ARTICLES
EFA Supplementation in Children with Inattention,
Hyperactivity, and Other Disruptive Behaviors
Laura Stevensa, Wen Zhanga, Louise Peckd, Thomas Kuczekb, Nels Grevstadb,
Anne Mahona, Sydney S. ZentallC, L. Eugene Arnolde, and John R. Burgessa,*
Departments of aFoods & Nutrition, bStatistics, and CEducational Studies, Purdue University, West Lafayette, Indiana 47907,
dDepartment of Food Science & Human Nutrition, Washington State University, Pullman, Washington 99164,
and eDepartment of Psychiatry, Ohio State University, Columbus, Ohio 43210
ABSTRACT: This pilot study evaluated the effects of supplementation with PUFA on blood FA composition and behavior in
children with Attention-Deficit/Hyperactivity Disorder (AD/HD)like symptoms also reporting thirst and skin problems. Fifty children were randomized to treatment groups receiving either a
PUFAsupplementproviding a daily dose of 480 mg DHA, 80 mg
EPA,40 mg arachidonic acid (AA), 96 mg GLA, and 24 mg (Xtocopheryl acetate,or an olive oil placebo for 4 mon of doubleblind parallel treatment. Supplementationwith the PUFA led to a
substantial increase in the proportions of EPA, DHA, and (Xtocopherol in the plasma phospholipids and red blood cell (RBC)
total lipids, but an increase was noted in the plasma phospholipid proportions of 18:3n-3 with olive oil as well. Significant improvementsin multiple outcomes(asrated by parents)were noted
in both groups, but a clear benefit from PUFA supplementation
for all behaviors characteristic of AD/HD was not observed. For
most outcomes, improvement of the PUFA group was consistently nominally better than that of the olive oil group; but the
treatment difference was significant, by secondary intent-to-treat
analysis, on only 2 out of 16 outcome measures:conduct problems rated by parents(-42.7 vs. -9.9%, n = 47, P= 0.05), and attention symptoms rated by teachers (-14.8 vs. +3.4%, n = 47,
P = 0.03). PUFA supplementationled to a greater number of participants showing improvement in oppositional defiant behavior
from a clinical to a nonclinical range compared with olive oil
supplementation (8 out of 12 vs. 3 out of 11, n = 33, P = 0.02).
Also, significant correlations were observedwhen comparing the
magnitude of change between increasing proportions of EPAin
the RBC and decreasing disruptive behavior as assessedby the
Abbreviated Symptom Questionnaire (ASQ) for parents (r =
-0.38, n = 31, P < 0.05), and for EPAand DHA in the RBCand
the teachers' Disruptive Behavior Disorders (DBD) Rating Scale
for Attention (r = -0.49, n = 24, P < 0.05). Interestingly, significant correlations were observed between the magnitude of increasein (X-tocopherolconcentrationsin the RBCand a decrease
in scoresfor all four subscalesof the teachers' DBD (Hyperactivity, r = -0.45; Attention, r = -0.60; Conduct, r = -0.41; Opposi*To whom correspondence should be addressed at Purdue University, Department of Foods and Nutrition, Purdue University, 700 West State St., GI,
West Lafayette, IN 47907. E-mail: [email protected]
Abbreviations: AA, arachidonic acid; AD/HD, Attention-Deficit/Hyperactivity Disorder; ALA, a-linolenic acid; ASQ, Conners' Abbreviated Symptom
Questionnaire; ASQ-P, Conners' Abbreviated Symptom Questionnaire for
Parents; ASQ- T, Conners' Abbreviated Sympton Questionnaire for Teachers; CPT, Conners' Continuous Performance Test; DBD Rating Scale, Disruptive Behavior Disorders Rating Scale; DGLA, dihomo-y-linolenic acid;
HRT, hit reaction time; ITT, intent-to-treat; RBC, red blood cells; WJ-R,
Woodcock-Johnson Psycho-Educational Battery-Revised.
CopyriRht
(> 2003 by AOCS Press
tional/Defiant Disorder, r= -0.54; n = 24, P< 0.05) as well as
the ASQ for teachers (r = -0.51, n = 24, P < 0.05). Thus, the results of this pilot study suggestthe need for further researchwith
both n-3 FA and vitamin E in children with behavioral disorders.
Paperno. L9175 in Lipids 38, 1007-1021 (October 2003).
Attention-DeficitlHyperactivity Disorder (AD/HD) is the most
prevalent psychiatric disorder in children. The behavioral
symptoms include inattention and/or impulsivity and hyperactivity (1). AD/HD is thought to arise from multiple causesinvolving both biological and environmental factors (2-8). In addition to their behavioral abnormalities, children with AD/HD
are also reported to exhibit a variety of other associatedproblems (3), including a higher frequency of health problems than
children without AD/HD (9,10). Persons with AD/HD have
also been reported to require more medical care than those
without AD/HD (11). Among the symptoms that children with
behavioral problems or AD/HD reportedly exhibit are several
that occur in EFA deficiency (12,13). These symptoms include
excessivethirst, frequent urination, dry skin, dry hair, dandruff,
brittle nails, and/or hyperfollicular keratoses.Thirst/skin symptoms and blood EFA abnormalities also have been reported in
patients suffering from allergy (14-19), rheumatoid arthritis
(20,21), end-stage renal disease (22-24), Sjogren's syndrome
(25,26), anorexia nervosa (27-29), and learning or psychiatric
disorders (30-36). Although abnormal FA metabolism has
been reported in all of these disorders, none of the EFA profiles, including anorexia nervosa (37), reveals a primary EFA
deficiency, which is characterized by low proportions of n-6
and n-3 PUFA and increased 20:3n-9 in the blood phospholipids. Patients have shown improvements in their thirst/skin
symptoms upon supplementation with n-6 and/or n-3 PUFA
(14,22-24,38,39).
In previous reports we demonstrated that about 40% of
children with AD/HD-type symptoms exhibited thirst/skin
symptoms (40,41), and the proportion of arachidonic acid
(AA) and DHA in their plasma phospholipids was significantly lower than that observed in other children with and
without AD/HD-type symptoms. However, there was no evidence that those children had blood levels of EFA consistent
with a primary EFA deficiency or that they consumed less
PUFA in their diets than controls (42).
1007
Lipids, Vol. 38, no. 10 (2003)
1008
STEVENSETAL.
Members of both the n-3 and n-6 families of long-chain
PUFA were found to be lower than controls in the plasma
phospholipids of the children with thirst/skin symptoms and
AD/HD-like symptoms (40). The n-3 FA are specifically implicated in maintaining central nervous system function and
are known to affect neurotransmitters, peptides, releasing factors, and hormones in the brain (43,44). Animal studies have
indicated that long-term n-3 FA deficiency is associated with
behavioral abnormalities including decreasedexploratory behavior in rats and increased total stereotypy and total locomotion in monkeys (45,46). The n-6 FA series serves as the
source for eicosanoid biosynthesis, and deficiency leads to
deleterious effects on growth, reproduction, and skin integrity
METHODS
Study participants. Girls and boys ages 6-13 were recruited
from the population of central Indiana within a l00-mi radius
of West Lafayette. All children were screened by telephone
interview with their parents or guardians. Both children and
parents gave written permission to participate in the study and
to send behavior questionnaires to the children's primary
teacil,ers. This study was approved by the Purdue University
Committee on the Use of Human Subjects in Research. Children were selected whose parents reported them as diagnosed
with AD/HD by their clinical psychologist, psychiatrist, or
pediatrician. Parents were asked whether their children had
any chronic health problems such as diabetes or kidney dis(47).
ease.
Children with chronic health problems were excluded
EFA status and supplementation have been studied in adult
from
the
study. Parents were also asked to evaluate the folpsychiatric disorders. Epidemiological studies in the United
lowing
combination
of symptoms: excessive thirst, frequent
States and other countries suggest an association between deurination,
dry
hair,
dry
skin, brittle nails, dandruff, and/or folcreased dietary n-3 FA intake and the increase in depression
licular
keratoses.
