Docosahexaenoic acid therapy in
Results of a double-blind, randomized trial
A.M. Paker, MD, MPH
J.S. Sunness, MD
N.H. Brereton, MS, RD
L.J. Speedie, PhD
L. Albanna, EdD
S. Dharmaraj, MD,
A.B. Moser, BA
R.O. Jones, PhD
G.V. Raymond, MD
Objectives: Peroxisome assembly disorders are genetic disorders characterized by biochemical
abnormalities, including low docosahexaenoic acid (DHA). The objective was to assess whether
treatment with DHA supplementation would improve biochemical abnormalities, visual function,
and growth in affected individuals.
Methods: This was a randomized, double-blind, placebo-controlled trial conducted at a single cen-
ter. Treatment groups received supplements of DHA (100 mg/kg per day). The primary outcome
measures were the change from baseline in the visual function and physical growth during the 1
year follow-up period.
Results: Fifty individuals were enrolled and randomized. Two were subsequently excluded from
study analysis when it was determined that they had a single enzyme disorder of peroxisomal ?
oxidation. Thirty-four returned for follow-up. Nine patients died during the trial of their disorder,
and 5 others were lost to follow-up. DHA supplementation was well tolerated. There was no
difference in the outcomes between the treated and untreated groups in biochemical function,
electroretinogram, or growth. Improvements were seen in both groups in certain individuals.
Conclusions: DHA supplementation did not improve the visual function or growth of treated indi-
viduals with peroxisome assembly disorders.
Classification of evidence: This interventional study provides Class II evidence that DHA supple-
mentation did not improve the visual function or growth of treated individuals with peroxisome
assembly disorders during an average of 1 year of follow-up in patients aged 1 to 144 months.
AA ? arachidonic acid; DHA ? docosahexaenoic acid; ERG ? electroretinogram; NALD ? neonatal adrenoleukodystrophy;
RBC ? red blood cell; VLCFA ? very long chain fatty acid; ZS ? Zellweger syndrome; ZSD ? Zellweger spectrum disorder.
Peroxisome assembly disorders are a genetically heterogeneous group of disorders characterized by
the disruption of peroxisomal protein importation.1The majority of these disorders result from
an abnormality of PTS1 targeting and importation and are referred to as Zellweger spectrum
disorder (ZSD). This disorder is seen in 1 per 50,000 births.2Clinically, individuals with ZSD
have hypotonia, development delay, retinal degeneration, sensorineural hearing loss, and other
organ system involvement. These are serious disorders, and many affected individuals die in
infancy or childhood, although survival to adulthood is documented.3
While genetically heterogeneous, the involvement of PTS1-mediated protein importation
results in a characteristic set of biochemical abnormalities. The list of involved pathways is
extensive but includes plasmalogen synthesis, long chain fatty acid degradation, and the synthe-
sis of docosahexaenoic acid (DHA).4DHA is a polyunsaturated fatty acid that has been deter-
mined to be important in brain and retinal development.5Individuals with ZSD have been
From the University of Texas Medical Branch (A.M.P.), Galveston, TX; Kennedy Krieger Institute (L.J.S., L.A., A.B.M., R.O.J., G.V.R.), Baltimore,
MD; Johns Hopkins Hospital (N.H.B.), Baltimore, MD; Greater Baltimore Medical Center (J.S.S.), Baltimore, MD; and Moorfields Eye Hospital
(S.D.), London, UK.
Study funding: Supported by the Food and Drug FD-R-001289 (G.V.) and the Johns Hopkins University School of Medicine General Clinical
Research Center M01 RR00052 from the National Center for Research Resources. Supply of agent and placebo were provided by Martek Biosciences
Corp, Columbia, Maryland.
Disclosure: Author disclosures are provided at the end of the article.
Address correspondence and
reprint requests to Dr. Gerald V.
Raymond, Department of
Neurogenetics, Kennedy Krieger
Institute, 707 N. Broadway,
Baltimore, MD 21205
Copyright © 2010 by AAN Enterprises, Inc.
determined to be deficient in DHA,6but the
exact role of this deficiency in the pathogene-
sis of the disorder is not known.
Preliminary studies7,8demonstrated the
ability to supplement DHA orally and in-
crease the levels in the blood stream. Whereas
other investigators8have reported improve-
ment in the clinical status, we have noted that
despite adequate supplementation in all pa-
tients studied in an open trial, the variation
and severity of preexistent deficits made the
experience difficult to interpret.
