amounts of the same
esize that the Omega 3–6 supplementation would interrupt FA being tested; 2) had been diagnosed with quadriparesis,
detrimental neurological pathways by reducing inflammation, deafness, a seizure disorder, blindness, a bleeding disorder,
a hallmark of ASD. or an autoimmune disorder (including Type I diabetes, Frag-
The first phase of the Omega Heroes study was to evaluate ile X, Rett, Angelman Syndromes, or Tuberous Sclerosis),
the efficacy of daily supplementation with a FA therapy that as these would have effects on both inflammation and neu-
included a combination of DHA + EPA + GLA versus placebo, rodevelopment, would theoretically pose safety concerns,
using doses based upon the child’s weight, on inflammatory or would mean children could not benefit from the interven-
responses in young children with ASD. It also aimed to evalu- tion; 3) had feeding problems precluding consumption of
ate safety and bioavailability of the FA supplement. the supplement or had a known allergy to any ingredient of
the investigational products as these would preclude tak-
ing the investigational products; or 4) had planned surger-
Methods ies scheduled within the timeframe of trial participation, as
these would potentially pose a bleeding risk.
Study Design and Setting A parent or legal guardian of potentially eligible chil-
dren was contacted via letter, phone, or email to confirm
Omega Heroes was a randomized, fully blind (research- eligibility and discuss possible participation. Interested
ers, parents, and children were blind to treatment assign- and eligible parents and children were scheduled for the
ment), placebo-controlled trial (NCT 03550209) reviewed baseline visit at the Center for Biobehavioral Health at

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5344 Journal of Autism and Developmental Disorders (2022) 52:5342–5355

Nationwide Children’s Hospital. A sample size goal of 18:2n − 6), 81 mg α-linolenic acid (ALA, 18:3n − 3),
66 (30 per group plus 10% attrition) was set based on the 590 mg oleic acid (18:1n − 9) per milliliter of placebo).
hypothesized effect size of at least Cohen’s d = 0.75 for The placebo was prepared with lemon oil flavor by the NCH
the change in the cytokines of interest from baseline to the Investigational Drug Service (IDS) to match the treatment
end of the trial, when comparing all children randomized product, and both products were packaged identically. Doses
to DHA + EPA + GLA to all children randomized to pla- were based on the child’s baseline weight and calculated to
cebo. The effect size was based on the differences observed deliver 50, 100, or 150 mg/kg/day in the proportions defined
by Masi et al. (2015) and preliminary data from our prior, by the manufacturer. Ninety days was selected because of
unpublished, trial (d = 0.75 for IL-1β, 0.77 IL-2, and 1.04 numerous previous trials of fatty acid supplements that
IFNγ). Additionally, we set a benchmark for what we consid- reported improvements in other behaviors within this time-
ered a potentially clinically meaningful effect size of d = 0.5 frame (Puri and Martins, 2014). IDS dispensed the products
to inform decisions about proceeding to a future trial exam- directly to families and provided a medicine syringe for par-
ining effects on behavior. ents to measure and administer the product to the child by
placing the liquid directly into the mouth. To promote com-
Randomization, Masking, and Intervention pliance, some parents added chocolate syrup to the measured
dose or mixed with food.
Children were allocated to one of six coded groups. Each
child was assigned to 1 of 3 different doses of treatment Data Collection
(25 “low”, 50 “medium”, or 100 “high” mg/kg/day of
GLA + EPA + DHA) or 1 of 3 doses of placebo (volumes The baseline study visit (day 0) included a parent question-
were calculated to be equivalent to the corresponding treat- naire to gather baseline child and family descriptions to char-
ment group low, medium, and high volumes) calculated acterize the sample and verify comparability across groups.
using baseline body weight. FA supplements have tradition- In addition, parents completed a FA-specific food frequency
ally been tested using a uniform dose, but recent evidence questionnaire as an interview to measure usual dietary intake
has highlighted the deficiency of this approach. Heavier of long-chain fatty acids (Kuratko, 2013). Finally, parents
children seem to require a higher dose to achieve the same were asked to report on their child’s current medications,
response in a recent study of omega-3 supplementation and upcoming surgeries, as well as use of complementary and
mood disorders (Arnold et al., 2017). The randomization behavioral therapies or programs. Child height and weight
scheme had varying block size of multiples of 6 and 12, were measured using a SECA scale and stadiometer. Par-
stratified by sex and age (2–3 years, 4- < 6 years), and with ents were asked about their intentions to complete the study,
equal allocation to treatment and placebo (Fig. 1). A pseu- attend the next study visit, and give their child the assigned
dorandom number generator in the statistical software R investigational product each day. They were counseled
was used to implement randomization. A statistician (J.R.) about how to give the investigational product and how to
prepared the scheme, assigned ID numbers, and prepared use study diaries to record compliance. Families were con-
opaque tamper-resistant envelopes. He had no partici- tacted by phone, email, or text message at regular intervals
pant contact. All investigators and staff remained masked at least 5 times to measure adherence, assess adverse events
throughout the trial. Children were assigned to either the using a modified version of the Safety Monitoring Uniform
treatment or placebo group using sealed, sequentially num- Report Form (SMURF), and address barriers to participation
bered envelopes. Each sequential envelope was pre-labeled (Greenhill et al., 2004). Group differences (entire treatment
with the next study ID number to prevent out-of-sequence group vs placebo group) in the total number and types of
assignment and was opened by a research assistant upon adverse events served as a prespecified primary endpoint for
written informed consent. safety for the trial. Study visits occurred at 45 ± 14 days and
The treatment group was assigned to 90 days of daily oral 90 ± 14 days to repeat the parent questionnaire/interview,
Complete Omega™ supplementation in the form of lemon measure adherence, assess adverse events and barriers to
oil flavored fish and borage oils (the supplement provided participation. At the last study visit, the parent/guardian was
185 mg total omega-3 fatty acids including 112 mg EPA, asked to guess their child’s treatment assignment. Families
67 mg DHA; 122 mg total omega-6 fatty acids including were compensated $75 for each visit, $2 for each 2-week
32 mg GLA; and 83 mg total omega-9 fatty acids per mil- study diary they submitted, plus parking and a small toy, and
liliter of supplement) (Complete Omega, Nordic Naturals, a $20 bonus if they completed all visits.
Inc., Watsonville, CA). Complete Omega is considered a
dietary supplement under U.S. Food and Drug Administra-
tion regulations. The placebo group was assigned to daily
canola oil (the placebo provided 188 mg linoleic acid (LA,

