Eur J Nutr

DOI 10.1007/s00394-017-1528-6

ORIGINAL CONTRIBUTION

Supplement intervention associated with nutritional deficiencies

in autism spectrum disorders: a systematic review
Yong‑Jiang Li1,2 · Ya‑Min Li3 · Da‑Xiong Xiang1,2 

Received: 5 July 2017 / Accepted: 9 August 2017

© Springer-Verlag GmbH Germany 2017

Abstract  and to improve the symptoms is little. More studies are

Purpose  Nutritional supplements have been used for cor-
rection of deficiencies that may occur in patient with autism
spectrum disorder (ASD) and to improve core symptoms.
We aim to provide current best evidence about supplements
for nutritional deficiencies and core symptoms in children
with ASD and to evaluate the effectiveness and safety.
Methods  A systematic literature search of scientific data-
bases was performed to retrieve relevant randomized con-
trolled trials. Risk of bias was assessed for each study.
Results  18 randomized controlled trials of five supple-
ments were included. B6/Mg was not helpful for improving
ASD symptoms (seven RCTs). Two RCTs of methyl B12
reported some improvement in ASD severity but the effects
on the correction of deficiencies were inconclusive. Two
RCTs of vitamin ­D3 both reported increased levels of mean
25(OH)D in serum but inconsistent results in behavioral out-
comes. Omega-3 fatty acid supplementation did not affect
ASD behaviors but may correct deficiencies (six RCTs). One
RCT of folinic acid reported positive results in improving
ASD symptoms measured by various behavioral scales.
Conclusions  Current evidence for the use of supplements
for correcting nutritional deficiencies in children with ASD
* Da‑Xiong Xiang
xiangdaxiong@163.com
1 deficiencies in ASD on symptoms of ASD. However, those
Department of Pharmacy, The Second Xiangya Hospital,
Central South University, Changsha 410011, Hunan, China
2 study design and methodological issues. In consideration
Institute of Clinical Pharmacy, Central South University,
Changsha 410011, Hunan, China
3 are often hard to learn the safety and effectiveness of such
Clinical Nursing Teaching and Research Section, The
Second Xiangya Hospital, Central South University,
Changsha 410011, Hunan, China
needed.
Keywords  Autism spectrum disorder · Core symptoms ·
Nutritional deficiency · Supplements
Introduction
Autism spectrum disorder (ASD) is defined as a class of
neurodevelopmental conditions characterized by impair-
ments in social communication, developmental delay, and
repetitive or ritualistic behaviors [1]. The etiological back-
ground of ASD is not clear. But genetic, neurologic, meta-
bolic, and immunologic factors are believed to be involved
in the complex pathophysiological process [2–4]. Currently,
no medication is available for the core symptoms of ASD.
Several interventions such as education and pharmacological
agents may have benefits [5–9], but their success is limited
because of safety, high variability of ASD, and difficulty in
behavioral evaluation. Therefore, there is an urgent need for
additional interventions [10].
Observational studies and anecdotal evidence have
confirmed that several nutritional deficiencies may occur
in some patients with ASD such as vitamins [11–14],
omega-3 fatty acids [15, 16], minerals [16], and folate [17,
18]. Hence, an increasing number of studies have investi-
gated the effects of supplements associated with nutritional
studies have yielded inconsistent outcomes due to diverse
of limitations in current research, families and caregivers
nutritional supplements.