Our aim in this study was to assess the effi-
cacy of DHA in improving visual function
and physical growth through a double-blind,
placebo-controlled, randomized trial. We also
investigated the relation between DHA sup-
plementation and biochemical peroxisomal
measures, including plasma very long chain
fatty acid (VLCFA) and red blood cell (RBC)
METHODS Participants. Eligible participants were chil-
dren aged 1 month to 10 years and diagnosed with Zellweger
syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and
infantile Refsum disease by characteristic biochemical profile,
which includes increased VLCFAs, increased plasma phytanic
acid, increased plasma pipecolic acid, decreased RBC plasmalo-
gen, and decreased plasma DHA levels.
Unit of the Johns Hopkins Hospital, Baltimore, Maryland.
Objectives. Our primary hypothesis was that DHA supple-
mentation improves visual function and physical growth (Class
II evidence) in children affected with peroxisome assembly disor-
ders. Our secondary hypothesis was DHA supplementation nor-
malizes biochemical peroxisomal measures, including plasma
VLCFA and RBC plasmalogen levels (Class II evidence).
Standard protocol approvals, registrations, and patient
consents. The study protocol was approved by the institutional
review board, and individual written informed consents were
obtained from patients’ families.
Study design and interventions. This was a randomized,
double-blind, placebo-controlled trial conducted at a single cen-
ter. Participants were randomly assigned to 2 interventions in a
1:1 ratio using a random number generator. Randomization was
performed by the investigational pharmacy of Johns Hopkins
Hospital. The treatment group received supplements of DHA
and arachidonic acid (AA). Assigned treatments were mailed to
patients’ homes through the investigational pharmacy at Johns
Hopkins Hospital; this pharmacy was the only unmasked entity.
Neither the investigators nor the patients were aware of the treat-
ment assignment during the trial period and until the evalua-
tions were completed and analyzed.
It had been previously determined that in otherwise healthy
premature infants, supplementation with DHA alone could result
in decreased AA levels and decreased growth. We have shown that
significantly in children with peroxisomal disorders (data not
tion is presently used in infant formula supplementation.
The prepared treatment was a microencapsulated powder
that contained DHA triglyceride (47% DHA) and AA triglycer-
ide (46% AA). Placebo was similarly prepared soybean oil. Doses
of these study medications were 100 mg/kg/d, which were based
on previous open study experience.9This study medication
could be mixed with food or infant formula. Both were similar in
appearance and comparable in taste and smell. Treatment com-
pliance was monitored by plasma DHA levels. Participants at the
time of enrollment were instructed in a low-phytanic diet,
mainly restricting whole milk consumption. The registered die-
tician was in regular contact with families to confirm adequate
intake of nutrients. This study was undertaken and completed
before the availability of commercial formulas that now rou-
tinely contain DHA with AA. Study agents were provided by
Martek Biosciences Corporation (Columbia, MD).
Outcome. Evaluations included neurologic, biochemical, oph-
thalmologic, growth, and monitoring of hematologic and blood
chemistry. Plasma VLCFA, and DHA and plasmalogen levels in
RBCs were evaluated at baseline and 3, 6, 9, and 12 months. The
remainders of the evaluations (described below) were performed at
baseline and 1-year follow-up. The standard ratio of 18:0 dimethy-
lacetals/18:0 in erythrocytes was used as a measure of plasmalogen.
formed in the Peroxisome Diagnostic Laboratories of Kennedy
Krieger Institution using standard methodologies.10-12
Visual function was evaluated using electroretinogram (ERG).
?V), c) low (?50 ?V), or d) moderate (?50 ?V). At follow-up,
and were categorized as worse, no change, or improved.
Physical growth was evaluated by following the Z scores of
weight and height over the course of the trial.
The primary outcome of the trial was change from baseline
in visual function and physical growth during the 1-year
All of our patients received neurologic and neurodevelop-
mental evaluations as part of the study, but the decision was
made to restrict analysis to quantitative outcomes. There was no
unusual developmental event on subjective evaluations in any of
The pretrial anticipated rate of improvement in the visual
function was 60% for the treatment group8and 20% for the
control group.2Power calculations were based on the natural
history data at Kennedy Krieger Institute. A sample of 22 sub-
jects per group was required for 80% power and a level of signif-
icance of 0.05. Sample size was further inflated by 10% to
address missing data or loss to follow-up. Power calculation was
also performed for change in weight and height Z scores, before
and after treatment with DHA. It was estimated that 25 patients
in each group would provide more than 90% power to detect 1
SD change in Z scores of weight and height.