This
combination
of symptoms was quantithat has been observed in recent years (48). Plasma and red
fied
using
a
questionnaire
in
which
each symptom was rated
blood cell (RBC) proportions of n-3 FA have been reported to
by
a
parent
on
a
4-point
scale:
0
=
not
at all, 1 = just a little, 2
be lower in depressed patients in comparison with healthy
=
pretty
much,
and
3
=
very
much.
The
total thirst/skin score
adults (49,50). Stoll et at. (51) reported that supplementing the
was
calculated
as
the
sum
of
the
scores
for each of the seven
diets of patients with bipolar disorder with n-3 FA-enriched
individual
symptoms.
Children
with
AD/HD
symptoms were
PUFA led to a significant improvement in symptoms. Scienselected
for
the
study
only
if
their
total
thirst/skin
score was 4
tists have also studied EFA status and supplementation in paor
greater.
A
score
of
4
or
more
was
chosen
as
the
cutoff to
tients with schizophrenia. Supplementing the diets of 20 pareflect
the
presence
of
one
or
more
severe
symptoms
or sevtients hospitalized with chronic schizophrenia with fish oil led
eral
mild
symptoms.
Furthermore,
in
a
previous
study
of
to a significant improvement in symptoms (52). However, not
plasma
phospholipids,
AA
and
DHA
concentrations
were
sigall studies using n-3 FA have shown positive effects. For exnificantly lower in children with scores of 4 or more than in
ample, supplementation with ethyl EPA did not improve
the controls or in children with AD/HD-like symptoms but
symptoms in a study of patients with schizophrenia (53).
few thirst/skin symptoms (40). In that study, 9.3% of controls
An early FA supplementation study using the n-6 GLArich primrose oil with children described as hyperactive re- had scores of 4 or more, whereas 39.6% of children with
ported minimal or no improvement (54). In two recently pub- AD/HD had scores of 4 or more (40). The thirst/skin scores
ranged from 0 to 8 in the controls and 0 to 12 in the children
lished double-blind, placebo-controlled intervention trials
with AD/HD (40).
using n-3 FA-enriched supplements to treat children with
In the study reported here, healthy children without
AD/HD or AD/HD-like symptoms, contrasting results were
AD/HD-like
symptoms and few thirst/skin symptoms were rereported. In the first, children with specific learning problems
cruited
to
serve
as a reference sample to compare blood FA
and AD/HD-like symptoms were given either a PUFA supand
tocopherol
values.
For the intervention, subject recruitplement, containing both n-6 and n-3 FA, or a placebo for 3
ment
included
193
inquiries
regarding the study. Of these, 126
mon, and improvements were reported in the supplemented
children
with
AD/HD-like
symptoms
were screened, and 93
group on 7 of 14 scales for cognitive and behavioral assessof
these
children
reported
frequent
thirst/skin
symptoms. Parments (55). In the second study, children accurately diagticipants
were
excluded
from
participation
in
the
study for the
nosed with AD/HD consumed DHA-rich supplements for 4
following
reasons:
age,
distance
from
the
test
site,
inability to
mon with no benefits reported, despite an average 2.6-fold inswallow
capsules,
lack
of
further
interest,
or
chronic
illness
crease in the proportion of DHA in the plasma (56).
(diabetes).
Fifty
participants
were
randomized
into
the
two
To further test whether PUFA supplementation might benefit children with behavioral problems, we report here the results treatment groups. Seventeenparticipants discontinued particiof a pilot intervention trial using a supplement enriched in n-3 pation in the study at different time points after randomization. These included 7 who received the PUFA and 10 who rebut also containing n-6 PUFA. The study sample included chilceived the placebo. Attrition of participants was due to diffidren under the care of a clinician for AD/HD and receiving
standardtherapies. Members of the sample were selectedbased culty contacting working parents, a change in family
on their higher frequency of skin/thirst symptoms because we circumstances (e.g., relocation, divorce), and the refusal or a
hypothesized that this sample would be mOre likely to show change in mind about taking supplements or having blood
benefits than a group of children not exhibiting any outward drawn. Analyses of the dropouts compared to the completers
for both supplementation groups revealed no statistical differsigns or symptoms associatedwith EFA deficiency.
lipids, Vol. 38, no. 10 (2003)
EFA SUPPLEMENJATION
IN CHILDREN WITH DISRUPTIVE BEHAVIORS
ences for any of the baseline characteristics, thirst/skin symptoms, teacher or parent behavior evaluation scores, or blood
FA proportions of AA, EPA, or DHA.
Intervention. The children with AD/HD and thirst/skin
symptoms were randomized into two treatment groups, A and
B, which were balanced for gender and medication status
[none, methylphenidate, methylphenidate plus antidepressant
(imipramine, amitriptyline), other medication (pemoline, dextroamphetamine salts, imipramine)]. In a double-blind design,
group A (n = 25) received eight capsules of PUFA a day and
group B (n = 25) received eight placebo capsules a day for a
parallel period of 4 mono The PUFA supplement (Efalex) was
supplied by Efamol Ltd. (Trowbridge, United Kingdom). The
FA content of the PUFA supplement (per capsule) was 60 mg
DHA, 10 mg EPA, 5 mg AA, 12 mg GLA, and 3 mg vitamin
E as a preservative. The placebo contained 0.8 g olive oil in
each capsule. Olive oil was chosen as the placebo because it
contains very little PUFA and consumption was not expected
to affect the blood FA profile. The odor and appearanceof the
PUFA capsules and the placebo capsules were comparable.
Compliance was monitored by using a separateform completed by the parents that indicated the number of pills consumed each day by their child. Based on the forms returned
at the end of the intervention, average compliance was 88%.
Assessment protocol. Fasting blood samples were drawn
at baseline and after 2 and 4 mon of treatment. Analysis of
the EFA composition of plasma and RBC membranes was
conducted as described previously (57-59) with one procedural difference. The methanolysis was carried out using
tetramethylguanidine (60). The a-tocopherol concentrations
in the plasma and RBC were determined as described previously (61). To assessthe effect of supplementation on thirst
and skin symptoms, a questionnaire was completed by the
parents at baseline and at 0.5, 1,2,3, and 4 mon of supplementation.
At baseline and at the end of the intervention period, both
parents and teachers completed the Conners' Abbreviated
Symptom Questionnaires (ASQ) (62) and the Disruptive Behavior Disorders (DBD) Rating Scale (65), which were the
primary outcome measures.Two additional neuropsychological tests were administered: the Conners' Continuous Performance Test (CPT) (66) and eight tests of cognitive ability
using the Woodcock-Johnson Psycho-Educational BatteryRevised (WJ-R) (67). The ASQ was chosen as a primary subjective outcome assessment tool because of its widespread
clinical use, utility in frequent assessment of progress, and
high validity (63,64). The parents' DBD Rating Scale was
also chosen as a primary subjective outcome measure because
this instrument contains questions that are phrased differently
from the ASQ. The DBD Rating Scale assessesfour types of
behavior: hyperactivity, attention, conduct, and oppositional
defiant behavior. If a child was on medication, parents were
asked to evaluate their child's behavior off stimulant medication but on any long-acting medications. Teachers also completed the DBD Rating Scale for children in the school environment, and their ratings were for children on medication for
1009
those subjects taking any psychostimulant or nonstimulant
treatment. For both the ASQ and the DBD Rating Scale,
higher scores are indicative of poorer performance.
The CPT and WJ-R Tests of Cognitive Ability were chosen as objective measures of outcome assessment.The CPT
is a computer task that requires the subject to hit the spacebar
every time a letter flashes onto the screen, except for the letter X. It was chosen as an objective measure of inattention
and impulsivity (3,66) and also becauseprevious studies have
shown that performance on this test is sensitive to interventions that improve attentionlbehavior (68). The test lasts for
over 14 min and children with AD/HD have a difficult time
maintaining attention throughout the entire test. The CPT
Index reflects overall performance (66); a score of less than 8
is in the normal range, 8 to 11 is borderline, and greater than
11 is considered poor performance. Hit reaction time (HRT)
is one of the components of the test that is measured; it represents the elapsed milliseconds between the letter flashing on
the screen and the pressing of the spacebar. This time is converted into aT-score that represents how each subject performed compared to a large comparative study group. Slow
speeds reflect abnormal performance and are indicated by Tscores that are low. Because the test is sensitive to medication status (69,70), we requested that participants refrain from
taking stimulant-type medications on the test day. Children
on long-term medications did not alter their dosage for the
test, and the number of subjects in each group was balanced.