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A recent systematic review has evaluated amino acids,
fatty acids, and vitamins/minerals. However, it only focused
on clinical aspects of ASD and did not discuss all supple-
ments [19]. Hence, the aim of our systematic review is to
comprehensively summarize current best evidence about the
safety and effect of supplements associated with nutritional
deficiencies in ASD.
Methods
Search strategy and study selection
A systematic literature search of scientific online databases
was conducted via PubMed, Embase, and Cochrane Library
from their inception to June 2017 using Medical Subject
Headings (MeSH), EMTREE headings, and relevant key
words to find studies published in English and related to
correction of nutritional deficiencies in ASD. The search
terms were as follows: ‘‘nutrition/dietary supplements/
vitamins/pyridoxine/ascorbic acid/magnesium/iron/miner-
als/fatty acids/folic acid/folinic acid” and “autism/autism
spectrum disorder/Asperger/autistic”. In addition, a second-
ary search was conducted by reviewing the reference lists
of included articles and other relevant reviews to further
identify additional studies that were potentially eligible for
inclusion criteria. The electronic literature search process
was performed by two independent investigators using a
standardized approach.
Study selection and inclusion criteria
Records were screened for eligibility of inclusion and a study
was included if it met all the following inclusion criteria:
1. The study was a randomized clinical trial;
2. The study population was pediatric or adolescent
groups;
3. The study measured symptoms of ASD as the primary
outcome;
4. The study must include at least ten participants;
5. The intervention duration was at least 2 weeks;
6. The study addressed supplements associated with nutri-
tional deficiencies in patients with ASD.
Quality of evidence
The assessment of overall methodological risk of bias of
each study was performed by refereeing the ASD-specific
assessment approach developed by the Vanderbilt Evidence-
based Practice Center for reviews of interventions for ASD
[20]. Quality levels were good, fair and poor in regards to
low, moderate, and high risk of bias ratings [21].
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Eur J Nutr
Assessment of risk of bias was based on eight domains:
(1) study design; (2) comparison; (3) diagnostic approach;
(4) inclusion and exclusion criteria; (5) description of inter-
vention; (6) outcome measures; (7) precision of effect; (8)
statistical analysis [20]. This process of risk of bias assess-
ment was independently performed by two reviewers. In
additon, the final score of each study was judged by the pri-
mary author after referring to the original article and the
opinions from the two reviewers.
Results
Description of studies
A total of 1914 records were identified in our initial search.
After removing duplicates and screening records, 15 publi-
cations (18 RCTs) that fully met the inclusion criteria were
retrieved (Fig. 1). There were five different supplements
for correction of nutritional deficiencies in ASD, including
vitamin B6/Mg [22–25], methyl B12 [26, 27], vitamin D3
[28, 29], omega-3 fatty acids [30–35], and folinic acid [36].
Several supplements, including iron [37] and folic acid [38],
that were associated with nutritional deficiencies in ASD but
have not been tested by RCT were excluded as well. Charac-
teristics of the 18 included RCTs are presented in Table 1.
The sample sizes ranged from 13 to 109 and supplement
intervention durations ranged from 2 to 35 weeks. In our
assessment of the risk of bias, three RCTs had a low risk of
bias, nine had a moderate, and six had a high risk (Table 2).
Vitamin B6/Mg
Vitamin B6 (pyridoxine) and magnesium (Mg) have been
shown to be involved in the synthesis of several important
neurotransmitters such as serotonin and dopamine, which
were reported to be abnormal in some patient with ASD
[39, 40]. Thus, supplementation of vitamin B6/Mg might
be useful for improving symptoms of ASD.
The first RCT was published in 1981 (high risk of bias)
[23]. The dose was B6: 625–1125 mg/day + Mg lactate:
400–500 mg/day. 13 responders and 8 nonresponders entered
the double-blind crossover with placebo (2 weeks each),
and 10 responders versus 2 nonresponders showed more
improvement in communication and in general responsive-
ness measured by Bretonneau II Clinical Scale on B6/Mg
supplementation than on placebo. However, a double-blind
RCT (moderate risk of bias) [22] reported no benefit in ten
children with autism treated with B6: 30 mg/kg/day + Mg
oxide: 10 mg/kg/day for 10 weeks. In another RCT study of
60-day hospital patients with ASD (high risk of bias) [24],
four crossover trials were performed with each trial lasting
8 weeks (2 weeks for baseline, 2 weeks for first treatment,
Eur J Nutr

Fig. 1  Flow diagram for selec-

tion of studies about the use

Idenficaon
of supplements for nutritional Records idenfied through Addional records idenfied
deficiencies in children with database searching through other sources
autism spectrum disorder (n = 1909) (n =5)

Records aer duplicates removed

(n = 1314)

Screening
Records of irrelevant
Records screened
topics excluded
(n = 1314)
(n =1194)
Eligibility

Full-text arcles excluded,

Full-text arcles assessed with reasons
for eligibility (n = 105)
(n = 120)  Open-label studies
(n = 8)

 Small sample size

(n = 1)

 Not original research

Included

(n = 38)

Papers included in  Not address outcome

qualitave synthesis of interest
(n =15) (n = 59)