Statistical methods. Baseline characteristics were compared by
randomized groups using t tests for continuous-level variables and
?2tests for proportions. Primary analyses were performed on 48
randomized participants based on intent-to-treat principle. The ?2
test was used to compare ERG outcomes. Repeated-measures anal-
ysis of variance was used to evaluate the difference in aspartate ami-
notransferase (AST), alanine aminotransferase (ALT), total
Neurology 75August 31, 2010
bilirubin, and change of weight and height from baseline to 1-year
follow-up. The criterion for statistical significance was set at ?0.05.
All analyses were performed using STATA version 8
(STATA Corp LP, College Station, TX).
RESULTS Because peroxisomal disorders are rare, we
informed all centers treating these patients about this
study. A total of 51 patients were screened, and 50 of
these were enrolled between February 1, 1997, and
April 30, 2001 (period of 4 years 3 months). Twenty-
five were randomly assigned to receive DHA, and 25
were randomly assigned to receive placebo. Two pa-
tients were excluded from study analysis when their di-
agnosis changed after complete biochemical analysis,
which was performed during the baseline visit (both in-
dividuals had single enzyme disorders affecting peroxi-
somal ? oxidation). All 48 patients received at least 1
dose of study medication and were included in efficacy
and safety analyses. The baseline characteristics of the 2
groups were similar, as shown in table 1. Three patients
in the DHA group and 2 patients in the placebo group
(10% of the enrolled participants) missed their
follow-up visit and could not be contacted. Nine pa-
tients died during the course of the trial secondary to
the natural progression of the disease. Eight of these 9
patients were diagnosed with ZS, and 1 was diagnosed
Figure Participant flow through the trial
DHA ? docosahexaenoic acid.
Table 1 Baseline demographic and clinical
characteristics of each group
mean (SD), mo
28.9 (32.8)25.04 (34.15) 0.691
Duration of follow-
up, mean (SD), mo
11.6 (3.2) 11.8 (2.3)0.843
No. of deaths
Abbreviations: ALD ? adrenoleukodystrophy; DHA ? doco-
Neurology 75 August 31, 2010
with NALD. The figure depicts the enrollment and
progress of patients throughout the trial.
The frequency of patients with improved ERG was
similar in 2 groups, as shown in table 2. The treatment
effect of DHA supplementation on other outcomes is
also shown in table 2. DHA was well tolerated, and
there was no serious adverse event reported that was
conclusively related to interventions.
DISCUSSION In this placebo-controlled, ran-
domized trial among children with peroxisome as-
sembly disorders, we found no overall effects of
oral DHA supplementation on visual function,
growth, or biochemical measures. We noted that
oral supplementation increases DHA blood levels
without any adverse effects. Surprisingly, visual
function deteriorated in only 1 patient of each
group who was able to return for follow-up; the
rest either improved or stayed the same. Similarly,
weight and height improved over time regardless
of intervention. DHA supplementation had no
significant impact on VLCFA or plasmalogen lev-
Table 2Baseline and endpoint biometric and biochemical data
DHA Placebop Value
Weight, mean Z score (SD)
?3.31 (2.1)0.714(?1.05 to 1.53)
?1.95 (1.1)0.824(?0.83 to 1.04)
Height, mean Z score (SD)
?1.99 (1.9) 0.97(?1.02 to 1.06)
?1.84 (1.4)0.147(?0.25 to 1.56)
C26:0, mean (SD)
1.68 (1.2)1.65 (0.92) 0.924(?0.59 to 0.65)
2.09 (1.5)1.8 (0.9) 0.452(?0.47 to 1.04)
DHA, mean (SD)
5.31 (7.7)4.29 (6.4)0.622(?3.13 to 5.18)
26.31 (20.8)6.74 (9.6)
?0.0001(9.55 to 29.58)
Plasmalogen, mean (SD)
0.041 (0.02) 0.038 (0.02)0.6551(?0.02 to 0.07)
0.039 (0.02)0.039 (0.01)0.981(?0.01 to 0.01)
ALT, mean (SD)
102.7 (81)85.4 (66) 0.499(?34.31 to 68.89)
74.6 (52) 133.6 (215) 0.294(?171.6 to 53.64)
AST, mean (SD)
244.8 (251)151.6 (118)0.134(?30.38 to 216.71)
160.56 (154)197.2 (292)0.651(?199.87 to 126.45)
Total bilirubin, mean (SD)
1.14 (1.4) 1.02 (2.1)0.841(?1.08 to 1.32)
1.09 (1.9)0.71 (1)0.425(?0.58 to 1.34)
Abbreviations: ALT ? alanine aminotransferase; AST ? aspartate aminotransferase; DHA ? docosahexaenoic acid.