The WJ-R Tests of Cognitive Ability were chosen to assess short-term memory, processing speed, auditory processing, and visual processing (71) because abnormal FA metabolism has been reported to affect perceptual function (72);
thus, it was hypothesized that these tests would be sensitive
to FA supplementation. Also, a deficit in EFA in primates has
been shown to result in impaired visual processing (73), and
a previous intervention study with hyperactive children indicated that supplementation with GLA improved the accuracy
of short-term memory and response time in a memory distraction task (54). Two different tests for each parameter were
conducted, and the results were evaluated in comparison to
normalized data for age categories. The results from the two
tests were combined to provide an evaluation presented as
standard scores, with scores of 100 representing optimal performance for a given age, gender, and educational level.
Parents of the children completed 3-d diet records at baseline and 4 mon after a detailed explanation by investigators
using food models to illustrate serving sizes. These records
were analyzed for macro and micro components by using the
University of Minnesota Nutrition Data System (NDS 95,
version 2.8; University of Minnesota, Minneapolis, MN).
Subjects and statistical analysis. Primary analyses were
conducted on those subjects who completed the 4-mon intervention and had a minimum compliance of 75%. The total
number of subjects at the conclusion of the study was 33-18
in the PUPA group and 15 in the placebo group. These results
are reported in the tables. Secondary analyses also were performed using an intent-to-treat (ITT) basis, with the last ob-
Lioids. Vol. 38. no. 10120031
STEVENSETAL.
1010
servationcarried forward for all subjectswho were randomized and who had taken at least the first doseof the supplements(74,75). Three of the 50 randomizedparticipantshad
not taken a single dose;thus, the ITT analysiswas basedon
47 participants.TheseITT results are reportedin the tables
and discussed,where appropriate,in the text. The baseline
characteristicsof the two treatmentgroupswerecomparedby
using Student'st-test for continuous variables and Fisher's
exact test for noncontinuousdata. Differences in blood FA
compositionbetweenthe intervention sampleand the referencesamplewerecomparedusing Student'st-test.The effect
of supplementson FA proportions and a-tocopherol was
tested by repeated-measures
ANaVA using general linear
modelsfollowed by post hoc testing on differencesbetween
meansusing Tukey's studentizedrangetest, which adjustsa
= 0.05 for multiple
comparisons. The assumptions of normal
distribution and homogeneityof variancewere met for these
analyses.Both the total thirst/skin scoresand the behavioral
datawere convertedto the percentagechangefrom baseline
scores,[(baseline- 4 mon)/baseline].IOO,and the effects of
FA supplementationwere tested using the Kruskal-Wallis
nonparametric test for group differences and the Wilcoxon
signed rank test for within-group differences. The associations betweenthe percentagechangein blood FA as well as
tocopherolandthe percentagechangein scoreson behavioral
assessment
testsat the conclusionof the studywereevaluated
using Spearmancorrelations.Thesestatisticalanalyseswere
carried out using SAS (SAS Institute, Cary, NC), with the
level of significanceat P < 0.05.
RESULTS
Sample description. Table 1 summarizes the characteristics at
baseline of those participants who were randomized and who
had consumed at least one dose of the supplement. No statistically significant differences were found between the two
treatment groups with respect to age, height, gender, medication status, frequency of thirst/skin symptoms, or nutrient intake. Mean weight was greater in the placebo group, but the
variation was large and no statistical differences were observed. In comparing the two groups for baseline behavioral
assessment scores, the ASQ for parents (ASQ-P), the DBD
Attention score for parents and the HRT for the CPT were
significantly different between the two treatment groups
(Table 1). As shown in Table 1, the remaining teacher and
parent tests, CPT Index, and WJ-R tests were not different.
Participants with AD/HD-1ike symptoms and reporting
thirst/skin symptoms had significantly lower mean proportions
of dihomo-y-1ino1enic acid (DGLA, 20:3n-6) (P < 0.001), AA
(20:4n-6) (P < 0.006), a-linolenic acid (ALA, 18:3n-3) (P <
0.001), EPA (20:5n-3) (P < 0.04), DHA (22:6n-3) (P < 0.002),
In-3 (P < 0.02), and the A6-desaturase index (P < 0.03) in
plasma polar lipids than in the reference sample. Also, the intervention sample differed from the reference sample in the RBC
FA composition (Table 2). The mean proportions of linoleic acid
(P < 0.03) and ALA (P < 0.02), the ratio of In-6/In-3
Lioids. Vol. 38. no. 10120031
(P < 0.001), and the il6-desaturase index (P < 0.001) were significantly lower in the children with AD/HD-like symptoms in
comparison with the control group. However, RBC AA (P <
0.001), 22:4n-6 (P < 0.001), 22:5n-6 (P < 0.008), Ln-6 (P <
0.001), EPA (P < 0.001), 22:5n-3 (P < 0.002), DHA (P <
0.001), and Ln-3 (P < 0.001) were significantly higher in the
intervention sample in comparison with the reference sample.
Effect of supplementation on blood FA composition. At
baseline, no significant differences between those participants
receiving the PUPA and those receiving the placebo were observed for plasma and RBC FA (Tables 3 and 4). Several significant changes occurred in plasma and RBC FA composition during the period of supplementation. For both treatment
groups, the proportion of plasma AA increased significantly
by the fourth month of supplementation with no difference
between groups. PUFA supplementation led to a dramatic decrease in both 22:4n-6 and 22:5n-6 by the fourth month. For
the n-3 FA series, the proportion of ALA increased almost
twofold, and a greater than twofold increase in DHA was observed in the plasma of the children in the PUFA-supplemented group. A great deal of variation was found in the proportion of EPA with supplementation. The ANaVA analysis
revealed within-group effects but no between-group effects.
The mean proportion ofEPA increased in both groups, but no
significant difference between the groups was found. In the
olive oil placebo group, the content of ALA in the plasma
phospholipids increased twofold by the end of the study, but
increases in both EPA and DHA were not significant. As a result of supplementation, the ratio of total n-6/total n-3 FA decreased in the PUFA-supplemented group but did not change
in the olive oil placebo group.
For saturated FA, both olive oil supplementation and
PUFA supplementation led to significant decreasesin the proportion of 16:0 but increases in the proportion of 18:0. Correspondingly, the proportion of 18:In-9 was found to be significantly lower at the end of the supplementation period in the
plasma phospholipids of both groups. This occurred in the
olive oil placebo group despite the fact that the children in
this group were consuming almost five additional grams of
oleic acid from the supplement.
For the RBC, similar changes in n-3 FA were observed in
the PUFA-supplemented group (Table 4), with twofold increasesobserved in the proportion of EPA and DHA, and approximately a 50% increase in the proportion of total n-3 FA.
Also, the proportion of total n-6 FA and the ratio of total
n-6/total n-3 in the RBC decreaseddramatically in the PUPAsupplemented group. However, in contrast to the plasma results, the proportion of AA in the RBC decreased in both
treatment groups, and the magnitude of the decreasewas significantly greater in the PUFA-supplemented group. Unlike
the plasma phospholipids, the proportion of 18:0 did not
change with supplementation and the proportion of 16:0 increased slightly rather than decreased.Also, no change in the
proportion of 18:0 was observed for either group.
Effect of supplementation on blood vitamin E concentration. The PUFA supplement contained 3 mg of added tocoph-
EFA SUPPLEMENTATION
TABLE 1
Characteristics
1011
IN CHILDREN WITH DISRUPTIVE BEHAVIORS
of Two Treatment Groups at Baseline"
Characteristic
Olive oil
(n=22)
Age (yr)b
Height (in.)