2 weeks for second baseline, and 2 weeks for second treat-
ment). Doses were B6: 30 mg/kg/day up to 1 g/day and/or
Mg lactate: 10–15 mg/kg/day. The first crossover involved
16 patients and showed improvement in overall behavior
rating scores of Behavior Summarized Evaluation (BSE)
for both the combination and for Mg lactate alone; the sec-
ond crossover involved 21 patients, they reported decreased
score for the categories of autistic symptoms in BSE for the
combination, but results for the placebo comparison was not
shown; in the third (B6 versus placebo, N = 35) and fourth
(Mg lactate versus placebo, N = 37) crossover, there was no
significant difference between treatment groups. In a double-
blind, asymmetric crossover RCT (high risk of bias) with
longer duration (5-week baseline and 3- and 10-week treat-
ment blocks) [25], the combination of B6 (200 mg/70 kg/
day) and Mg gluconate (100 mg/70 kg/day) showed no treat-
ment effect on the severity of symptoms of ASD.
Although no significant adverse events have been
observed to occur with vitamin B6/Mg in ASD in above
RCTs, the risk of neuropathy from B6 and the risk of diar-
rhea from Mg when high doses administrated have been
identified [41, 42].
Methyl B12
Studies have reported laboratory changes such as lower
plasma methionine, S-adenosylmethionine (SAM), homo-
cysteine, cystathionine, cysteine, and total glutathione,
which were associated with transsulfuration metabolism and
reduced antioxidant capacity, in children with ASD [43, 44].
Therefore, vitamin B12 has been used for elevating methyla-
tion capacity and improving the ‘‘redox status’’ of children
with ASD as it is a vital cofactor for the regeneration of
methionine from homocysteine by providing methyl groups
for the transmethylation and transsulfuration metabolic path-
ways [45].
In a pilot study (moderate risk of bias), Bertoglio et al.
[26] first published the results of a RCT of methyl sup-
plementation on 30 children with ASD. They observed
clinically significant improvement on the Clinical Global
Impression (CGI) Scale. But no statistically significant
mean difference was identified in other clinical measures
or in glutathione status between active and placebo groups.
Recently, they published their new findings on the RCT
(low risk of bias) of 57 children aged 3–7 years with ASD
[27]. The results on core symptoms of ASD were consistent

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Table 1  Summary of RCTs evaluating the effect of supplements for nutritional deficiencies in ASD
References Study design Subjects (N, Supplements Control Duration Laboratory outcomes Behavior outcome Behavior benefits

13
age in years) measure

Vitamin B6/Mg
 Lelord et al. [23] Double-blind, 21, 3–16 B6: 625–1125 mg/ Placebo Crossover; 2 weeks Not reported Bretonneau II Potential improvement
crossover RCT day each Clinical Scale in communica-
Mg lactate: tion and in general
400–500 mg/day responsiveness
 Martineau et al. Double-blind, 16, 3–14 B6: 30 mg/kg/day Mg lactate Crossover; 2 weeks Decrease of the urinary BSE Decreased score in
[24] crossover RCT Mg lactate: each HVA level overall behavior rat-
10–15 mg/kg/day ing scale
 Martineau et al. Double-blind, 21, 3–14 B6: 30 mg/kg/day Placebo Crossover; 2 weeks Significant decrease of BSE Decreased score for
[24] crossover RCT Mg lactate: each the urinary HVA level the categories of
10–15 mg/kg/day autistic symptoms
 Martineau et al. Double-blind, 35, 3–14 Mg lactate: Placebo Crossover; 2 weeks No change in the urinary BSE None
[24] crossover RCT 10–15 mg/kg/day each HVA level
 Martineau et al. Double-blind, 37, 3–14 B6: 30 mg/kg/day Placebo Crossover; 2 weeks No change in the urinary BSE None
[24] crossover RCT each HVA level
 Tolbert et al. [25] Double-blind, 15, 6–18 B6: 200 mg/70 kg/ Lactose as 35 weeks Not reported R-F None
asymmetric day placebo
crossover RCT Mg gluconate:
100 mg/70 kg/day
 Findling et al. [22] Double-blind, 12, 3–17 B6: 30 mg/kg/day Placebo Crossover; 4 weeks Not reported CARS, CGI, CPRS, None
crossover RCT Mg oxide: 10 mg/ each OCS, PRS, TRS
kg/day
Methyl B12
 Bertoglio et al. Double-blind, 30, 3–8 B12: 64.5 μg/kg, Saline as Crossover; 6 weeks No statistically sig- PIA-CV, CGI-I, Better CGI-I score, no
[26] crossover RCT every 3rd day placebo each nificant in glutathione CARS, PPVT-III, significant improve-
status Stanford Binet ment in other
Fifth Edition measures
Routing Subsets,
ABC, CBCL and
MCDI
 Hendren et al. [27] Double-blind RCT 57, 3–7 B12: 75 μg/kg every Saline as 8 weeks Increases in plasma CGI-I, ABC, SRS Better CGI-I score,
3rd day placebo methionine, decreases no improvement in
in SAH and improve- ABC and SRS
ments in the ratio of
SAM to SAH
Eur J Nutr
 Table 1  (continued)
Eur J Nutr

