Life Sciences 145 (2016) 205–212

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Life Sciences
journal homepage: www.elsevier.com/locate/lifescie

Increased ω-3 polyunsaturated fatty acid/arachidonic acid ratios andupregulation of signaling mediator in individuals with autism
spectrum disorders
Kunio Yui a,⁎, George Imataka b, Yohei Kawasak c, Hiroshi Yamada caResearch Institute of Pervasive Developmental Disorders, Ashiya University, 13-22 Rokurokusocho, Ashiya 659-8511, Hyogo, Japan
Department of Pediatrics, Dokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu 321-0293, Tochigi, Japanc
Department of Drug Evaluation and Informatics, School of Pharmaceutical Science, University of Shizuoka., 52-1 Yada, Shizuoka 422-8526, Japanba r t i c l e

i n f oArticle history:
Received 27 July 2015
Received in revised form 15 November 2015
Accepted 22 December 2015
Available online 24 December 2015
Keywords:
Autism spectrum disorders
Competitive interaction
Arachidonic acid
Omega-3 polyunsaturated fatty acids
Signaling mediator
Ceruloplasmina b s t r a c t
Aims: The investigation of links between the ratio of omega-3/omega-6 PUFAs and neuronal signaling is a
research priority in autism spectrum disorders (ASD).
Main methods: We examine the relationships between the plasma ratios of docosahexaenoid acid (DHA)/arachidonic acid (AA) and eicopentaenoic acid (EPA)/AA and biomarkers of AA-related signaling mediators such as
ceruloplasmin, transferrin and superoxide dismutase, in the behavioral symptoms of 28 individuals with ASD(mean age 13.5 ± 4.6 years) and 21 age- and gender-matched normal healthy controls (mean age 13.9 ±5.7 years). Behavioral symptoms were assessed using the Aberrant Behavior Checklists (ABC). We conducted
controlling for dietary intake and assessed the dietary intake of nutrients.
Key ﬁndings: There were no signiﬁcant differences in intake of nutrients such as omega-3 and omega-6 PUFAs,
saturated and unsaturated fatty acid, DHA, AA, iron and copper. Plasma EPA, DHA, and arachidic acid levels,and plasma DHA/AA and EPA/AA ratios were signiﬁcantly higher, while plasma AA and adrenic acid were significantly lower in the 28 individuals with ASD than in the 21 normal controls. The ABC scores were signiﬁcantly
higher in the ASD group compared to the control group. The plasma ceruloplasmin levels in the ASD groupwere signiﬁcantly reduced compared to those in the control group.
Signiﬁcance: Increased plasma DHA/AA and EPA/AA ratios may be related to low plasma levels of ceruloplasmin
which has neuroprotective properties. Reduced plasma ceruloplasmin levels may diminish the protective capacity against brain damage, and may contribute to the pathophysiology of behavioral symptoms in individuals
with ASD.
© 2016 Elsevier Inc. All rights reserved.

1. Introduction
Studying alterations in the composition of PUFAs and related signal
mediators may be a major strategy in understanding the pathophysiology of autism spectrum disorders (ASD) [1]. The omega-3 PUFAs
docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) and theomega-6 PUFA arachidonic acid (AA) are important in nervous system
function [2], signal transduction [3], proper synapse formation [4] andneurotransmission [4]. Previous studies reported a marked reductionin plasma DHA levels [5,6] with changes in the plasma omega-3/
omega-6 ratio [7]. The balance between omega-3 and omega-6 PUFAs
is well known to be critical for normal brain function and development[8–10]. A recent clinical study reported that the relationship between
omega-3 PUFAs and AA is antagonistic to maintaining homeostasis at⁎ Corresponding author at: Research Institute of Pervasive Developmental Disorders,Ashiya University, Ashiya, Japan.
E-mail address: yui16@bell.ocn.ne.jp (K. Yui).

http://dx.doi.org/10.1016/j.lfs.2015.12.039
0024-3205/© 2016 Elsevier Inc. All rights reserved.