10.1 %2.0
59.0 %10.2
Weight(Ib)
91.2 % 39.4
Medication (# of subjects)
None
Methylphenidate
Methylphenidate + other
Other
Gender (% female)
Total thirs!iskin symptoms scoreb
Nutrient intakeb,c
Energy(kcal)
Total fat (g)
Total carbohydrate (g)
Total protein (g)
Iron (mg)
Zinc (mg)
SFA(g)
MUFA (g)
PUFA(g)
Linoleic acid (g)
Linolenic acid (g)
Behavioral assessmentsb
ASQ-Parents
ASQ-Teachers
DBD Questionnaires (Parents)
Hyperactivity
Inattention
0.0001
Conduct
Oppositional/Defiant Disorder
DBD Questionnaires (Teachers)
Hyperactivity
Attention
Conduct
Oppositional/Defiant Disorder
Attention performance: Conners' CPT
5
12
3
2
13.3,
8.8 %3.5
PUFA
(n= 25)
p
NS
NS
NS
5
13
3
4
11.1
8.4:t
3.2
NS
NS
NS
NS
NS
NS
2248 %690.5
79.7 %24.1
313.4 % U5.7
77.7 %26.7
17.6 % 7.8
11.3%4.7
29.0 %9.4
30.3 %9.1
14.6 % 5.3
13.1 %4.8
1.3 %0.5
2054.7 :I: 484.2
77.9 :I: 21.7
270.6:1: 73.8
73.8 :I: 25.1
15.3 :I: 6.7
10.5 :I: 4.0
28.9 :I: 7.7
29.4:1: 7.7
14.1 :I: 5.6
12.8:1: 5.3
1.1 :I: 0.4
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
19.9 %4.5
13.1 % 7.7
16.5:1:4.9
10.5:1:7.4
0.004
NS
23.1 %5..1
20.6
24.2 %2.9
19.5 :I: 4.2
8.2 %5.3
17.3 %5.1
7.7:1: 5.2
14.5 :I: 5.4
NS
NS
10.1 % 7.7
15.1 %9.3
1.8 %2.2
6.9 %6.0
10.5*
11.7*
1.9 *
5.6 *
NS
NS
NS
NS
:I: 4.7
8.9
8.8
3.9
5.9
8.9% 6.3
8.4:t: 8.2
Hit reaction time (ms)
459.0 %74.7
415.8:t:64.4
Hit reaction time (T-scores)
35.7 % 12.0
45.7:t: 8.8
Cognitive ability: Woodcock-JohnsonTestsof Cognitive Ability'
Processingspeed
93.2 :I: 15.5
99.8 :!: 22.6
Short-term memory
95.9 % 15.9
101.4 :!: 17.0
Visual processing
104.8 % 14.2
107.1 * 16.1
Auditory processing
96.4:1: 12.4
98.6:!: 12.8
CPTOverallIndex
NS
NS
0.03
0.002
NS
NS
NS
NS
'Scoreson Conners' Abbreviated Symptom Questionnaire (ASQ)-Parents, Disruptive Behavior Disorders (DBD) Inattention (Parents) and Conners' Continuous Performance Test (CPT) hit reaction time
were also significantly different between groups using a completers-only analysis. No other score
was significantly different.
bMean :!: SD. MUFA, monounsaturated FA; SFA, saturated FA; NS, not significant.
CNutrient analysis of 3-d diet records. Placebo, n = 19; PUFA, n = 23.
dStandard scores.
erol acetatein addition to naturally occurring a-tocopherol
(0.16 mg) and y-tocopherol (0.10 mg) in each gel capsule.
The olive oil placebocontainedno tocopherolacetatebut did
contain small amountsof a-tocopherol (0.22 mg)and y-tocopherol (0.009 mg). Supplementationwith PUPA led to a
greaterthan 50% increasein plasmaand RBC a-tocopherol
concentrationand a significant decreasein y-tocopherolcon-
centration in comparison with supplementation
with the olive
oil placebo (Table 5).
Effect of supplementation on thefrequency of thirst/skin
symptoms. Parents of children in both the PUFA andolive oil
groups reported a decrease in total thirst/skin symptomsbut
the percentage change from baselinewasnot significantlydifferent based on either a comp1etersor In analysis(Table6).
lipids, Vol. 38, no. 10(2003;
L. STEVENSETAi.
1012
TABLE 2
Blood FA Composition and Tocopherol Content (mean :t SO) of Children With AO/HO-like
and Frequent ThirsUSkin Symptoms vs. Control Children Without AO/HO-like Symptoms
and Few Thirst/Skin
Symptoms
Symptoms
RBC (area%)
Plasma(area%)
Reference
sample 1a
P-value
(n = 24)
(n=24)
16:0
18:0
18:1
18:2n-6
20:3n-6
20:4n-6
22:4n-6
22:5n-6
18:3n-3
20:4n-3
20:5n-3
22:5n-3
22:6n-3
In-6
In-3
~6-Desat indexc
In-6/I.n-3
Tocopherols
(X-Tocopherol (~g/ml)
y-Tocopherol (~g/ml)
16.04 ~: 5.37
15.70
11.70
22.91
3.67
10.52
0.86
0.64
0.46
0.49
0.35
0.95
2.16
38.58
4.55
1.54
9.52
~: 2.60
~:2.77
~:4.26
~: 1.41
~: 1.60
~: 0.46
~: 0.35
~: 0.23
~:0.66
~: 0.36
~: 0.51
~: 0.64
~: 3.99
~: 1.92
~:0.50
~:2.97
7.77:t:2.15
1.59 :t: 0.56
Reference
sample 1a
I: 3.73
I: 2.44
I: 6.27
I: 3.12
1:0.67
1:2.52
I: 0.33
I: 0.26
I: 0.17
I: 0.28
t: 0.19
I: 0.28
1:0.48
I: 4.64
I: 0.92
I: 1.19
I: 2.36
8.33 :I: 2.59
1.81 :I: 0.49
0.001
NS
NS
0.07
0.001
0.006
0.04
NS
0.001
NS
NS
0.04
0.002
0.001
0.01
0.03
NS
:t: 1.02
:t: 0.62
:t: 0.63
:t: 1.00
:t: 0.25
18.16 :t: 1.31
4.74 :t: 0.61
1.13 :t: 0.14
0.13 :t:0.08
0.17 :t: 0.23
0.31 :t: 0.13
2.22 :t: 0.28
2.88 :t:0.40
40.29 :t: 1.32
5.72 :t: 0.66
0.55 :t: 0.06
7.14 :t: 0.92
NS
NS
5.11 :I: 0.97
1.63 :I: 0.40
15.82
14.69
14.41
14.11
2.14
"Recruited reference sample without Attention-Deficit/Hyperactivity
Disorder (AD/HD)-like
thirst/skin symptoms. RBC, red blood cells; for other abbreviation see Table 1.
bRecruited sample with AD/HD-like symptoms and thirst/skin symptoms for intervention.
CRatioof 18:2n-6/(18:3n-6 + 20:3n-6 + 20:4n-6 + 22:4n-6 + 22:5n6).