References Study design Subjects (N, Supplements Control Duration Laboratory outcomes Behavior outcome Behavior benefits
age in years) measure

Vitamin D3
 Saad et al. [28] Double-blind RCT 109, 3–10 D3: 300 IU/kg/ Placebo 4 months Mean serum levels of 25 ABC, CARS, Significant improve-
day, not to exceed (OH)D were sig- ATEC, SRS ments in all meas-
5000 IU/day nificantly elevated from ures
26.3 to 45.9 nmol/L in
the treatment group, but
not in placebo group
 Kerley et al. [29] Double-blind RCT 38, mean 7.1 D3: 2000 IU/day Placebo 20 weeks There was a significant ABC, SCQ, DD- Significant improve-
increase in mean CGAS, SRS ment in self-care on
25(OH)D from 54.2 to DD-CGAS, but not
83.8 nmol/L in the in other measures
treatment group, but not
in placebo group
Omega-3 fatty acids
 Amminger et al. Double-blind RCT 13, 5–17 EPA: 840 mg/day 1 g of coco- 6 weeks Not reported ABC No significant
[30] DHA: 700 mg/day nut oil as improvement in
placebo ABC
 Bent et al. [31] Double-blind RCT 27, 3–8 EPA: 700 mg/day Safflower oil 12 weeks Significant increases ABC, PPVT, EVT, No significant differ-
DHA: 460 mg/day as placebo of DHA and EPA in BASC, SRS, ences in all measures
the treatment group; CGI-S
Significant difference of
TNF-α between groups
 Yui et al. [35] Double-blind RCT 13, 6–28 ARA: 120 or Olive oil as 16 weeks Significant difference in ABC, ADI-R, SRS Significant improve-
240 mg/day placebo the change in plasma ment in all measures
DHA: 120 or TF levels; no significant
240 mg/day differences in plasma
levels of DHA or ARA;
no significant differ-
ences in the plasma
levels of DHA, ARA,
SOD, or TF at the end
of the study
 Voigt et al. [34] Double-blind RCT 48, 3–10 DHA: 200 mg/day 250 mg of 6 months A significant increase in CGI-I, ABC, CDI, No significant
corn oil and plasma phospholipid BASC improvement in
250 mg of DHA levels CGI-I across groups
soybean oil and in all other
as placebo measures across
groups

13


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Table 1  (continued)
References Study design Subjects (N, Supplements Control Duration Laboratory outcomes Behavior outcome Behavior benefits
age in years) measure

 Bent et al. [32] Double-blind RCT 57, 5–8 EPA: 700 mg/day Safflower oil 12 weeks Not reported ABC, SRS, CGI-I Significant improve-
DHA: 460 mg/day as placebo ment in stereo-
typy and lethargy
subscales of ABC,
no significant dif-
ferences in other
measures
 Mankad et al. [33] Double-blind RCT 37, 2–5 EPA: 1.5 g/day Olive oil and 6 months Not reported PDDBI, BASC-2, No significant
DHA: 1.5 g/day medium CGI-I, VABS-II, improvement in all
chain tri- PLS-4 measures
glycerides
as placebo
Folinic acid
 Frye et al. [36] Double-blind RCT 48, mean 7.3 Folinic acid: 2 mg/ Placebo 12 weeks Not reported CELF-preschool-2, Significant improve-
kg/day, max CELF-4, PLS-5, ment in verbal com-
50 mg/day OACIS, VABS, munication, VABS,
ABC, SRS, ABC, ASQ, BASC,
BASC, AIM, and better for FRAA-
ASQ positive participants