high EPA and DHA concentrations. The incorporation of omega-3
PUFAs lowers the AA concentration [11,12], thereby down-regulating
the synthesis of eicosanoids, the mediators of AA signaling in the cell
membrane [12]. Dietary omega-3 PUFAs may attenuate tissue AA levels
and eicosanoid formation [13]. The antagonism of AA signaling may
have important effects upon cell signaling in the central nervous system[15,16], modifying biomedical and behavioral effects [15]. This competitive interaction between omega-3 PUFAs and AA reﬂects an increase in
the plasma omega-3/AA ratios [8,9,11].
In respect to signaling mediators in ASD, alterations in ceruloplasmin(Cp) [17], superoxide dismutase (SOD) [18] and transferrin (Tf) [19]have been reported as pathophysiological factors. Cp is an important
copper signaling biomarker of neuronal function [20] and a natural neuroprotective protein [21,22]. Cp reduces the synthesis of AA-derived eicosanoid mediators, such as leukotrienes [23] and cyclooxygenase-2[24]. SOD is a biomarker of copper signaling [25]. AA [26] and essential
PUFAs [27] increased the activity of SOD. Moreover, protecting SOD activity has been related to the excess production of the AA-derived

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K. Yui et al. / Life Sciences 145 (2016) 205–212

eicosanoid family prostaglandin F2 [28]. Tf is an iron-signaling mediator[29]. The Tf receptor protein is elevated by DHA-enrichment [30].
Collectively, Cp, SOD and Tf are related to the functions of AA, DHA
and PUFAs. Changes in the blood levels of SOD, TF and CP have beenshown to indicate altered antioxidant status [31–33], vulnerability
to oxidative stress [34,35], and copper dyshomeostasis [36,37] in
ASD patients. However, the importance of the interaction between thealtered composition of PUFAs and the AA-related signaling mediators(Cp, Tf and SOD) remains unclear.
We hypothesized that increased plasma DHA/AA and EPA/AA ratios
are related to the down-regulation of AA-dependent signaling mediators in the behavioral symptoms of individuals with ASD. Thus, thelevels of 23 fatty acid fractions and three AA-related signaling mediatorsin the plasma were determined. Plasma fatty acid levels were expressedas the percentage of total fatty acids and in μg/ml to examine the correlation among the fatty acids. Because there are multiple subfamilies ofAA-derived eicosanoid signaling mediators [38] and these mediators
are affected by multiple factors [39], we examined plasma Cp, SOD
and Tf levels as known AA-related signaling mediators. We conducted
controls for dietary intake and to assess the intake of nutrients.
2. Methods
2.1. Participants
A total of 49 young, physically healthy individuals who were fromthe Kansai area (Hyogo and Osaka Prefectures) of Japan participatedin this study. They were recruited from medical care facilities of the
Research Institute of Pervasive Developmental Disorders of Ashiya
University by the order of their submission to medical consultation
between January 2012 and July 2014. Diagnoses were performed
based on the DSM-IV-TR criteria and were additionally conﬁrmed by
the Autism Diagnostic Interview-Revised (ADI-R) [40]. Among the 49
individuals, 28 had an independent clinical diagnosis of ASD (20 males
and 8 females, mean age: 13.5 ± 4.7 years old, age range: 6–21 years
old), and the remaining 21 were normal healthy controls (15 males
and 6 females, mean age: 13.9 ± 5.7 years old, age range: 5–21 yearsold). The 28 ASD individuals and the 21 individuals in the control
group were matched in respect to home environment, feeding habits,
age, gender and full intelligent quotient (IQ) scores (Table 1). Two

psychiatrists specializing in ASD and developmental disorders diagnosed all 28 individuals with ASD. These individuals had the core symptoms of the DSM-IV-R diagnostic criteria for ASD without any abnormal
neurological symptoms (e.g., seizure or movement disorders). The 21
normal controls were considered to be physically and mentally healthy
based on initial physical and mental screening tests. At the initial
screening, physical (resting blood pressure and heart rate) and clinicallaboratory examinations (hematology and plasma chemistry, including
plasma fatty acids, cholesterol and triglycerides) were performed on all
49 participants. These 49 participants did not have any abnormalities intheir physical examinations and laboratory ﬁndings. The weight andheight of the 49 individuals were within average values for both 6–
17 years old [41] and 6–22 years old (http://paro2day.blog122.fc2.
com/blogentry9html). The IQs of the individuals were estimated using
the Wechsler Intelligence Scale for children and adolescents aged 6–
16 years old (WISC-III) [42] or the respective scale for adults (WAISR) [43] (Table 1). Comorbid psychiatric disorders were evaluated by
the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID).
None of the ASD or control individuals had any history of neurologicalconditions, including seizure, movement disorders, head injury,
Attention-Deﬁcit Hyperactivity Disorder or learning disorders. The
additional inclusion criteria were as follows: (a) the absence of any
other medical or comorbid psychiatric disorders; (b) a baseline verbal
or full IQ greater than 70 as calculated by the WISC-III [42] or the respective scale for adults (WAIS-R) [43] because subjects with highfunctioning pervasive developmental disorders were required to havea total IQ of at least 70 [44]; (c) no treatment with antidepressants, anxiolytic medications or neuroleptics within the three months prior to
the study (the treatment of ADHD symptoms with stimulants wasallowed during this study, provided that the patient's dosage was stable
for at least 3 months before and during the study).
The present work has been carried out in accordance with theCode of Ethics of the World Medical Association (Declaration of
Helsinki) for experiments involving humans (http://www.wma.net/
en/30publications/10policies/b3/index. html). This study was performed with the approval of the Ethics Committee of the Fujimoto
Medical Clinic in Kobe City, Japan. This ethics committee is registeredwith the Pharmaceuticals and Medical Devices Agency of Japan to register the IRB information (http://www.info. pmda.go.jp/). Written informed consent was obtained from the participants and/or their parents.