However,within the olive oil groupthe changein scoresfrom
Intervention
sample 2b
(n= 50)
P-vall
12.48 ~: 4.29
15.01 ~:1.38
14.13 ~:0.92
13.32~ : 1.51
2.15 ~: 0.52
20.15 ~:2.39
5.66 ~: 1.11
1.29 ~: 0.27
0.09 ~:0.06
0.52 ~: 0.62
0.39 ~: 0.07
2.61 ~: 0.57
3.52 ~: 0.83
42.56 :I: 3.07
7.12 ~: 1.55
0.46 ~:0.09
6.2 :I: 1.11
4.59
:t 1 .08
1.45 :t 0.38
0.001
NS
NS
0.02
NS
0.001
0.001
0.008
0.02
0.009
0.001
0.002
0.001
0.001
0.001
0.001
0.001
0.05
NS
symptoms and without
ual thirst/skin symptoms were also analyzed for treatment and
baselineto 4 mon decreased
28% (P < 0.005), andin the within-group changes. At baseline there were no differences
PUFA group the decreasewas 44.3% (P < 0.0002). lndividTABLE 3
FA Composition
in the frequency of any thirst/skin symptoms between groups.
in Plasma at Baseline, 2 mon, and 4 mon (area% of total FA)a-c
Olive oil (n=15)
PUFA (n = 18)
10.62:i:3.41Y
20.75 JI: 3.67"
11.31:t:3.38Y
16:0
16.58 :i: 1.25Y
16.80 :t: 1.52Y
14.93 ~I: 1.43"
18:0
8.65:i: 1.49Y
9.94:t: 1.36
12.02 JI: 3.55
18:1n-9
21.37~4.07
22.12 :t:2.38
20.05 JI: 3.77
18:2n-6
3.60:i: 1.02Y
3.80:t:0.76Y
2.80 J1:0.57"
20:3n-6
10.63:i: 1.24b,y
12.53:t: 1.88"'Y
9.82 JI: 2.27"
20:4n-6
0.15:i: 0.25b,y
0.66 :t: 0.34"
22:4n-6
0.75 JI: 0.23
0.02 :i: 0.08b,z
0.44 :t: 0.27"
0.59 J,0.15
22:5n-6
0.47 :i: 0.26Y
0.59:t:0.31Y
0.28 J: 0.21"
18:3n-3
0.70 :i: 0.52Y
0.61 :t: 0.49
0.39 J,0.23
20:5n-3
0.46:i: 0.49b
0.81 :t:0.40"
22:5n-3
0.83 J: 0.29
7.65:i: 3.17b,z
1.90 J: 0.62
2.70:t:0.54"
22:6n-3
35.76 :i: 3.67
39.55 :t: 2.78Y
34.01 J:4.50"
Ln-6
9.77 :i: 3.05b,z
5.17:t: 1.21"
4.12 ~: 0.96
Ln-3
4.06 :i: 1.56b,y
8.19:t: 2..73"'x
8.61 ~: 0.04"'Y
Ln-6/n-3
aMean:I: so. Variables were analyzed using repeated-measures ANOVA. Tukey's studentized range test was used to determine statistical differences between means for time and treatment effects. Treatment comparisons are indicated by a,band time comparisons by x-z within each group. Means with the
same superscripts are not different. P < 0.05.
bCompleters analysis (n = 33) GROUP"TIME interactions: 18:0, P < 0.04; 18:1, P < 0.04; 22:4n-6, P < 0.06; 22:5n-6, P < 0.009; 22:6n-3, P < 0.0001; Ln-3,
P< 0.0001; Ln-6/Ln-3, P< 0.0001.
Clntent-to-treat analysis (n = 47) GROUP"TIME interactions: 18:0, P < 0.08; 18:1, P < 0.07; 22:4n-6, P < 0.03; 22:5n-6, P < 0.002; 22:6n-3, P < 0.0001;
Ln-3, P < 0.0001; Ln-6/Ln-3, P < 0.0001.
Lipids, Vol. 38, no, 10(2003)
EFASUPPLEMENTATIONIN CHILDREN WITH DISRUPTIVEBEHAVIORS
1013
TABLE 4
FA Composition of RBCat Baseline,2 mon, and 4 mon (area% of total FA)a-c
RBC FA
16:0
18:0
18:1n-9
18:2n-6
20:3n-6
20:4n-6
22:4n-6
22:5n-6
18:3n-3
20:5n-3
22:5n-3
22:6n-3
rn-6
rn-3
rn-6lrn-3
14.68 :I: 3.03
14.61 :I: 0.76
14.19 :I: 0.95
13.22 :I: 1.72
1.91 :I: 0.28
19.39 :I: 1.66
5.28 :I: 0.77
1.20 :I: 0.153
0.10 :I: 0.07"
0.39 :I: 0.04
2.38 :I: 0.32
3.39 :I: 0.78
41.00:1: 2.383
6.72 :I: 1.13
6.25 :I: 0.99
15.39 :~ 1.35
14.63 :~ 1.04
14.58 :~ 0.63
13.30 :~ 1.26
1.87 :~ 0.17
18.58 :~1.61a
5.05 :~ 0.61 a
1.10: ~ 0.11 a
16.56 % 1.09
14.47%0.82
14.85 % 1.24
13.78 % 1.54
1.89%0.23
18.17%1.30"
4.75 % 0.44"
1.03 % 0.09"
0.21 :~ 0.04Y
0.38, ~ 0.07a
2.19 j ~ 0.34
3.36 j ~ 0.54a
39.91 j ~ 2.20a
6.31 j ~ 0.81a
6.43 j 0 0.89a
11.30 % 4.80x
14.96%1.78
14.22 % 1. 15x
13.04 % 1.55x
2.10%0.50"
20.89%3.01x
5.94 % 1.27x
1.36 % 0.31 b,x
0.;29 % 0.17Y
0.40 % 0.08"
2.17 % 0.33
3.13
0.10 % 0.07x
0.41 % 0.08x
2.79 % 0.72x
3.67 % 0.83x
% 0.54"
39.62 % 1.67"
6.15%0.74"
6.54 % 0.93"
43.32 % 3.80b,x
7.45 % 1.62x
6.02 % 1.07x
15.25:t0.99Y
14.33 :t 0.59
13.58 :t 0.99Y
13.51 :t 0.95x,y
1.73:t0.31Y
16.71 :t 1.22b,y
3.77 :t 0.51 b,y
0.90:t0.l1b,y
0.16 :t 0.07x,y
0.69 :t 0.17b,y
2.00 :t 0.23Y
7.18:t1.04b,y
36.61 :t 1.99b,y
10.03:t 1.17b,y
3.71 :t 0.55b,y
16.66 :i: 0.83Y
14.32 :i: 0.77
14.36 :i: 0.84Y'z
13.97:i:l.05Y
1.77:i:0.34Y
15.92:i:l.21b,y
3.16:i: 0.52b,y
0.78:i: 0.09b,y
0.17 :i: 0.07Y
0.75:i: 0.19b,y
1.91 :i: 0.20Y
8.08 :i: 1.56b,z
35.59 :i: 1.96b,y
11.14 :i: 1 :62b,y
3.28 :i: 0.64b,y
"Mean % SO. Variables were analyzed using repeated-measures ANOVA. Tukey's studentized range test was used to determine statistical differences between means for time and treatment effects. Treatment comparisons are indicated by ",b and time comparisons by x-z within each group. Means with the
same superscripts are not different. P < 0.05. For abbreviation see Table 2.
bCompleters analysis (n = 33) GROUP'TIME interactions: 16:0, P< 0.027; 18:1n-9, P< 0.006; 20:3n-6, P< 0.0006; 20:4n-6, P< 0.0001; 22:4n-6, P<
0.0001; 22:5n-6, P < 0.0001; 20:5n-3, P < 0.0001; 22:5n-3, P < 0.0004; 22:6n-3, P < 0.0001; Ln-6, P < 0.0001; Ln-3, P < 0.0001; Ln-6/'I.n-3, P < 0.0001.
Clntent-to-treat analysis (n = 47) GROUP'TIME interactions: 16:0, P < 0.07; 18:1, P < 0.01; 20:3n-6, P < 0.001; 20:4n-6, P < 0.001; 22:4n-6, P < 0.0001;
22:5n-6, P < 0.0001; 20:5n-3, P < 0.0001; 22:5n-3, P < 0.0001; 22:6n-3, P < 0.0001; Ln-6, P < 0.001; Ln-3, P < 0.0001; Ln-6/'I.n-3, P < 0.0001.
A treatment effect was observed for frequent urination scores
that decreased more in the PUFA group (29.7%) (P < 0.05),
but no other group differences were observed. Within group
changes, dry skin scores improved 45.6% in the olive oil
group (P < 0.004) and 50.1 % in the PUFA group (P <
0.0002). Dry hair scores improved 36.5% (P < 0.13) in the
olive oil group and improved 42.6% (P < 0.004) in the PUFA
group. Although excessive thirst scores improved 33.3% in
the PUFA group, this was not significant (P < 0.06). There
were no within-group differences for dandruff, brittle nails,
or follicular hyperkeratosis.