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Journal of Autism and Developmental Disorders (2022) 52:5342–5355 5345

Fig. 1  CONSORT diagram

Fatty Acid Biomarkers cell (RBC) FA levels at baseline and at the last study visit.
Plasma and RBCs were separated by centrifugation at the
Blood was collected via venipuncture to examine red blood time of collection and stored at -80 °C until analysis. FA

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5346 Journal of Autism and Developmental Disorders (2022) 52:5342–5355

were measured in patient RBCs by standard protocols using our primary outcomes, we combined all doses to form one
gas chromatography (Eder, 1995). Analysis was performed treatment group and one placebo group. No interim analyses
on a Shimadzu GC-2010 gas chromatograph equipped with were conducted. Sample characteristics at baseline for the
an AOC-20i autosampler and with flame-ionization detec- treatment and placebo groups are shown in Table 1. The
tion. Fatty acid methyl esters were quantified using experi- baseline characteristics in Table 1 were examined across the
mentally derived standard curves. The following FA were treatment and placebo groups as predictors of missingness at
included in the analysis: myristic (14:0), palmitic (16:0), posttest for the outcomes of interest; none were statistically
palmitoleic (16:1n − 7), steric (18:0), oleic (18:1 n − 9), significant (p > 0.05).
linoleic acid (18:2 n − 6), α-linolenic acid (18:3 n − 3), The cytokines and fatty acid biomarkers were analyzed
γ-linolenic acid (GLA,18:3 n − 6), arachidic acid (20:0), as continuous outcomes. Analyses for both parameters com-
arachidonic (AA, 20:4n − 6), eicosapentaenoic acid (EPA, pared the change in the particular measurement of interest
20:5 n − 3), and docosahexaenoic acid (DHA, 22:6 n − 3). between the treatment and placebo groups, controlling for
Differences in the bioavailability of EPA and DHA (each FA baseline scores, using mixed effects regression (based on
as a percent of total RBC FA at the end of the trial) between an approach analogous to analysis of covariance Coffman
the Omega 3–6 group and the placebo group served as a et al., 2016; Winkens et al., 2007). This method leverages
prespecified primary endpoint for the trial. maximum likelihood to account for missing data. Treatment-
by-time interaction terms were included as fixed effects and
Cytokines served as estimates of treatment effect; no participant char-
acteristics were included as covariates in the models. Group
Three specific biological signatures indicative of inflam- mean differences divided by the standard deviation of the
mation were chosen a priori based on prior studies indi- residuals from an analogous analysis of covariance model
cating increases in these markers in children with ASD were calculated as standardized effect sizes for each out-
(Masi et al., 2015). The three primary biological signa- come, i.e., an analysis of covariance version of Cohen’s d
tures included the cytokines IL-1β, IL-2, and IFNγ. The (Lai and Kelley, 2012). Accuracy in parameter estimation
change in these cytokines from baseline to the end of the for ANCOVA and ANOVA contrasts: Sample size planning
trial (entire treatment group vs placebo group) served as a via narrow confidence intervals (Lai and Kelley, 2012). A
prespecified primary endpoint for the trial. Six additional clinically meaningful effect size was pre-defined as Cohen’s
cytokines were selected as exploratory after the trial began d ≥ 0.5 or equivalent. P-values < 0.05 were considered sta-
but before assays were conducted in order to provide a more tistically significant. Analyses for the fatty acid biomarkers
complete understanding of possible changes in inflamma- compared the posttest for the particular fatty acid biomarker
tory profiles resulting from the Omega 3–6 supplementa- between the treatment and placebo groups, controlling for
tion. These included TNFα, IL-4, IL-6, IL-8, IL-10, and baseline scores, using mixed effects regression (based on
IL-12p70. These cytokines were measured using an ELISA an approach analogous to analysis of covariance; Coffman
based technology offered by Meso Scale Diagnostics (Meso et al., 2016; Winkens et al., 2007)). Finally, we carried out
Scale Diagnostics, Rockville Maryland). pre-specified exploratory analyses to examine potential
dose effects using the same modeling approach as above to
examine change in the cytokines between the treatment and
Compliance placebo groups which had the same dose.