Table 1
Subject characteristics and plasma levels of signaling mediators, and the ABC subscale scores in the 28 individuals with ASD and 21 normal controls.
Variables
Age (Year)
Mean ± S.D
Sex (male/female)
Full IQ
Scores of Autism Diagnostic
Interview-Revised
Domain A (social)
Domain B (communication)
Domain C (stereotyped)
Plasma biomarkers levels
Cp (mg/dl)
Tf (mg/dl)
SOD (U/ml)
Subscale scores of the ABC
Irritability
Social withdrawal
Stereotypy
Hyperactivity
Inappropriate speech
Total

ASD (n = 28)

Controls (n = 21)Up valuecorr p value

13.5 ± 4.7
7/21
94.7 ± 9.6

13.9 ± 5.7
6/15
98.0 ± 6.6

272.50χ2 = 0.00
70.00

0.66
1.00
0.47

21.1 ± 7.0
13.7 ± 5.0
5.8 ± 5.4

N/A
N/A
N/A

23.92 ± 4.39
262.36 ± 42.54
3.36 ± 2.76

27.86 ± 5.12
268.24 ± 37.37
3.78 ± 2.94

163.00265.50229.000.007⁎⁎
0.56
0.18

0.001⁎
NS
NS

14.36 ± 9.45
21.61 ± 9.37
5.79 ± 5.67
19.04 ± 11.59
5.14 ± 4.46
65.96 ± 28.96

0.67 ± 0.91
0.86 ± 2.00
0.24 ± 0.62
0.90 ± 2.10
0.29 ± 0.64
2.95 ± 5.11

9.50
1.50
68.50
10.00
50.00
1.00

0.000⁎⁎⁎
0.000⁎⁎⁎
0.000⁎⁎⁎
0.000⁎⁎⁎
0.000⁎⁎⁎
0.000⁎⁎⁎

0.00⁎⁎

Values are mean ± SD. corr p value, p value after the Bonferroni correction; ABC, Aberrant Behavior Cheklist; Cp, ceruloplasmin; Tf, transferrin, SOD, superoxide dismutase.
⁎ p b 0.05 versus normal controls.
⁎⁎ p b 0.01 versus normal controls.
⁎⁎⁎ p b 0.001 from normal controls.

K. Yui et al. / Life Sciences 145 (2016) 205–212

2.2. Controlling for dietary intake and assessment of nutrient intake
As plasma fatty acid levels may be confounded by prior dietaryintake, many prior studies have used the “Dietary Approaches to Stop
Hypertension (DASH)” to control for dietary intake [45,46]. In thisstudy, all 49 participants received the “Japanese Food Guide” [47],which outlines the daily intake guide of nutrients and food based on
the “Overview of Dietary Reference Intake for Japanese (2010)” [48].
All 49 individuals' parents were provided a sample of the diet mealplan and menu (KAWASAKI FOODMODEL) (http://item.rakuten.co.jp/
foodmodel/751741/), which was edited according to the “Japanese
Food Guide” [47]. Moreover, to assess the daily food and nutrient intake,a semi-constructive questionnaire for the Japanese (DHQ) was performed using the junior high school version (DHQ15) (DHQ Support
Center, http://www.ebnjapan.org/). The DHQ15 consisted of 72 questions on the frequency of intake of 150 various food and beverage
items and cooking methods. DHQ15 was administered one month before the study on randomly selected subsamples of 18 individuals
with ASD and 10 normal controls by the order of their submission to
medical consultation during January 2013 and June 2014. The foodand beverage items and portion sizes in the questionnaire were derived
primarily from the data in the Overview of Dietary Reference Intake forJapanese [49]. The DHQ15 sheets were checked by the researcher (KY).
If there was any missing or unclear information recorded on the sheetby the guardian, the researcher (KY) questioned the guardian byphone or e-mail. The validity of the DHQ15 has been veriﬁed [50]. The
estimated intake of nutrients was calculated using a dedicated program
for the DHQ system (DHQ Support Center, Tokyo, Japan) [51,52].