After 1 year of follow-up, weight and height improved in both groups, and treatment effect of DHA was not significant.
Plasma DHA levels were below normal in all patients at baseline (normal range 26.01–46.95 ?g/mL). C26:0 levels and
C26:0/C22:0 ratios were increased in all patients at baseline (normal range C26:0 0.26 ? 0.15 ?g/mL, C26:0/C22:0
0.01 ? 0.004). DHA supplementation did not reduce the C26:0 levels. At baseline, plasmalogen levels (18:0 dimethylac-
etals/18:0 ratio) were low in both groups. DHA treatment did not affect the plasmalogen levels. ALT (U/L), AST (U/L), and
total bilirubin (mg/dL) levels were not changed by DHA supplementation, and there was no treatment effect. Confidence
intervals are 95% confidence intervals of difference in means between DHA and placebo groups.
Neurology 75August 31, 2010
els, which were characteristic peroxisomal bio- Download full-text
chemical abnormalities in our patients.
In previous open studies,7,8it had been stated that
DHA supplementation was associated with improve-
ments in vision and growth of these children. In a
similar fashion, we also noticed improvement in vi-
sion, weight, and height in both groups, but these
effects were not related to DHA supplementation be-
cause they were present also in the untreated group.
Additionally, unexplained biochemical improve-
ments were also reported. Most likely these changes
in VLCFAs and plasmalogens may reflect mild varia-
tions with age and survival. In light of these findings
of maturational effects in untreated individuals,
other alleged benefits, including changes in myelin,
are suspect without a comparison group.13
This study, in comparison with previous open tri-
als, highlights the limitations of presuming to know
the natural history of a rare disorder. It is apparent
that the children who did not die during the study
period maintained stable features and in some in-
The limits of our study are that there was no uni-
formity in the genetic etiology of the affected chil-
dren. It is possible that certain mild genotypes of
particular PEX genes may receive more benefit from
postnatal supplementation with DHA, but given the
rarity of these conditions, such a limited study is not
feasible. Similarly, the restricted sample size and vari-
ability in age of participants may have missed a small
treatment effect, but prior open case reports did not
limit their claims of efficacy, which were not shown
to be valid here.
Unfortunately, therapies for peroxisomal disor-
ders remain limited. Peroxisome assembly disorders
involve multiple systems and have their onset during
fetal development. Many of the pathologic features
are set in motion at this developmental stage, and
any therapy instituted after birth is unlikely to offset
this. Although supplementation with DHA is with-
out significant adverse effect in this population, its
therapeutic use cannot be endorsed at this time.
Statistical analyses were performed by Asif M. Paker, MD, MPH.
The authors thank Dr. Irene Maumenee and the fellows, residents, and
staff who assisted in the evaluations. The authors also are indebted to
Polly Green, Willie Foreman, and Dr. Hugo Moser (all deceased), who
were instrumental in the design and carrying out of this project.
Dr. Paker reports no disclosures. Dr. Sunness serves on scientific advisory
boards for Potentia Pharmaceuticals and Acucela Inc.; has received fund-
ing for travel from Acucela Inc., Merck Serono, Jerini AG, and Genen-
tech, Inc.; has received publishing royalties from Ryan Retina Online (low
vision course); serves as a consultant for SYTERA, LLC, Eyetec GmbH,
Alcon, Merck Serono, Johnson & Johnson, Othera Pharmaceuticals, Inc.,
Acucela Inc., Jerini AG, Taligen Therapeutics, Genentech, Inc., Pfizer
Inc, Potentia Pharmaceuticals, Avnet, Inc., Health Advances, LLC, Ad-
vanced Vision Therapies, Inc., Ophthotech Corporation, Shire plc, and
Neurotech; has received research support from the NIH (R03 EY14148
[PI]); and holds stock options in Potentia Pharmaceuticals. Ms. Brereton
is a research nutritionist of the Institute for Clinical and Translational
Research, which is funded by the NIH. Dr. Speedie, Dr. Albanna, Dr.
Dharmaraj, Ms. Moser, and Dr. Jones report no disclosures. Dr. Ray-
mond received a speaker honorarium from Garrod Association of Canada;
receives research support from the NIH (NICHD R01 HD39276 [PI],
R01 FD003030 [PI], and NICHD R01 HD057136 [PI]), the European
Leukodystrophy Association, and the Myelin Project; and has served as a
consultant for the Department of Health and Human Services in the
Division of Vaccine Injury compensation.
Received April 3, 2009. Accepted in final form May 13, 2010.
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