Compliance was assessed by parent-completed daily diaries

(the proportion of days the supplement/placebo was con- Results
sumed divided by the total days enrolled) and measurement
of the amount of remaining oil in bottles returned to IDS Recruitment and Retention
(the amount of missing oil divided by the expected amount).
Based on ASD clinical diagnostic information in the medical
Statistical Analysis records, 1,325 children were prescreened for eligibility. Car-
egivers of children who were found to be potentially eligi-
All analyses used SAS software (v9.4, SAS Institute) and ble (n = 481) were mailed recruitment materials, and phone
were conducted according to intent-to-treat (all randomized contact was attempted to complete the remaining eligibility
children were included). The study was designed as an early screening and to invite enrollment if found to be eligible.
phase trial to identify differences in inflammatory markers One hundred forty-two refused to participate. An additional
and assess safety and compliance; consequently, it was not 267 were either unreachable (nonresponsive or unable to
powered to identify differences across varying doses. For locate), determined to be ineligible after speaking with the

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Journal of Autism and Developmental Disorders (2022) 52:5342–5355 5347

Table 1  Participant baseline characteristics, Omega Heroes Trial (Ohio, USA) (n = 70)
Characteristic Total (n, %) Omega 3–6 (n, %) Placebo (n, %)
(n = 70) (n = 37) (n = 33)

Child sex
Male 57 (81) 29 (78) 28 (85)
Female 13 (19) 8 (22) 5 (15)
Child age
2–3 years 36 (51) 21 (57) 15 (45)
4–6 years 34 (49) 16 (43) 18 (54)
Child race
Black or African-American 13 (19) 10 (27) 3 (9)
White 47 (67) 20 (54) 27 (82)
Other or multiple races 9 (12) 6 (17) 3 (9)
Missing 1 (1) 1 (3) 0 (0)
Child ethnicity
Hispanic 6 (9) 4 (11) 2 (6)
Non-Hispanic 64 (91) 33 (89) 31(94)
Caregiver marital status
Single/never married 12 (17) 4 (11) 8 (24)
Married or living with partner 46 (66) 27 (73) 19 (58)
Partner not living together, separate, divorced, widowed 11 (16) 6 (17) 5 (15)
Missing 1 (1) 0 (0) 1 (3)
Household income
< $833 per month ($10,000 per year) 7 (10) 1 (3) 6 (18)
$833–$1666 per month ($10,000–$19,999 per year) 11 (16) 6 (16) 5 (15)
$1667–$2499 per month ($20,000–$29,999 per year) 10 (14) 5 (14) 5(15)
$2500–$3332 per month ($30,000–$39,999 per year) 11 (16) 9 (24) 2 (6)
$3333–$4166 per month ($40,000–$49,999 per year) 12 (17) 7 (19) 5 (15)
$4167–$4999 per month ($50,000–$59,999 per year 3 (4) 2 (5) 1 (3)
$5000–$6666 per month ($60,000–$79,999 per year) 6 (9) 2 (5) 4 (12)
$6667–$7499 per month ($80,000–$89,999 per year) 2 (3) 2 (5) 0 (0)
$7500–$8332 per month ($90,000–$99,999 per year 2 (3) 0 (0) 2 (6)
> $8333 per month ($100,000 per year) 5 (7) 2 (5) 3 (9)
Missing 1 (1) 1 (3) 0 (0)
Caregiver employment
Employed 44 (63) 24 (65) 20 (61)
Unemployed or disabled 7 (10) 5 (14) 2 (6)
Homemaker or student 19 (27) 8 (22) 11 (33)
Caregiver education
High school or less 17 (24) 9 (24) 8 (24)
Some college 27 (39) 13 (35) 14 (42)
Associate's degree 13 (19) 5 (14) 8 (24)
Bachelor's degree + 13 (19) 10 (27) 3 (9)
Caregiver age (years) Mean = 32.7 SD = 5.4 Mean = 32.5 SD = 4.7 Mean = 32.9 SD = 6.2
Health insurance status
Private only 24 (34) 13 (35) 11 (33)
Public insurance 41 (59) 23 (62) 18 (55)
Military 2 (3) 0 (0) 2 (6)
Uninsured 3 (4) 1 (3) 2 (6)