207

inter-assay coefﬁcients of AA were 110.14 μg/ml (standard deviation(SD), 3.87; coefﬁcient of variation (CV), 5.28%) and 100.63 μg/ml (SD,5.51; CV, 5.48%), respectively, and those of DHA were 73.87 μg/ml (SD,2.30; CV, 3.11%) and 68.07 μg/ml (SD, 2.30; CV, 3.33%), respectively.
The plasma levels of the fatty acids were expressed as both themean ± SD weight (percentage) of the total fatty acids and themean ± SD in μg/ml.
2.4.3. Plasma levels of Cp
A Bering BN IINephelometer (Siemens Healthcare Diagnostics K.K.,
USA) was utilized to estimate plasma CP levels. The assay sensitivity
was 3.0 mg/dl. The intra- and inter-assay coefﬁcients were 10.2 mg/dl
and 10.1 mg/dl, respectively.
2.4.4. Plasma levels of SOD
Plasma SOD levels were estimated from the rate of decrease in nitrite
produced by hydroxylamine and the superoxide anions based on the nitrite method, using a VersaMax instrument (Molecular Devices Co,
Tokyo, Japan). Human plasma was assayed using an SOD Assay Kit(Takara Bio, Tokyo) according to the cytochrome c method. The plasma
SOD levels are expressed as units per milliliter. The assay sensitivity was
0.3 U/ml. The intra-assay and inter-assay coefﬁcients were 2.11 and2.10 U/ml, respectively.
2.4.5. Plasma levels of Tf
A standard turbidimetric assay and an automated biochemical
analyzer (JCA-BM8000 series, JEOL Ltd., Tokyo, Japan) were utilized toestimate plasma Tf levels. The intra- and inter-assay coefﬁcients were108.1 mg/dl and 107.4 mg/dl, respectively.

2.3. Assessment of behavioral symptoms
2.5. Statistical analyses
The ABC was used to assess the behavioral symptoms of the 28 individuals with ASD and the 21 normal controls. The ABC is primarily
intended to evaluate treatment responses in psychopharmacologicaland behavioral intervention trials for children and adolescents withmental retardation [53] and normal IQ levels [54]. The subscales are as
follows: (1) irritability (15 items); (2) social withdrawal (16 items);(3) stereotypic behavior (seven items); (4) hyperactivity (16 items);and (5) inappropriate speech (four items). The ABC is a broad assessment that captures a wide variety of behavioral problems [55]. This
test also appears to be capable of discriminating between several
syndromes, such as disruptive behavior disorders and the behavioral
symptoms of ASD [55].
2.4. Assays of plasma levels of PUFAs, Cp, SOD and Tf
2.4.1. Blood sampling procedures
Whole-blood samples were collected in EDTA tubes by venipuncture
and immediately placed on ice in a refrigerator. After a clotting time of
20–25 min, the plasma was obtained by centrifugation for 20 min at
3000 × g at room temperature (22 °C). The plasma samples wereprepared by spinning a tube of fresh blood containing an anticoagulant
in a centrifuge until the blood cells fell to the bottom of the tube. To
decrease the effects of circadian variation, the blood collection was
performed at 11:00–12:30 pm in a quiet laboratory room after supine
rest for 20 min. The samples were frozen at −80 °C until analysis. The
specialists at SRL, Inc. (Tokyo, Japan) measured the plasma levels of
PUFAs, Cp, Tf and SOD.