ASD autism spectrum disorder, RCT randomized controlled trial, HVA homovanillic acid, SAM S-adenosylmethionine, SAH S-adenosyl-l-homocysteine, DHA docosahexaenoic acid, ARA ara-
chidonic acid, EPA eicosapentaenoic acid, SOD superoxide dismutase, TF transferrin, TNF-α tumor necrosis factor-α, ABC Aberrant Behavior Checklist, ADI-R autism Diagnostic Interview-
Revised, AIM autism impact measure, ASQ Autism Symptoms Questionnaire, ATEC Autism Treatment Evaluation Checklist, BASC Behavior Assessment Scale For Children, BSE behavior
summarized evaluation, CARS Childhood Autism Rating Scale, CBCL Child Behavior Checklist, CDI Child Development Inventory, CELF clinical evaluation of language fundamentals, CGI-I
Clinical Global Impression Scale of Improvement, CGI-S Clinical Global Impression Scale of Severity, CPRS Children’s Psychiatric Rating Scale, DD-CGAS Developmental Disabilities—Chil-
dren’s Global Assessment Scale, EVT Expressive Vocabulary Test, MCDI MacArthur Communication Developmental Inventory, OACIS Ohio Autism Clinical Impression Scale, OCS NIMH
Global Obsessive Compulsive Scale, R-F Ritvo-Freeman Real Life Rating Scale for Autism, PDDBI Pervasive Developmental Disorders Behavioral Inventory, PIA-CV Parent Interview for
Autism–Clinical Version, PLS Preschool Language Scale, PPVT Peabody Picture Vocabulary Test, PPVT-III Peabody Picture Vocabulary Test-Third Edition, PRS Conners Parent Rating Scale,
SCQ Social Communication Questionnaire, SRS Social Responsiveness Scale, TRS Conners Teacher Rating Scale, VABS Vineland Adaptive Behavior Scale
Eur J Nutr
Eur J Nutr
with their prior published results, methyl B12 supplemen-
tation improved symptoms of ASD measured by the CGI-I
score but not in the parent-rated Aberrant Behavior Check-
list (ABC) or in the Social Responsiveness Scale (SRS).
However, the better CGI-I score was found to be positively
correlated with increased levels of methionine, decreased
S-adenosyl-l-homocysteine (SAH) in plasma, and a better
ratio of SAM to SAH, which indicated an improvement in
cellular methylation capacity.
No serious adverse event was observed. A number of mild
adverse events were similar in methyl B12 group (21) and in
the placebo group (24) [27].
Vitamin D
A growing number of studies have reported decreased vita-
min D levels in patients with ASD [46, 47]. In addition,
the therapeutic potential for vitamin ­D3 has been reported
by anecdotal evidence [11]. Two possible mechanisms for
vitamin ­D3 that help prevent and treat ASD have been sum-
marized in recent comprehensive reviews [12, 48]. The first
one is its anti-inflammatory effects in the brain [49]; the
second one is associated with serotonin [50].
In the first RCT (moderate risk of bias) on vitamin D ­ 3
supplementation [28], 109 children aged 3–10 years with
ASD completed the trial and the dose was 300 IU/kg/day,
not exceed 5000 IU/day. As a result, mean 25(OH)D in
serum in the treatment group was significantly increased
(26.3–45.9  nmol/L), but not in the placebo group after
4 months of supplementation of vitamin ­D3. For clinical
aspects, rated scores of behavioral scales, including Aberrant
Behavior Checklist (ABC), Childhood Autism Rating Scale
(CARS), Autism Treatment Evaluation Checklist (ATEC),
and Social Responsiveness Scale (SRS), were all signifi-
cantly better in children receiving treatment of vitamin ­D3.
The other RCT (moderate risk of bias) tested a lower dose
of vitamin ­D3 (2000 IU/day) for 20 weeks and 38 children
with ASD completed the trial [29]. In addition, there was a
significant increase in serum 25(OH)D (54.2 –83.8 nmol/L)
in the treatment group but not in the control group. However,
results showed no effect on the primary endpoint (ABC),
only an improvement in self-care on Developmental Dis-
abilities—Children’s Global Assessment Scale (DD-CGAS).
The adverse events observed for high dose of vitamin ­D3
supplementation were mild and transient with no harms to
the participants [28].
Folinic acid
Several abnormalities in the metabolism of folic acid have
been linked to ASD [51], and studies have evidenced cer-
ebral folate deficiency in autism [18]. Folic acid cannot be
synthesized by human body and thus is dependent on dietary
sources or supplementations, while folinic acid is a reduced
form of folate thus it can readily enter the folate cycle with-
out reduction and is more suitable for supplementation [52].
Besides, folinic acid is able to cross the blood–brain barrier,
which may block folic acid, through reduced folate carrier.
One recent double-blind placebo-controlled RCT (low
risk of bias) has tested the effect of high-dose folinic acid
on ASD [36]. In that study, 48 children with ASD and lan-
guage impairment were randomized to receive folinic acid
(2 mg/kg/day, maximum 50 mg/day; n = 23) or placebo
(n = 25). Folate receptor-α autoantibody (FRAA) status of
the children were subtyped. The investigators used a variety
of outcome measures of verbal communication, including
Clinical Evaluation of Language Fundamentals (CELF) and
Preschool Language Scale (PLS), and various behavioral
assessments. After 12 weeks of supplementation of folinic
acid or placebo, significant improvements were found in
verbal communication, and core symptoms of ASD in those
receiving folinic acid as compared with those receiving pla-
cebo; what is more, the improvements were significantly
greater for FRAA-positive participants.
No serious adverse event was observed for folinic acid
supplementation. A number of mild adverse events were
similar in folinic acid group (21) and in the placebo group
(25) [36].
Omega‑3 fatty acids
Omega-3 fatty acids are of fundamental importance for brain
structure and function because they are orthomolecules and
their functional sites are exclusively cell membranes [53]
and thus have been studied for the treatment of ASD [54]
and other psychiatric disorders [55–57]. In addition, reduced
concentrations of omega-3 fatty acids have been observed in
children with ASD [15], suggesting supplementation inter-
vention might be of help for treatment of ASD.
There have been six double-blind, placebo-controlled tri-
als, with inconsistent results. Amminger et al. (moderate
risk of bias) [30] first tested 840 mg/day eicosapentaenoic
acid (EPA) + 700 mg/day docosahexaenoic acid (DHA) for
6 weeks, and found improvements in reducing stereotypy,
inappropriate speech and hyperactivity compared with pla-
cebo, though their statistical analyses for ABC subscales
indicated no significant differences between treatment
groups. In 2015, Mankad et al. (moderate risk of bias) [33]
published a replication study on omega-3 fatty acid sup-
plementation of the same dose as Amminger et al. but with
longer duration (12 weeks). However, they did not observe
any improvement on core symptoms of ASD. In a pilot
trial (low risk of bias), Bent et al. [31] used 700 mg/day
EPA + 460 mg/day DHA for 12 weeks for the treatment of
ASD in 27 children, their initial results did not show any
statistically significant benefit from omega-3 fatty acids.
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 Eur J Nutr