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5348 Journal of Autism and Developmental Disorders (2022) 52:5342–5355

caregiver, or were scheduled to enroll but never attended. observed in clinical settings. Roughly equal numbers of
Seventy-two children were enrolled and randomized (Omega participants were enrolled in the two age strata (2–3 years
3–6 treatment n = 37; Placebo n = 35). Two participants, who and 4- < 6 years). About two-thirds of the sample identified
were allocated to receive placebo, were immediately with- as White, 19% reported Black or African American, 12%
drawn by the investigators because they were found to be reported another or multiple races, and 9% reported His-
ineligible just after randomization. Thus, the final enrolled panic ethnicity. Most caregivers were married or living with
and randomized sample included in the analyses is 70. Six a partner, employed, and had attended at least some college.
participants withdrew themselves from the trial; reasons for A majority of participants received public health insurance,
withdrawal were not related to study design (Table 2). Two and 34% had private health insurance.
participants, who were allocated to receive Omega 3–6 treat-
ment, were lost to follow-up. Sixty-two children attended the Participant Withdrawals
last study visit. Overall, 89% of 70 who were randomized
and eligible were retained to the end of the trial and included The six families who withdrew themselves were asked about
in the final analysis (Fig. 1). Recruitment occurred between reasons for withdrawal. All stated reasons related to family
June 2018 and September 2019. All participant follow-up issues and time constraints, not the investigational product or
and data collection were completed in January 2020 due to study procedures. The reasons caregivers gave were typical
reaching pre-specified recruitment goals. of other trials we have conducted, with the possible excep-
tion that we had more families than usual who were expe-
Participant Characteristics riencing divorce or separation and resulting family conflict
that interfered with the child’s ongoing participation (e.g.,
The participant characteristics are presented in Table 1. non-resident father not willing to give the child the supple-
More males were enrolled than females. ASD is diagnosed ment when visiting) (Table 2).
in males more frequently than females, and our enroll-
ment population mimicked the sex-distribution commonly

Table 2  Reasons for Withdrawal from the Trial (n = 6) and Adherence Based on Measured Volumes and Study Diaries, Omega Heroes Trial
(Ohio, USA)
Reasons for ­Withdrawala
Category Reason n

Family related Family not supportive of child participation 2

Family wanted child to discontinue supplement 1
Personal/family problems getting in the way of involvement 3
Time related Problems with time commitment 4
Too many personal/research appointments at hospital 2
Requirements interfere with daily life 2
Scheduling concerns 1
Study design related Maintaining study diary is inconvenient 0
Concerns with blood draw 0
Preferring not to share personal information 0
Study visits too stressful for child 0
Inadequate compensation 0
Cannot choose child’s treatment group 0
Interactions with new people too stressful for child 0
Study related phone calls and mail were bothersome 0
Treatment adherence
Treatment Omega 3–6 Placebo

Low Dose Medium Dose High Dose All

Pharmacy Volume 125% 89% 65% 92% 75%
Study Diaries 77% 62% 61% 68% 77%
a
Results reflect data for 6 families, caregivers could offer more than one reason

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Journal of Autism and Developmental Disorders (2022) 52:5342–5355 5349

Adherence Fatty Acid Levels in RBCs

Based on volume dispensed and returned, children in this We observed an absolute change of 1.40 mol% for EPA and
study took 92% of the product dispensed to them. Based 1.62 mol% for DHA in the Omega 3–6 group after 90 days
on diaries, children took 73% of the product dispensed to of supplementation, compared to baseline (Table 3). In terms
them. We believe the former figure is somewhat overesti- of the prespecified endpoint related to bioavailability, at the
mated because some bottles of product were returned that end of the trial, the Omega 3–6 group displayed a 1.5 mol%
appeared to have been spilled or dumped (e.g., see estimate (p = 0.002) greater EPA level and a 1.8 mol% (p < 0.0001)
for low dose group) (Table 2). The latter figure is subject greater DHA level than the placebo group. As expected, and
to a handful of families failing to return a complete set of similar to Keim et al. (2018), we observed no statistically
diaries. No statistically significant differences between the significant increase in the other key FA in the supplement,
groups were detected for either adherence measure, but the GLA, because it is rapidly metabolized to longer-chain fatty
data are suggestive that adherence was higher for the lowest acids.
dose and lowest for the highest dose.
Biological Signatures
Primary Outcomes
Statistical analyses per our prespecified analysis plan
Safety revealed a statistically significant decrease in IL-2, with
Cohen’s d = -0.62 (the negative sign here signifies a decrease,
Two hundred and ninety-five adverse events were docu- or improvement, in inflammatory profile as desired, p = 0.02)
mented (167 in the treatment group, average 4.51/child; 128 (Table 4). For IL-1β, while not reaching our pre-defined cut-
in the placebo group, average 3.88/child; no statistically sig- off, we did observe Cohen’s d = -0.36 (p = 0.18). No mean-
nificant difference, t-test value = -1.03, p = 0.31). Most were ingful effect was observed for IFNγ. In terms of the post
upper respiratory infections, diarrhea, or appetite changes. hoc selected cytokines, for TNFα, we observed a moderate
The PIs and Study Doctor reviewed each event and found suggestive improvement as well (d = -0.41, p = 0.13).
none to be serious and related to the treatment.