Because the data were not normally distributed, the non-parametricMann–Whitney U test for multiple comparisons was used to determine
the signiﬁcant differences in the plasma levels of PUFAs, the plasma
ratios of DHA/AA and EPA/AA, and the plasma levels of the three biomarkers (Cp, Tf and SOD) as well as the ﬁve subscale and total ABC
scores and the intake of nutrients between the ASD and the normalgroups. Considering the potential false positive rate incurred by multiple comparisons of the most important variables, which were necessary
to prove the relationship between the ratios of the omega-3 PUFAs/AA
and the AA-related signaling mediators, we applied the Bonferroni correction method to adjust the p values for these important variables(e.g., the plasma DHA/AA and EPA/AA ratios, the levels of the three signaling mediators and the total ABC scores). p values b0.05/6 (0.0083)
indicated statistical signiﬁcance after the Bonferroni correction [58].
Spearman's rank correlation coefﬁcients (r) were used to determine
the correlations between the plasma DHA/AA and EPA/AA ratios, theplasma levels of the signaling biomarkers (Cp, Tf and SOD), and theﬁve subscale and total ABC scores in the entire population. Moreover,multiple linear regression was used to conﬁrm the relationship between
the plasma DHA/AA and EPA/AA ratios and the other main variables,
adjusting for the two subject groups, the three signaling biomarkers
and the total ABC scores (Table 5). We conducted statistical analyses
using SPSS version 18.0 (IBM Tokyo, 2009).
3. Results
3.1. Study population

2.4.2. Plasma levels of PUFAs
The fatty acid composition of the PUFA fraction from the plasma ofeach patient was determined as previously described [56]. In summary,the total lipids were extracted from the plasma according to [57]. Aftertransmethylation with HCl-methanol, the PUFA composition was
analyzed using gas chromatography (GC2010 Shimadzu Co., Japan).
A total of 24 long-chain fatty acids were identiﬁed. The intra- andThe behavioral symptoms of the 28 young individuals with ASD
included restricted, repetitive and stereotyped patterns of behavior, interests and activities (n = 9), irritability and crying (n = 3) and stereotyped behavior (n = 16). The mean total ABC score was 65.96 ± 28.96.
An earlier study reported a total ABC score of 85.6 ± 27.3 for children
and adolescents with ASD who were treated with neuroleptics [59]208

K. Yui et al. / Life Sciences 145 (2016) 205–212(Table 1). Thus, our patients suffered from moderate ASD symptoms,
including restricted repetitive and stereotyped patterns of interestsand behaviors. The ages did not differ signiﬁcantly between the twogroups (p = 0.66).

As shown in Table 4, the plasma DHA/AA and EPA/AA ratios weresigniﬁcantly correlated with all ﬁve ABC subscale scores and the total
scores (all r values were greater than 0.36, p b 0.05, p b 0.01 or
p b 0.001) for the whole population. In the case of the levels of plasma
fatty acids expressed as the mean ± SD μg/ml, the Spearman correlation
coefﬁcients indicated similar results to the above described ﬁndings.

3.2. Plasma levels of PUFAs and biomarkers
3.3. Assessment of nutrient intake
The Mann–Whitney U test revealed that plasma EPA, DHA, DPA andarachidic acid levels and the plasma DHA/AA and EPA/AA ratios weresigniﬁcantly higher, while the plasma AA, adrenic acid and Cp levels
were signiﬁcantly lower in the 28 individuals with ASD than in the 21
normal controls, as expressed as the mean ± SD weight (percentage)of the total fatty acids (Table 2). In the case of plasma fatty levels
expressed as the mean ± SD in μg/ml, the signiﬁcant difference in theplasma levels of DHA and arachidic acid of the two groups disappeared(Table 3).
There was no signiﬁcant correlation between the plasma levels of
PUFAs and plasma levels of Cp (r = 0.08–0.253, p = 0.16–0.64), andTf and SOD(r = 0.02–0.19, p = 0.33–0.92).
Even after the Bonferroni correction, the plasma ratios of DHA/AA(p = 0.0002) and EPA/AA (p = 0.0012), the level of Cp (p = 0.001),
and the total ABC scores (p = 0.000) were signiﬁcantly higher in the
ASD group compared with the control group (Tables 1 and 2). The
ABC subscale scores for irritability (p = 0.000), social withdrawal,
stereotypic behavior, hyperactivity and inappropriate speech and thetotal ABC scores were signiﬁcantly higher in the ASD group than in thecontrol group (Table 1).