Table 2  Assessment of risk of bias for individual studies

References Study design Comparison Diagnostic Inclusion and Description Outcome Precision Statisti- Overall risk of
approach exclusion of interven- measures of effect cal analy- ­biasa
criteria tion sis

Vitamin B6/Mg
 Lelord et al. ★ – – – – – – ★ High
[23]
 Martineau et al. ★ – – – – – – ★ High
[24]
 Tolbert et al. ★ – ★ – ★ – – ★ High
[25]
 Findling et al. ★ – ★ ★ – – ★ ★ Moderate
[22]
Methyl B12
 Bertoglio et al. ★ – ★ ★ ★ ★ ★ ★ Moderate
[26]
 Hendren et al. ★ ★ ★ ★ ★ ★ ★ ★ Low
[27]
Vitamin D3
 Saad et al. [28] ★ ★ ★ ★ – ★ ★ ★ Moderate
 Kerley et al. ★ ★ ★ ★ – ★ ★ ★ Moderate
[29]
Fatty acids
 Amminger ★ ★ ★ – ★ – – ★ Moderate
et al. [30]
 Bent et al. [31] ★ ★ ★ ★ ★ ★ ★ ★ Low
 Yui et al. [35] ★ – ★ ★ ★ ★ ★ ★ Moderate
 Voigt et al. [34] ★ ★ – ★ ★ ★ ★ ★ Moderate
 Bent et al. [32] ★ ★ ★ ★ ★ – ★ ★ Moderate
 Mankad et al. ★ ★ ★ ★ ★ – ★ ★ Moderate
[33]
Folinic acid
 Frye et al. [36] ★ ★ ★ ★ ★ ★ ★ ★ Low