Table 3  Change in red blood cell fatty acid levels (mol%), comparing the Omega 3–6 group to the placebo group, after 90 days of supplementa-
tion, Omega Heroes Trial (Ohio, USA)
Fatty Acid Baseline (mean (SD), mol%) End of Trial (mean (SD), mol%) Difference in Pre-Post Change
(Omega 3–6—Placebo)
(mol%, 95% CI, p-value)a
Omega 3–6 n = ­36b Placebo n = 33 Omega 3–6 n = 29 Placebo n = 30

C12:0 0.81 (0.14) 0.80 (0.11) 0.80 (0.08) 0.81 (0.17) − 0.02 (− 0.09, 0.06); p = 0.65
C14:0 1.07 (0.26) 1.05 (0.22) 1.09 (0.31) 1.05 (0.21) 0.04 (− 0.10, 0.18); p = 0.59
C16:0 24.71 (1.26) 24.60 (1.46) 25.01 (2.13) 24.52 (2.57) 0.35 (− 0.87, 1.56); p = 0.57
C16:1 0.98 (0.20) 1.03 (0.21) 0.98 (0.16) 0.95 (0.13) 0.04 (− 0.03, 0.11); p = 0.29
C18:0 14.44 (1.13) 14.79 (1.12) 14.44 (1.21) 15.06 (1.48) − 0.56 (− 1.27, 0.16); p = 0.12
C18:1n9c 17.41 (1.74) 17.51 (1.62) 16.62 (2.07) 17.65 (2.59) − 0.92, − 1.99, 0.15); p = 0.09
C18:2 19.74 (2.66) 19.68 (2.34) 18.73 (2.24) 19.45 (2.24) − 0.63 (− 1.78, 0.52); p = 0.28
C20:0 0.67 (0.08) 0.68 (0.12) 0.67 (0.07) 0.68 (0.08) − 0.01 (− 0.05, 0.03); p = 0.71
C18:3n6 0.59 (0.10) 0.60 (0.09) 0.59 (0.07) 0.70 (0.63) − 0.11 (− 0.35, 0.13); p = 0.37
C18:3n3 0.83 (0.17) 0.82 (0.15) 0.78 (0.12) 0.88 (0.13) − 0.11 (− 0.17, − 0.04); p = 0.002
C20:4 15.39 (1.66) 15.31 (1.72) 13.72 (2.37) 14.98 (1.75) − 1.29 (− 2.27, − 0.310; p = 0.01
C20:5 0.79 (0.19) 0.80 (0.27) 2.30 (1.86) 0.80 (0.33) 1.43 (0.77, 2.09); p < .0001
C22:6n3 2.57 (0.70) 2.33 (0.37) 4.26 (1.53) 2.46 (0.92) 1.52 (0.89, 2.15); p < .0001
a
Model-based estimates
b
One child in the Omega 3–6 group was missing data at baseline

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5350 Journal of Autism and Developmental Disorders (2022) 52:5342–5355

Table 4  Change in biological signatures due to omega 3–6 supplementation, Omega Heroes Trial (Ohio, USA)
Cytokinea Baseline End of Trial Difference in Pre- Group Difference in Change p-value Cohen’s d
(mean (SD), pg/mL) (mean (SD), pg/mL) Post (Omega 3–6— (Omega 3–6 – Placebo) (95%
Placebo) CI)b
Omega 3–6 Placebo Omega 3–6 Placebo Omega 3–6 Placebo
n = ­37c n = 33 c n = 29 c n = 30 c