Table 2
Plasma levels of fatty acid fractions.
Variables

ASD(n = 28)

Controls(n = 21)U

p valuecorr pvalue

Plasma PUFA levels (%)
Omega-3 series
C18:3ω3 (ALA)
0.73 ± 0.38
0.59 ± 0.20 226.50 0.17
C20:5ω3 (EPA)
1.33 ± 0.86
0.74 ± 0.41 142.50 0.002⁎⁎
C22:5ω3 (DPA)
0.54 ± 0.16
0.46 ± 0.10 169.50 0.01⁎
C22:6ω3 (DHA)
3.59 ± 1.20
2.92 ± 0.93 189.00 0.03⁎
Omega-6 series
C18:2ω6 (LA)29.49 ± 3.60 29.79 ± 3.46 290.50 0.94
C18:3ω6 (GLA)
0.31 ± 0.14
0.33 ± 0.13 288.50 0.91
C20:2ω6 (DGLA)
1.17 ± 0.42
1.32 ± 0.24 290.00 0.94
C20:2ω6
0.19 ± 0.04
0.19 ± 0.04 259.00 0.48
C20:4ω6 (ARA)
5.71 ± 1.25
6.62 ± 1.30 190.50 0.04⁎
C22:4ω6 (adrenic
0.20 ± 0.14
0.22 ± 0.07 193.00 0.04⁎
acid)
Ratios of plasma levelsof PUFAs
DHA/AA
0.64 ± 0.23
0.44 ± 0.14 123.00 0.001⁎⁎ 0.0002⁎
EPA/AA
0.24 ± 0.16
0.12 ± 0.07 109.50 0.000⁎⁎⁎ 0.000⁎⁎
C14:1ω5
0.02 ± 0.03
0.05 ± 0.05 180.00 0.05
C16:1ω7
1.18 ± 0.82
1.74 ± 0.69 291.00 0.95
C18:1ω9
20.09 ± 3.37 21.16 ± 2.99 216.00 0.12
C20:1ω90.16 ± 0.05
0.14 ± 0.03 247.00 0.34
C20:3ω90.08 ± 0.04
0.08 ± 0.02 214.50 0.10
C22:1ω90.04 ± 0.03
0.03 ± 0.02 261.50 0.49
C24:1ω9
1.20 ± 0.32
1.12 ± 0.12 259.50 0.49
Saturated fatty acids
C12
0.14 ± 0.13
0.15 ± 0.24 284.50 0.85
C14
0.92 ± 0.47
0.82 ± 0.33 274.50 0.69
C16
23.18 ± 2.27 21.17 ± 4.97 255.00 0.43
C18
7.81 ± 0.57
7.50 ± 0.52 201.00 0.06
C20 (arachidic
0.31 ± 0.51
0.27 ± 0.04 145.00 0.003⁎⁎
acid)
C22
0.71 ± 0.17
0.66 ± 0.09 226.00 0.17
C24
0.61 ± 0.16
0.56 ± 0.09 216.50 0.12
Values are mean ± SD. corr p value, p value after the Bonferroni correction.
⁎ p b 0.05 versus normal controls.
⁎⁎ p b 0.01 versus normal controls.
⁎⁎⁎ p b 0.001versus normal controls.