Effects of supplementation on behavior and cognitive functions. The effects of PUFA supplementation on scoresfor multiple behavioral measuresare summarized in Table 6. The pri-
mary subjective behavioral outcome measureschosen were the
ASQ-P and the DBD Rating Scale for parents. Both treatment
effects and within-group changes were evaluated. There was a
significant decrease in the ASQ-P score from baseline within
both the olive oil group (16.4 %, P < 0.02) and the PUPA group
(23.2%, P < 0.006), but no treatment effect was observed.
A significant improvement in scores at 4 mon compared
with baseline was observed within both supplementation
groups for the DBD assessment by parents of hyperactivity
(37.8-40.2% decrease),attention (29.3-34.7% decrease),and
oppositional/defiant behavior (24.3-28.8% decrease), but no
treatment effect was observed (Table 6). In contrast, for
parents' DBD assessment of conduct, only the PUFA-supplemented group showed significant improvement (42.7%
TABLE5
Tocopherol Comparisons in Plasmaand RBCat Baseline,2 mon, and 4 mon <1Ig/mL)a-C
Tocopherols
8.55:t2.12
9.02
:t2.91
1.74:t:O.6SX
0.98 % 0.48a,y
4.65 :j: 1.12
6.21 :I: 2.46
1.40:!: 0.37
1.54:tO.51a
1.85x
3.38 :t 3.80b,y
13.05 :t 2.87b,y
2.03 :t 0.45x
0.61 % 0.39b,y
0.30 % 0.23b,y
5.57:1: 1.593
4.23 :t O.87x
7.86:!: 2.48Y
6:70:1: 1.63b,y
1.81 :t O.49a
.44:t 0.40'
0.83:1: 0.31b,y
0.92
8.12 :t 2.6Sa
.01 :I: 0.51 a,y
8.37:t
"Mean :t SO. Variables were analyzed using repeated-measures
ANOVA.
Tukey's
tween means for time and treatment effects. Treatment comparisons
are indicated
studentized
range test was used to determine statistical
by a,b and time comparisons
by x-z within each group.
same superscripts are not different. P < 0.05. For abbreviation
see Table 2.
bCompleters
analysis (n = 33) GROUP"TIME
interactions:
plasma a-tocopherol,
P < 0.0001;
0.0001; RBC y-tocopherol,
P<O.OOOl.
Clntent-to-treat
analysis (n = 47) GROUP"TIME
0.0005;
RBC y-tocopherol,
interactions:
plasma
a-tocopherol,
P < 0.001;
:I: 0.30b.y
differences beMeans with the
plasma
y-tocopherol,
P< 0.0001;
RBC a-tocopherol,
P<
plasma
y-tocopherol,
P < 0.0001;
RBC a-tocopherol,
P<
P < 0.0001.
Lioids. Vol. 38. no. 10 (2003}
STEVENSETAL.
1014
TABLE6
PercentageChange in Scoresfrom Baselineto 4 mon for PUFA and Olive Oil for Completersa
P for difference
Absolute change (range)
Parentscores
Thirst/skin symptoms total score
Olive oil
PUFA
ASQ-Parents
Olive oil
PUFA
DBD Hyperactivity
Olive oil
PUFA
Percentagechangeb
pfor difference within group
between
groups
,
2..9(-2 to14)
2.9 (-1 to 9)
28.0
44.3
0.005
0.0002
0.25 (0.08)
2.9(-3009)
4.3 (-3 to12)
16.4
23.2
0.02
0.006
0.29 (0.19)
8.5 (1 to 20)
7.9 (3 to 19)
37.8
40.2
0.0001
0.0001
0.68 (0.28)
29.3
34.7
0.0002
0.0001
0.46 (0.24)
9.9
42.7
0.09
0.0005
0.08 (0.05)
24.3
28.8
0.02
0.002
0.88 (0.55)
-2.5
12.3
0.71
0.29
.0(0.94)
44.3
44.9
0.92
0.32
0.71 (0.97)
-3.4
14.8
1.00
0.009
0.09 (0.03)
1.9
-18.2
1.00
0.74
0.69 (0.41)
-2.3
-1.3
0.96
0.53
0.53 (0.19)
0.006
0.99
0.28 (0.13)
0.43
0.05 (0.05:
(ITT)C
DBD Attention
Olive oil
6.6 (0 to 18)
PUFA
6.8 (0 to 15)
DBD Conduct
Olive oil
2.1 (-3 to 9)
PUFA
3.4 (0 to17)
DBD Oppositional/Defiant Disorder
Olive oil
4.5 (-7 to14)
PUFA
4.4 (-4 to 20)
Teacher scores(n = 26)d
ASQ-Teachers
Olive oil
1.9 (-3 to 8)
PUFA
1.4(-9to11)
DBD Hyperactivity
Olive oil
-0.4 (-14 to 6)
PUFA
2.5 (-9 to18)
DBD Attention
Olive oil
0.2 (-6 to 9)
PUFA
4.6 (-2 to17)
DBD Conduct
Olive oil
O.O(-ltol)
PUFA
0.2 (-5 to 5)
DBD Oppositional/Defiant Disorder
Olive oil
0.1 (-4 to 4)
PUFA
0.8 (-10 to14)
Conners' CPT (n = 32)
CPT Indexe
Olive oil
-3,9 (-12 to 6)
PUFA
-0.2 (-15to21)
Hit reaction time (ms)
Olive oil
-12.3 (-85.5 to 71.8)
PUFA
25.1 (-63.2 to 105.8)
Hit reaction time (T-score)
Olive oil
3.1 (-11.3 to 11.2)
PUFA
-3.2 (-19.0 to 13.2)
Woodcock-Johnson Psycho-EducationalBattery-Revised
Processingspeed
Olive oil
-0.1 (-Bto13)
PUFA
0.9 (-11 to17)
Short-term memory
Olive oil
-0.8 (-10 to17)
PUFA
-1.4 (-20 to 26)
Visual processing
Olive oil
-4.9 (-23 tol0)
PUFA
-3..7 (-21 to16)
Auditory processing
Olive oil
-5.1 (-23to14)
-5.4 (-21 to 7)
PUFA
-2.4
5.6
0.08
12.6
-9.08
0.12
0.16
0.02 (0.12)
-"1 :0
0.9
0.69
0.56
0.54 (0.66:
0.9
-1.5
0.96
0.68
0.55 (0.96)
-3.7
-4.3
0.09
0.19
0.69 (0.88:
-5.3
0.09
0.86 (0.74]
-5.8
0.02
an = 33 unless otherwise stated. For abbreviations see Table 1.
bpercentagechange is calculated as (value at baseline - value at 4 mon)/(value at baseline) x 100, using meanvalues.A positive percentagechangeindicates improvement, except for CPT(T-scoresonly) and Woodcock-Johnson tests, which are basedon standardscores.
cP-valuecompleters(P-value intent-to-treat).
rfreacher evaluations were completed at 2.5-3 mono
esimple change rather than percentage change due to some zero values at baseline.
Lipids, Vol. 38, no. 10(2003)
EFA SUPPLEMENTATION
1015
IN CHILDREN WITH DISRUPTIVE BEHAVIORS
decrease, P < 0.01); the treatment effect fQr the comp1eters
analysis was not significant (P < 0.08), whereas the ITT
analysis was significant (P < 0.05).
The teachers' assessment of behavior using the ASQ for
teachers (ASQ- T) and DBD questionnaires were chosen as
secondary outcome measures for several reasons. First, the
teachers interacted mostly with the participants on medication; thus, their evaluation of each child's behavior did not
agree with that of the parents. Second, the teachers' final assessments during the intervention occurred at the end of the
school year, which was a month prior to the end of the intervention period. Finally, data collection at the end of the study
was incomplete for the teachers. A treatment effect for the
teachers' DBD attention subscale was not significant for the
completers but was significant based on the ITT analysis (P <
0.03). Within the PUFA group, the attention score improved
14.8% (P < 0.009), with no change in the olive oil group.