IL-1β 0.11 (0.15) 0.10 (0.06) 0.09 (0.09) 0.11 (0.07) − 0.02 0.01 − 0.021 (− 0.053, 0.01) 0.18 − 0.36
IL-2 0.36 (0.56) 0.48 (0.47) 0.29 (0.16) 0.49 (0.44) − 0.09 0.01 − 0.17 (− 0.31, − 0.02) 0.02 − 0.62
IFNγ 9.99 (43.17) 16.72 (7.94) 8.20 (7.43) 8.02 (5.25) − 3.1 − 9.77 0.36 (− 2.99, 3.72) 0.83 0.06
TNFα 4.01 (0.90) 3.78 (0.88) 3.61 (0.82) 3.76 (0.84) − 0.39 0.03 − 0.28 (− 0.65, 0.08) 0.13 − 0.41
IL-6 0.58 (0.51) 0.69 (0.71) 0.49 (0.35) 0.74 (1.22) − 0.15 0.04 − 0.24 (− 0.71, 0.24) 0.32 − 0.26
IL-8 4.36 (2.30) 4.07 (1.31) 4.02 (1.62) 4.20 (1.52) − 0.02 0.09 − 0.15 (− 0.90, 0.61) 0.70 − 0.10
IL-10 0.79 (0.49) 0.74 (0.60) 0.64 (0.38) 0.81 (0.78) − 0.19 0.06 − 0.19 (− 0.51, 0.13) 0.23 − 0.32
IL-12p70 0.16 (0.07) 0.25 (0.45) 0.16 (0.06) 0.22 (0.36) 0.003 − 0.04 0.02 (− 0.014, 0.05) 0.27 0.29
a
Most values for IL-4 were below limits of detection, results not shown
b
Model-based estimates
c
8 children in the Omega 3–6 group and 3 children in the placebo group were missing data at the end of the trial for all cytokines because of
inability to collect adequate volumes of blood. Values were below the limits of detection for IL-1β for 4 children in the Omega 3–6 group at
baseline and 9 at the end of the trial

Exploratory Analysis of Biological Signatures by Dose FA supplements are among the most popular comple-
of Omega 3–6 mentary health interventions utilized by children with ASD.
Their use is driven by anecdotal evidence of benefit and a
While this study was not powered to analyze treatment few small RCTs that demonstrated improvements in some
effects by dose of Omega 3–6, some differences were externalizing behaviors in this population (Amminger et al.,
observed in our exploratory analyses (Table 5). The reduc- 2007; Bent et al., 2011, 2014). Overall, there is little evi-
tion in IL-2 was greater in the medium dose treatment group dence for efficacy in treating the actual core symptoms of
than the placebo group (p = 0.01) and met our Cohen’s d ASD, and little is known about the physiological pathways
effect size benchmark (d = 1.10 > 0.5). Several cytokines that contribute to this disorder. Our choice of specific FAs
showed greater reductions in one or more treatment groups was related to the biological functions of each and their
than in the placebo group (e.g., IL-1β, medium and high; relevance to neurological health. DHA, the primary neuro-
TNFγ, low and medium), but no others met our effect size active FA, is critical to neurotransmitter function, synap-
benchmark and had a statistically-significant p-value. togenesis, gene expression, membrane fluidity, neurogen-
esis, neuroplasticity, and regulating inflammation (Bazan,
2006; Chalon, 2006; Coti Bertrand et al., 2006; Kitajka
Discussion et al., 2002; Salem et al., 2001; Simopoulos, 2002). EPA is
often included in omega-3 supplements and has triglyceride
Omega Heroes is the first randomized controlled trial to lowering and anti-inflammatory properties similar to DHA
test the effects of a DHA + EPA + GLA (Omega 3–6) FA (Mozaffarian & Wu, 2012; Wei & Jacobson, 2011). GLA
combination from fish and borage oil in children with ASD. is an omega-6 FA commonly found in borage and evening
Our hypothesis was that the Omega 3–6 combination would primrose, is biologically available, and is quickly converted
reduce inflammation, a common feature of ASD, thereby to dihomo-γ-linolenic acid (DGLA), which also has sub-
potentially serving as a pathway to improving the core fea- stantial anti-inflammatory properties (Sergeant et al., 2016).
tures of ASD.(Granpeesheh et al., 2009; Martinez, 1994) DHA is the most widely investigated FA for treating men-
This early phase trial was designed to establish safety, adher- tal health disorders; however, DHA alone has demonstrated
ence, and changes in prescribed inflammatory markers. We little or no benefit in treating aberrant behavioral symptoms
observed no serious adverse outcomes and adherence was (Johnson et al., 2010). Adding EPA to DHA has been evalu-
high compared to previous trials of FAs in children diag- ated in 4 rigorous RCTs for ASD. One trial (n = 13) showed
nosed with ASD (Bent et al., 2014; Mankad et al., 2015). benefit to language and maladaptive behaviors (Amminger
We also observed a significant decrease in IL-2 and TNFa et al., 2007). Two others reported benefits limited to hyper-
levels in children receiving Omega 3–6 compared children activity (Bent et al., 2011, 2014). Another (n = 36) reported
receiving placebo. worsening externalizing behaviors (Mankad et al., 2015).