All 49 participants received a control for their dietary intake according to the “Japanese Food Guide” [47]. As shown in the Table 3, there
were no signiﬁcant differences in weight, height, energy intake, and intake of cholesterol, protein, carbohydrate, fat, animal fat, saturated fatty
acids and unsaturated fatty acids. Importantly, the intake of omega-6(p = 0.19) and omega-3 PUFAs (p = 0.34), AA (p = 0.34), EPA (p =0.96), DHA (p = 0.87), iron (p = 0.78) and copper (p = 1.00) did not
signiﬁcantly differ between the ASD and control groups (Table 5).
3.4. Predictor variables
The multiple linear regression analysis demonstrated that the plasma ratios of DHA/AA (R2 = 0.336, p = 0.003) and EPA/AA (R2 =0.265, p = 0.018) signiﬁcantly contributed to the variables after
adjusting for the two subject groups, the three signaling biomarkers
and the total ABC scores (Table 5). The use of group assignments andthe plasma Cp levels as the dependent variable signiﬁcantly contributed
to the plasma DHA/AA ratio (group, unstandardized coefﬁcients,
B = −0.268 ± 0.098, β = −0.621, and p = 0.009; Cp, unstandardized
coefﬁcients, B = 0.013 ± 0.006, β = 0.295, and p = 0.046). When thegroups were used as the dependent variable, a trend toward the statistical signiﬁcance of contribution to the plasma EPA/AA ratio was shown(group, unstandardized coefﬁcients, B = −0.122 ± 0.098, β = −0.430,
and p = 0.08) (Table 6). In the case of the plasma fatty acid levels
expressed as the mean ± SD in μg/ml, the multiple linear regression
analysis indicated similar results to the above described ﬁndings.
These ﬁndings indicated that the plasma ratios of DHA/AA as well
as EPA/AA allowed for the prediction of these variables in the ASD andcontrol groups.
4. Discussion
Plasma PUFA levels have been shown to reﬂect changes in brain
PUFA levels [60]. In this study, the plasma EPA, DHA, DPA and arachidic
acid levels and the plasma DHA/AA and EPA/AA ratios were signiﬁcantly
higher, while the plasma AA, adrenic acid and Cp levels were signiﬁcantly lower in the 28 individuals with ASD than in the 21 normal controls(Table 1). In the case of the fatty acid levels in the plasma expressedas the mean ± SD in μg/ml, the signiﬁcant difference in the plasma
levels of DHA and arachidic acid between the two groups disappeared(Table 3). Plasma levels of DHA [61] expressed in μg/ml were different
from those expressed as a percentage [61]. Similarly, plasma arachidic
acid levels expressed in μg/ml were different from those expressed asa percentage [61,62]. Of reference, the time to reach peak concentrationof plasma DHA levels expressed in μg/ml (about 40–100 h [63]) waslater than those in a percentage (about 6 h [64]). Thus, low plasma
DHA levels expressed in μg/ml might remain until the time of blood
sampling, inducing disappearance of signiﬁcant difference. Moreover,in the metabolism of EPA and DHA, there is limited conversion of EPA
to DHA [65], potentially reﬂecting the lack of signiﬁcant difference inthe plasma DHA level between the two groups. Plasma AA levels
expressed as μg/ml increased more rapidly than those expressed as percent after food intake [62]. Considering that arachidic acid is known tobe formed by the hydrogenation of AA [66], high plasma levels ofarachidic acid expressed in μg/ml might be refractory of rapid increasein plasma AA levels, inducing disappearance of signiﬁcant differenceof plasma arachidic acid.

K. Yui et al. / Life Sciences 145 (2016) 205–212

209

Table 3
Plasma levels of fatty acid fractions.
Variables
Plasma PUFA levels (μg/ml)
Omega-3 series
C18:3ω3 (ALA)
C20:5ω3 (EPA)
C22:5ω3 (DPA)
C22:6ω3 (DHA)
Omega-6 series
C18:2ω6 (LA)C18:3ω6 (GLA)
C20:2ω6 (DGLA)
C20:2ω6
C20:4ω6 (ARA)
C22:4ω6 (adrenic acid)
Ratios of plasma
DHA/AA
EPA/AA
C14:1ω5
C16:1ω7
C18:1ω9
C20:1ω9
C20:3ω9
C22:1ω9
C24:1ω9
Saturated fatty acids
C12
C14
C16
C18
C20 (arachidic acid)
C22
C24

ASD (n = 28)

Controls (n = 21)Up value

18.45 ± 12.47
32.26 ± 17.87
0.54 ± 0.16
84.76 ± 26.51

16.85 ± 6.69
20.75 ± 13.01
0.46 ± 0.10
80.12 ± 28.59

280.5072.00
287.50275.000.78
0.014⁎
0.89
0.70

699.23 ± 150.00
7.28 ± 4.22
36.23 ± 36.69
4.73 ± 1.40
133.50 ± 32.00
4.23 ± 1.48

795.24 ± 182.00
8.72 ± 3.82
35.76 ± 9.42
5.01 ± 0.90
180.41 ± 38.91
5.75 ± 1.72

199.0
219.50192.50259.00101.00155.00

0.91
0.13
0.04⁎
0.48
0.00⁎⁎⁎
0.05

0.65 ± 0.22
0.25 ± 0.17
0.46 ± 1.03
40.62 ± 23.67
494.40 ± 222.53
4.00 ± 2.31
1.77 ± 0.93
1.60 ± 1.26
27.83 ± 5.52