None of the other teacher DBD and ASQ- T outcome measures was affected by supplementation.
For the CPT tests, differences were observed within each
group. The average CPT Index score for the children on PUFA
supplementation did not change from baseline to 4 mon,
whereas that for the placebo group increased (P < 0.006). A
deterioration in performance on this objective outcome measure was not unexpected, as the CPT often shows a negative
practice effect (66). No treatment effect was observed on the
CPT Index. Of the components of the test compiled in the
index, only HRT showed a treatment effect (P < 0.05, compIeters and ITT). HRT is a highly sensitive index of how
quickly targets are processed (66), and a slow HRT is indicative of inattention (76). For the WJ-R tests of cognitive skills,
auditory processing improved 5.8% (P < 0.02) in the PUFA
group. No other group or within-group differences were found.
Effect of supplementation on clinical outcomes. In addition to total scores, the DBD Rating Scale results were also
evaluated based on diagnostic criteria, which is typically how
these assessment tools are used in clinical practice. The results are summarized in Table 7. At baseline there were no
significant differences between the groups in terms of the proportion of participants with scores in the clinical range for
each subscale for either the parents or the teachers. With supplementation, treatment effects were observed for the parents'
Oppositional/Defiant Disorder subscale. No other clinical
outcome was affected by PUFA supplementation.
Association between FA composition and performance on
behavioral measures. Because this pilot study was designed,
in part, to evaluate the relationship between proportions of
PUFA in the blood and the severity of behavioral symptoms,
we compared the changes in the proportion of FA and tocopherol in the erythrocytes with the changes in scores on behavioral outcomes. This was done using correlations between the
percentage change from baseline, [(baseline - 4 mon)/baseline]. 100, for each PUFA and behavioral assessment.Pearson
correlations were conducted on normally distributed data and
Spearman correlations for the rest not normally distributed,
as indicated in Table 8. A negative correlation between a FA
or a-tocopherol and a behavioral score indicates that a positive change in the biochemical parameter is associated with a
negative change in the behavioral parameter, whereas a positive correlation indicates the reverse. Negative changes signify improvement for all parameters except the WoodcockJohnson tests. For this outcome measure, increasing values
signify better performance. The percentage change in ASQ-P
scores was negatively correlated with the percentage change
in RBC EPA and positively correlated with RBC AA. Although no correlations were significant for the parents' DBD
TABLE7
Effects of Supplementation on Clinical Outcomes
ParentsDBDd
Hyperactivity
Attention
Conduct
Oppositional/Defiant Disorder
12
13
4
11
(80)
(86.7)
(26.7)
(73.3)
n=9
14 (78)
13 (72)
5 (27.8)
12 (66.7)
n = 17
NS
NS
NS
NS
5
4
3
9
9
5
8
NS
0.09
NS
0.02
1
1
0
1
4
4
0
le
NS
NS
NS
NS
3 monb
Teachers DBDd
Hyperactivity
Attention
Conduct
Oppositional/Defiant Disorder
1 (11.1)
4 (44.4)
0(0)
2 (22.2)
5 (29.4)
6 (35.3)
0(0)
2 (11.8)
NS
NS
NS
NS
'Participantsmeeting the criteria for clinical diagnoses based on parent and teacher DBD subscales at baseline.
~he number of participants who improved enough that they no longer met the criteria at the end of the study.
CStatistical analysis using Fisher's exact test.
dDiagnostic criteria were as follows: parents' or teachers' responses of 2 ("pretty much") or 3 ("very much") for at least six
questions on the hyperactivity rating scale, at least six questions on the attention rating scale, at least three questions on the
conduct rating scale, or at least four questions on the Oppositional/Defiant Disorder rating scale. For abbreviations see
Table 1.
'Three participants did not meet criteria at baseline but did at 3 mono
lipids, Vol. 38, no. 10 (2003)
L. STEVENSETAL.
1016
TABLE 8
Correlationsa Between Percentage Change in EFA and Tocopherols in RBC and Percentage Change in ThirsUSkin
Symptoms Score, Behavioral Assessments, and Cognitive Skills at 4 mon (n = 31)
FA
Total thirst/skin symptoms score
ASQ-Parents
Parents DBD rating scale
Hyperactivity
Attention
Conduct
Oppositional/Defiant
Disorder
ASQ- Teachers (n = 24)
Teachers DBD Rating Scale (n = 24)
Hyperactivity
Attention
Conduct
Oppositional/Defiant Disorder
CPT (n = 30)
Overall Index
Hit reaction time
-0.05
0.36a
0.16
0.18
0.16
0.05
0.01
-0.07
0.11
-0.01
0.12
0.17
0.19
0.34
-0.04
0.32
-0.15
-0.19
0.08
-0.06
-0.20
-0.32
-0.14
0.03
-0.20
-0.13
0.08
0.18
-0.07
0.07
-0.21
-0.15
-0.20
-0.38
-o.49a
-0.15
-0.32
-0.02
-D.21
-0.08
-0..10
-0.03
-0.28
-0.39
0.14
0.39
0.13
0.33
-0.19
-0.31
0.08
0.31
-0.51"
-0.45"
-O.GOb
-0.41
"
-o.54b
-0.27
-0.46"
'Spearman correlation coefficients: .p S 0.05, bp S 0.01. Pearson correlations were used when both blood and behavi
data were normally distributed. AA, arachidonic acid; for other abbreviations see Table 1.
Rating Scale,all of the subscalesof the teachers'DBD Rating Scalecorrelated negatively with the changein RBC atocopherolconcentration.This was also true for the ASQ-T.
Additionally, the Attention subscale of the teachers' DBD
Rating Scalewas negatively correlatedwith the percentage
changein the proportion of RBC DHA. RBC a-tocopherol
scoreswere negatively correlatedwith the HRT of the CPT.
Also, a positive correlation was found for the processing
speedcomponentof the WJR andthe RBC n-6/n-3ratio.
DISCUSSION
The purposeof this study was to determinewhether supplementationwith n-3 FA-enrichedPUFA might benefitchildren
with AD/HD-like symptomswho reporteda high frequency
of thirst and skin symptoms.We had previously shown that
children with AD/HD-like and thirst/skin symptoms had
lower proportions of AA, EPA, and DRA in their plasma
phospholipidsin comparisonwith otherchildrenwith AD/HD
not exhibiting thesethirst/skin symptoms(42). Data from the
literaturefor the PUFA compositionof plasmaphospholipids
for healthychildren vary acrosspublications.For AA, for instance, proportions range from a mean of about 6% for
healthy Polish children (77) to a high of about 12.5% for
healthy children from the United States(78). For DRA, the
proportionsin the literaturefor healthychildrenrangefrom a
low of about 1.9% for American children (78) to a high of
about 2.6% as reported from a study of apparently healthy
Germanchildren (79). For FA proportions in erythrocytes,
AA rangesfrom a low of about 16.4% for healthy German
childrento a high of 26.3%for normal girls and boys in New
Zealand(80). Similarly, proportionsof DRA in erythrocytes
havebeenreportedto vary from about2.2% (40) for healthy
children in the U.S. Midwest to 6.3% for children from New
Lipids. Vol. 38. no. 10 (2003)
Zealand (80). In these different publications, the range in the
proportions of the two main families of PUFA varies considerably across populations, as much as 100%. Factors that contribute to this variation include differences in methodology as
well as genetic background and food intake patterns (81). The
variation within a reference sample is much smaller, generally 10-20%; thus, the most appropriate sample for a study is
one drawn from the local population having similar characteristics and food intake patterns. For these studies we recruited a reference sample of the same age range and with a
similar dietary intake. Proportions of plasma phospholipid FA
proportions were statistically lower for the intervention sample in comparison with the reference sample for AA (8.9 vs.