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 Table 5  Change in biological signatures due to omega 3–6 supplementation: -exploratory analyses by dose, Omega Heroes Trial (Ohio, USA)
Cytokinea Baseline End of Trial Group Difference in Change (Omega 3–6–placebo),
(mean)(pg/mL) (mean)(pg/mL) Cohen’s d, p-value b
Omega 3–6 Placebo Omega 3–6 Placebo Low Medium High
Low Med n = ­10c High n = ­12c Low n = ­10c Med n = ­10c High n = ­12c
n = ­10c

IL-1β 0.10 (0.05) 0.16 (0.25) 0.07 (0.05) 0.10 (0.06) 0.10 (0.05) 0.13 (0.15) 0.07 (0.04) 0.11 (0.07) − 0.01 − 0.02 − 0.03
(d = − 0.18, p = 0.64) (d = − 0.34, p = 0.38) (d = − 0.51, p = 0.16)
IL− 2 0.31 (0.12) 0.55 (0.96) 0.23 (0.10) 0.48 (0.47) 0.32 (0.13) 0.26 (0.12) 0.28 (0.22) 0.49 (0.44) − 0.10 − 0.29 − 0.11
(d = − 0.37, p = 0.34) (d = − 1.10, p = 0.01) (d = − 0.41, p = 0.26)
Journal of Autism and Developmental Disorders (2022) 52:5342–5355

IFNγ 8.95 (6.40) 11.22 (10.18) 9.80 (7.35) 16.72 (43.17) 6.76 (5.01) 6.47 (3.66) 10.79 (10.57) 8.02 (5.25) − 1.04 − 1.43 2.99
(d = − 0.17, p = 0.67) (d = − 0.23, p = 0.56) (d = 0.48, p = 0.19)
TNFα 4.37 (0.97) 3.55 (0.70) 4.09 (0.88) 3.78 (0.88) 3.76 (1.00) 3.27 (0.86) 3.77 (0.60) 3.76 (0.84) − 0.41 − 0.33 − 0.15
(d = − 0.58, p = 0.14) (d = − 0.47, p = 0.23) (d = − 0.21, p = 0.56)
IL-6 0.71 (0.77) 0.45 (0.12) 0.60 (0.44) 0.69 (0.71) 0.66 (0.54) 0.39 (0.16) 0.44 (0.24) 0.74 (1.22) − 0.10 − 0.32 − 0.29
(d = − 0.11, p = 0.79) (d = − 0.35, p = 0.37) (d = − 0.32, p = 0.38)
IL-8 5.21 (3.39) 4.50 (1.44) 3.45 (1.30) 4.07 (1.31) 3.57 (1.25) 3.67 (1.25) 4.67 (2.01) 4.20 (1.52) − 0.74 − 0.73 0.83
(d = − 0.56, p = 0.16) (d = − 0.55, p = 0.17) (d = 0.63, p = 0.09)
IL-10 0.71 (0.57) 0.86 (0.49) 0.80 (0.43) 0.74 (0.60) 0.62 (0.18) 0.69 (0.56) 0.62 (0.36) 0.81 (0.78) − 0.21 − 0.17 − 0.19
(d = − 0.34, p = 0.38) (d = − 0.28, p = 0.47) (d = − 0.32, p = 0.38)
IL-12p70 0.17 (0.08) 0.15 (0.04) 0.14 (0.07) 0.25 (0.45) 0.16 (0.08) 0.14 (0.05) 0.17 (0.05) 0.22 (0.36) 0.0001 0.01 0.04
(d = 0.003, p = 0.99) (d = 0.21, p = 0.60) (d = 0.61, p = 0.09)
a
Most values for IL-4 were below limits of detection, so results not shown
b
Model-based estimates
c
3 children in the low Omega 3–6 group, 3 in the medium group, 3 in the high group, and 3 children in the placebo group were missing data at the end of the trial for all cytokines because of
inability to collect adequate volumes of blood. Values were below the limits of detection for IL-1β for 2 children in the medium Omega 3–6 group and 2 in the high group at baseline, and 3 chil-
dren in the high Omega 3–6 group at the end of the trial

13
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5352 Journal of Autism and Developmental Disorders (2022) 52:5342–5355