A star was given for each positive score to the domain

a
 Positive scores on all domains to receive a rating of low risk of bias, five to seven positive scores to receive a rating of moderate risk of bias,
and less than five positive scores to receive a rating of high risk of bias

Several years later, Bent et al. (moderate risk of bias) pub-
lished their new findings from the trial with a larger sample
size (n = 57) [32]. As a result, they observed a significant
improvement in stereotypy and lethargy subscales of ABC,
but no significant differences in other measures such as SRS
and CGI-I. In another pilot study (moderate risk of bias),
Yui et al. [35]. used a 12 week, 240 mg/day DHA + 240 mg/
day arachidonic acid (ARA) intervention for ASD on 13
patients. Significant improvements were observed in social
withdrawal subscale of ABC, in stereotyped and repetitive
behaviors of Autism Diagnostic Interview-Revised (ADI-R)
and in communication subscale of SRS, this was the only
study that reported significant improvements in all behavior
measures. Voigt et al. (moderate risk of bias) [34] measured
the effect of long-term (6 months) intake of low dose DHA
(200 mg/day) on 48 children with ASD, but they did not find
a significant difference in CGI-I or in other measures across
treatment groups.
No severe adverse events were observed for omega-3 fatty
acids of the dose range administrated in studies mentioned
above, however, several mild adverse events such as gastro-
intestinal symptoms, rash, and agitation were the most often
reported [31–33].
Discussion
Although the five supplements were all used for correcting
nutritional deficiencies in ASD, their potentials for improv-
ing ASD symptoms were based on different neurobiological

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Eur J Nutr

mechanisms as each nutrient has unique metabolic patterns

and biological activities. Briefly, vitamin B6/Mg involved

DHA: 460 mg/day (3–8 years)

64.5–74 μg/kg every third day

300 IU/kg/day, not to exceed

Doses suggested (age range)

5000 IU/day (3–10 years)
in the synthesis of neurotransmitters such as serotonin and

2 mg/kg/day, not to exceed

50 mg/day (3–10 years)
dopamine, and both serotonin transporter and dopamine
transporter binding were both found to be abnormal in brains

EPA: 700 mg/day
of autistic individuals [40]. The effects of methyl B12 and

(3–7 years)
folinic acid on ASD were related to the transsulfuration
pathway which was impaired in ASD [58]. Omega-3 fatty

None
acids play fundamental roles in brain as cell membrane func-
tional sites that may help to prevent development of ASD
[53]. Last, the potential treatment effect of vitamin D­ 3 may

Improvement in core symptoms Effectiveness inconclusive but

Effectiveness inconclusive but

Less effective but acceptable

be from its anti-inflammatory effects [49] or may be associ-
ated with serotonin [50].
The use of nutritional supplements as a complementary

Not recommended
Recommendation
therapy for ASD is of high prevalence, but little evidence
supports their effectiveness. Among the five supplements

acceptable
acceptable

Improvement in verbal commu- Promising

reviewed, none was recommended for improvement of
behavioral symptoms of ASD according to current best evi-
dence (Table 3).
First, B6/Mg was not recommended. RCTs of B6/Mg

Improvement in stereotypy and

Potential improvement in com-

nication, repetitive behavior,

Positive outcomes reported (if

and inappropriate speech (in

Improvement in overall ASD

ASD autism spectrum disorder, DHA docosahexaenoic acid, EPA eicosapentaenoic acid, RCT randomized controlled trial
supplementation for treatment of ASD were all published

hyperactivity (in 2 RCTs)

munication and repetitive
two decades ago, and most were rated as high risk of bias in

behavior (in 3 RCTs)

behavior (in 2 RCTs)

of ASD (in 1 RCT)

our quality assessment. Besides, high dose B6/Mg supple-
mentation may increase the risk of neuropathy and diarrhea.
Second, our conclusion for methyl B12 was acceptable as
it may reverse the deficiency but further investigations are