10.5%, Table 2) and DHA (1.7 vs. 2.2%), and this result is
similar to our previous report (40). Paradoxically, the proportions of AA and DHA in RBC were greater in the intervention sample compared with the reference sample of healthy
children with no behavioral problems (AA: 20.2 vs. 18.2%;
DHA: 7.1 vs. 5.7%), an observation that differs from what we
previously found. Additionally, a comparison of these values
with literature data revealed that the proportions were not outside those reported for some samples of healthy children. Perhaps focusing on the absolute proportions of n-6 and n-3 FA
in the plasma and/or the RBC is not as important as focusing
on whether a change in the proportions of these FA leads to
an improvement in outcome measures.
We chose a supplement that contained the specific preformed PUFA that were low in the plasma phospholipids of
the reference sample from a previous study (42). The supplement was enriched in n-3 relative to n-6 FA, but contained
both families of EFA.. The PUFA supplement contained a
greater proportion ofDHA than EPA (6:1), and this was desired because we hypothesized that the lower proportion of
DHA in the blood was reflecting a decreased proportion of
EFA SUPPLEMENTATION
IN CHILDREN WITH DISRUPTIVE BEHAVIORS
this important FA in the brain and thus might mediate the abnormal neuronal signaling that results in aberrant behaviors
(82). This was based on previous studies demonstrating that
changes in the proportion of EFA in plasma and RBC were
also reflected in the brain (83). However, although this assumption may be true in children for muscle tissue (84), recent studies suggest it may not be true for brain tissue (85).
The relationship between plasma phospholipids, proportions
in RBC, and brain chemistry needs to be explored further in
these children.
We predicted that the proportions of AA, EPA, and DHA
in the blood would increase by supplementing the diet with
PUFA. Several studies have shown that proportions of PUFA
in the blood can be increased by consumption of preformed
PUFA. Consumption of marine oils, rich in n-3 PUFA, has
been shown to lead to an increase in the proportions of EPA
and DHA in the serum and blood cells of healthy adults (86).
Similarly, a study with newborn infants demonstrated that increasing the concentration of DHA in the formula led to an increase in the proportion of this FA in the blood (87). A previous study of school-age children with behavior problems
tested the effects of supplementing an oil source rich in the n-6
FA GLA, and found that increasing the proportion of this FA
in the diet led to an increase in the amount in the blood (54).
The results from this study show that daily oral supplementation with 480 mg DHA and 80 mg EPA led to at least a
doubling in the mean proportion of these two FA in both the
plasma phospholipids and the RBC. Supplementation with a
combination of the n-6 FA GLA, at 96 mg/d, and AA, at 40
mg/d, along with the n-3 PUFA, led to approximately a 25%
increase in the proportion of AA in plasma, but approximately
a 25% decrease in the RBC. There was also a decrease in the
composition of two other long-chain n-6 FA, 22:4n-6 and
22:5n-6. This decreasein the proportion of n-6 FA in the RBC
was probably due to the large increase in the proportion of the
n-3 FA in the RBC membranes.
Not only are the proportions of each n-6 and n-3 FA key to
influencing membrane function but the ratio between total n-6
and total n-3 is also important (43). The n-6/n-3 ratios in
plasma and RBC were significantly influenced by supplementation, which led to significant decreasesin the PUFA group
(9.35 to 4.06 in plasma, 6.02 to 3.28 in RBC), whereas the
ratio in the placebo group remained constant over the 4 mono
This antithetical relationship between n-3 and n-6 PUFA has
been noted previously (88,89).
We assessedthe frequency of thirst/skin symptoms in selecting a subpopulation of children with behavioral problems
on which to test the effects of supplementation. We hypothesized that children exhibiting thirst/skin symptoms would be
more likely to show effects of PUFA supplementation than
those not reporting these EFA-deficiency-related symptoms.
As noted previously, it is unlikely that the thirst/skin symptoms resulted from a primary EFA deficiency in these children. Similar types of thirst/skin symptoms have been reported to occur in patients suffering from a variety of different conditions (14-36). Although improvements have been
Vol. 38. no. 10 (2003:
1018
l. STEVENS ET AL.
beenas clear (93,94). Proportions of n-3 FA in membranes
havebeenreportedto influenceaggressivebehaviors(95-97),
as well as to moderatethe severity of depressivesymptoms
(51). Few studieshaveaddressedthe relationshipbetween(Xtocopherolconcentrationsin membranesand psychiatricdisorders.However, a recentpaper (98) reportedimprovement
in schizophrenicsusing EFA supplementsalong with vitamins E and C. Thus, further studies are warranted into the
mechanisticrelationshipbetweenthe membranecomposition
of AA, EPA, DHA, and (X-tocopheroland the neurological
pathwaysthat influencethesebehaviors.
The preliminary interventionstudyreportedherehad several confounding variables or limitations that should be
avoided in future studies.First, participants were accepted
into the studybasedon the parents'confirmationof a clinical
diagnosisby a qualified doctor,but the diagnosiswas not reconfirmed by a collaborating psychiatrist or psychologist.
Barkley (3) hasreportedthat symptomsof AD/HD are similar to manyotherconditionsincluding visual or hearingproblems, lead poisoning, anemia,sleepdisorders,unrecognized
learning disabilities, mild mental retardation,pervasivedevelopmentaldisorders,depression,and anxiety disorders.In
a study of children who had beendiagnosedwith AD/HD by
their own physicians,only 72% qualified as having AD/HD
using a structured diagnostic interview (99). Thus, participantscan be consideredas only "exhibiting AD/HD symptomology." Therefore,future studiesshould confirm the diagnosis basedon DSM-IV criteria (56). Second,the olive oil
placebowas "active" in that the supplementdid not maintain
the baseline plasma phospholipid PUFA composition. To
more accuratelyconduct a treatmenteffect comparison,the
useof a different oil, suchas liquid paraffin oil (100), asthe
placeboin supplementationstudieswould be more appropriate.Another limitation was the timing of the interventionrelative to the school year.Becausethe recruiting and baseline
testing processtook 6 mon, the intervention componentdid
not begin until March. This resultedin the final testing outcomeassessments
being completedat different times for the
teachersand parents.Thus, future studiesshouldbe designed
to start earlier in the school year and be completedprior to
the onsetof summervacation.Another factor that may have
affectedthe resultsis the useof medication.The studyprotocol did not interferewith eachparticipant'streatmentregimen
and, as such, somechildren stoppedmedication during the
summermonths,which wasthe 4-montestingtime point. Statistical testing did not reveal confoundingevidencefor medication or a changein medicationfor the behavioroutcomes,
but this practice may still have have obscured or overwhelmeda treatmenteffect of the PUFA. Finally, the sample
size was small and did not provide adequatepower in all
cases.Future studiesshouldinclude a larger samplesize and
strive for improved retention to provide adequatestatistical
powerfor assessingbehavioralchange.
In summary,children with AD/HD-type symptomswho
reportedfrequentthirst/skin symptomsand undera variety of
standardtreatmentsfor their behavioralproblemsparticipated
Lipids, Vol. 38, no. 10 (2003)
in a double-blind intervention trial comparing a PUFA supplement enriched in DHA, EPA, and (X-tocopheryl acetate vs.
olive oil placebo. Supplementation with the PUFA led to up
to a twofold increasein the proportionsof EPA, DHA, and(Xtocopherol in the plasma phospholipids and RBC total lipids,
but a small increase was noted in the plasma phospholipid
proportions of n-3 FA with olive oil as well. A clear benefit
from PUFA supplementation for all behaviors characteristic
of AD/HD was not observed; however, treatment effects for
conduct (parents) and attention (teachers), together with clinical improvements in Oppositional/defiant behavior, along
with significant associations between the magnitude of the
change in RBC FA and vitamin E vs. improvements for some
behavioral outcome measures support further research into
these relationships.
ACKNOWLEDGMENTS
This trial wasfundedby grantsfrom the NationalInstituteof Mental
Health (# RO3 MH56414) and from Scotia Pharmaceuticals,Ltd.
Wen ZhangandAnne Mahonweresupportedin part by a grantfrom
the National FisheriesInstitute. We thank Dr. Mary Campbell for
her work in administering learning tests to the participants with
AD/HD.
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