However, EPA + DHA was moderately efficacious in reduc- causes deficits in learning and memory processing in animal
ing symptoms of ADHD in large RCTs (meta-analysis stand- studies. Alternatively, IL-2 regulates immunological homeo-
ardized mean difference = 0.31, 95% CI 0.16, 0.47) (Bloch stasis, self-tolerance, and T cell development and function;
& Qawasmi, 2011). One recent trial tested DHA, EPA, and thus implying dysregulation may play a significant role in
evening primrose oil (GLA, AA) in an open-label, non-con- neurological autoimmune diseases (Petitto et al., 2012; Xu
trolled design and showed improvements in parent-reported et al., 2015). Interferon gamma (IFNγ) is produced primar-
social and attention-related outcomes (Ooi et al., 2015). In ily by epithelial cells and is the acute phase cytokine pro-
this early phase trial, we did not test benefit in ASD-related duced in response to viral infections. (Lazear et al., 2015;
behaviors but will perform extensive cognitive and behavior Sweeten et al., 2004; Tostes et al., 2012). Tumor Necrosis
assessments for this purpose in the next phase of research. Factor alpha (TNFα) is an acute phase cytokine involved in
One significant shortcoming to the past trials and innate responses. A recent meta-analysis revealed that TNFα
addressed in our current study is the lack of consideration was elevated in most studies of ASD patients versus typi-
for dosing by weight. Many individuals with ASD have cally developing individuals (IL-2 was not evaluated)(Masi
comorbidities that impact health and functioning: at least et al., 2015). Our prior, unpublished trial observed lower
34% have gastrointestinal problems (constipation), 34% are levels of IL-1β, TNFα, IL-2, and IFNγ, in the Omega 3–6
overweight, and 18% are obese (de Vinck-Baroody et al., group compared to the placebo group at the end of the trial
2015; Ibrahim et al., 2009). Theoretically, these may mod- and these findings were the basis for our choices and power
ify the effect of FA supplementation on ASD symptoms by calculations for Omega Heroes. The results of our Omega
inhibiting gastrointestinal absorption of the supplement or Heroes trial revealed a significant decrease (improvement)
by sequestering a portion of ingested FAs in adipose tissue. in IL-2 with a Cohen’s d = -− 0.62. While not reaching our
In fact, differences in treatment response have been reported pre-defined effect size benchmark, we did observe Cohen’s
by weight and BMI in at least one prior omega-3 trial for d = -− 0.36 for IL-1β and d = -− 0.41 for TNFα (Table 5).
mood disorder (Christian et al., 2017). We observed no Decreases in these three cytokines could have important
adverse effects with dosing by weight. implications in suppressing inflammation in neurological
Children with ASD often exhibit altered FA metabolism, tissues and thus effect behaviors. The additional cytokines
specifically lower levels of DHA, EPA, and AA. The rea- (IL-4, IL-8, IL-6, IL-10, IL-12p70) have been shown to be
sons are unclear but may be due to diet deficiencies, altered elevated in children diagnosed with ASD in individual trials
intestinal absorption, and defects in FA synthesis related and are likely important and relevant to our outcomes with
to polymorphisms in FA desaturase genes (e.g., FADS2) a larger sample size.
or other metabolic alterations (Brigandi et al., 2015; Innis, Our enrollment and retention were excellent, and despite
2008; Vancassel et al., 2001; Yui et al., 2016). Since all of potential oral sensitivity and feeding difficulties among
these FAs contribute to important biological functions, and young children with ASD, compliance and adherence was
influence anti-inflammatory pathways, we speculate that high (Fig. 1, Table 2). The form of the investigational prod-
decreases or deficiencies in these FAs directly contribute to uct (a liquid oil that has a strong taste) and the study popula-
increased inflammation in ASD. As observed in our previ- tion (young children with ASD) combine to make adherence
ous study (Preemie Tots), no increases in RBC GLA were particularly challenging. Compounding these concerns is the
indicated; but we did measure increases in ALA, AA, EPA, fact that the eligibility criteria required that children were
and DHA pre- versus post-treatment (Table 3). This find- recently diagnosed with ASD. This means that the children
ing provides evidence that increases in these important FAs were not yet in therapy programs which often coach parents
offer essential substrate for (anti) inflammatory responses on how to improve feeding challenges and to address sensory
and normal growth and neurodevelopment. issues.
Inflammation is well characterized in youth with ASD Omega Heroes is unique in several ways. We are one of
both in the circulation and CSF (Theoharides et al., 2016). the first studies to test the efficacy of a combination therapy
Interleukin (IL)-1β is an acute response cytokine (Xu et al., of fish and borage oil derived DHA + EPA + GLA using
2015), can cross the blood–brain barrier inducing increased changes in inflammatory biomarkers as a primary outcome.
permeability, or be produced by astrocytes and microglia in Secondly, our dosing was based on weight, which can be
the brain. Increases in IL-1β in neurological tissues causes a significant variable in the ASD population and with age
increased exocytosis of several neurotransmitters, resulting in early childhood. The study population was indicative of
in increased excitability, anxiety, and activation of the HPA the population in our geographical area and consisted of a
(Anisman et al., 2002; Vezzani & Viviani, 2015). Interleukin similar racial and ethnic distribution (Table 1).
(IL)-2 has been extensively studied in the context of brain The greatest weaknesses were the relatively small sample
development and T-cell fate (Petitto et al., 2012). IL-2 acts size, moderate loss to follow-up or withdrawal of partici-
in a trophic manner during development and loss of IL-2 pants, and the single site setting. At this time, we were also

13
Journal of Autism and Developmental Disorders (2022) 52:5342–5355 5353

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