1 RCT)
needed for confirming its effectiveness on ASD. Of the two
any)

RCTs of methyl B12 reviewed, sample sizes were small and

laboratory testing was inadequate though they were rated
Moderate: 2
Moderate: 1

Moderate: 5
Moderate: 1

Moderate: 0
Risk of bias

as low and moderate risk of bias. Third, effect of vitamin D

High: 0
High: 6

High: 0
High: 0

High: 0
Low: 0
Low: 0

Low: 1
Low: 1

Low: 1
is also inconclusive. Findings from the first RCT suggested
that oral vitamin D3 supplementation could safely correct
vitamin D deficiency and improve in serum 25(OH)D lev-
and diarrhea from Mg when

els and could significantly improve core symptoms of ASD

7 double-blind RCTs Risk of neuropathy from B6

was significant. The other trial observed increased 25(OH)D

Potential adverse effects

levels in the treatment group but failed to confirm the thera-

high doses used

­ 3 may be
peutic effect in clinical aspects. However, vitamin D
recommended for children with ASD with vitamin D defi-
2 double-blind RCTs Not reported

Omega-3 fatty acids 6 double-blind RCTs Not reported

2 double-blind RCTs Not reported

Not reported

ciency for its relative safety and cheapness. Fourth, omega-3

Table 3  Summary of evidence and recommendations

fatty acids may be less effective but acceptable. Among six

RCTs reviewed, five showed that omega-3 fatty acid sup-
plementation did not improve core symptoms of ASD in
Number of evidence

1 double-blind RCT

overall behavioral measures, and the study that reported the

most positive results was clearly limited in sample size [35].
Besides, a recent meta-analysis of five RCTs has quanti-
tatively assessed the association, and the results indicated
that there was no statistically significant improvement in
ASD symptoms [59]. Current best evidence did not support
omega-3 fatty acid supplementation for treatment of core
Vitamin B6/Mg

symptoms of ASD in children. However, omega-3 fatty acids

Supplement

Methyl B12

Folinic acid
Vitamin ­D3

were generally safe and were well tolerated, and mounting

evidence support that they may still be of potentials for the
neurodevelopment in children [60]. Fifth, folinic acid is also

13

a promising supplement. It improved core symptoms of ASD
significantly and was well tolerated in the trial. In addition,
the results demonstrated that FRAA status in patients was
predictive of response to treatment. Limitation of the trial
was small sample size and short treatment duration. Folinic
acid may become increasingly used to treat ASD in the
future, but safety should be further ensured.
Risk of bias
In our assessment of the risk of bias, only three studies were
rated as low [27, 31, 36] and most studies were of moder-
ate or high risk of bias. Numbers of included studies also
influence our confidence in conclusions. Although findings
from RCT of folinic acid supplementation were encouraging,
data were inadequate to make a valid conclusion as there
was only one RCT. Therefore, our conclusion on folinic acid
supplementation was promising.
Limitations
Several limitations should be mentioned for our review pro-
cess. First, only five supplements for nutritional deficiencies
in ASD were reviewed, several other nutritional deficiencies
were not discussed because no relevant study has been pub-
lished or because we focused on the best evidence according
to our inclusion criteria. Second, we excluded open-label
trials because of potential placebo effect, thus the number
of included studies was limited. Third, significant heteroge-
neity were found in interventions and in outcome measures
that precluded quantitative meta-analysis. Therefore, stud-
ies were reviewed qualitatively. Fourth, the tool we used to
assess risk of bias and rate overall quality of studies may not
be valid, though questions of interest were specific for ASD
and the domains were similar to those recommended by the
Cochrane Collaboration.
Conclusions
In conclusion, evidence was little and our confidence is not
high. Of five supplements for nutritional deficiencies in ASD
that were systematically reviewed, none was recommended.
Although the administration of nutritional supplements may
correct specific nutritional deficiencies in children with
ASD, their effects on clinical aspects of ASD need further
investigation. However, several supplements might be help-
ful and further researches are encouraged as most studies
were limited in sample size and intervention durations.
Clinicians and families should be cautious before making
a decision on taking a supplement as a safe and effective
alternative approach for reversing deficiencies as well as for
improvement of ASD.
13
Eur J Nutr
Acknowledgements  No funding was received in support of this work.
Author contributions  Y-JL and D-XX defined the focus of the review.
All authors searched and screened the papers for inclusion. Y-JL sum-
marized included papers. All authors contributed to quality assessment of
included papers. Y-JL drafted the manuscript. All authors contributed to
reviewing the manuscript and read and approved the submitted version.
Compliance with ethical standards 
Conflict of interest  The authors have no relevant interests to declare.
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