0.45 ± 0.15
0.10 ± 0.07
1.03 ± 1.10
44.95 ± 17.41
536.97 ± 185.86
3.73 ± 0.94
2.22 ± 0.86
1.38 ± 0.89
30.23 ± 6.15

117.0081.00
164.50219.00202.00251.00200.50272.50223.500.00⁎⁎
0.00⁎⁎⁎
0.04⁎
0.13
0.06
0.38
0.06
0.65
0.15

3.68 ± 3.65
12.13 ± 16.99
563.77 ± 216.21
188.85 ± 54.99
7.37 ± 1.98
16.11 ± 5.99
13.70 ± 5.13

4.18 ± 7.08
21.63 ± 9.88
604.13 ± 127.61
199.82 ± 37.65
7.08 ± 1.20
17.67 ± 3.51
15.16 ± 3.06

289.00265.50
197.0021,050289.50226.00249.00corr p value

0.0002⁎0.000⁎⁎⁎

092
0.57
0.05
0.09
0.93
0.27
0.36

Values are mean ± SD. corr p value, p value after the Bonferroni correction.
⁎ p b 0.05 versus normal controls.
⁎⁎ p b 0.01 versus normal controls.

Only plasma Cp levels were signiﬁcantly different between the twogroups. Considering that Cp has no capacity to bind fatty acids [67]and that the serum Cp levels are not signiﬁcantly correlated to the nutrient levels [68], no signiﬁcant correlation between the plasma levels of
PUFAs and plasma Cp levels in this study appears to be reasonable.
We conducted controls for the dietary intake of all 49 participants.
Moreover, the assessment of daily nutrients revealed no signiﬁcantdifferences in the intake of fat, omega-3 and omega-6 PUFAs, AA, EPA,
DHA, iron and copper between the random subsamples of 18 individuals with ASD and the 10 healthy controls. Thus, the altered composition of PUFAs detected in this study may not be due to dietary foodand nutrient intake.
Excessive omega-3 PUFAs signiﬁcantly inhibit the conversion ofomega-6 PUFAs as a result of substrate competition [8,10,11]. BothDHA and EPA compete for the enzymes and products of AA metabolism[8,13,14]. A concurrent increase in the dietary omega-3 PUFAs, a reduction in plasma AA levels [11,12], and the levels of AA-derived eicosanoids are decreased by these omega-3 PUFAs in a dose-dependent

manner [14]. A previous clinical study of 1979 healthy male and female
subjects reported an antagonistic relationship between omega-3 PUFAs
and AA in the maintenance of homeostasis [9]. Drawing these facts
together, it is reasonable to note that the present ﬁndings revealed, forthe ﬁrst time, that increased plasma DHA/AA and EPA/AA ratios,
which are indicative of the competitive interaction between high plasma DHA and EPA levels and low plasma AA levels, may be correlatedwith reduced plasma levels of Cp, which is a neuroprotectant [21,22]
involved in the etiology of central nervous system diseases [22,69], inthe 28 young individuals with ASD. Plasma DHA/AA and EPA/AA ratios
were signiﬁcantly correlated with all ﬁve ABC subscales and the total
ABC scores. Notably, the multiple linear regression analysis identiﬁed
signiﬁcant correlations between the plasma DHA/AA and EPA/AA ratios
and the variables, after adjusting for the two subject groups, the threesignaling biomarkers and the total ABC scores in the whole population(Table 4). These ﬁndings revealed that the plasma DHA/AA and EPA/
AA ratios ﬁt models that distinguished the ASD group from the control
group and signiﬁcantly predicted these important adjusted variables.

Table 4Spearman's correlation coefﬁcient between the plasma ratios of DHA/AA and EPA/AA and signaling mediators, and the Aberrant Behavior Checklist scores in the 28 individuals with ASD
and the 21 normal controls.
Irritability

DHA/AA
EPA/AA
Cp
Tf
SOD⁎⁎⁎

Social withdrawal

Stereotypy

Hyperactivity

Inappropriate speech

Total

r

p

r

p

r

p

r

p

r

p

r

p

0.43
0.45
−0.18
−0.05
−0.25

0.001⁎⁎
0.001⁎⁎
0.21
0.70
0.07

0.45
0.50
−0.13
−0.02
−0.12

0.001⁎⁎
0.000⁎⁎
0.37
0.89
0.39

0.35
0.42
−0.08
0.13
−0.22

0.01⁎
0.002⁎⁎
0.90
0.36
0.12

0.41
0.42
−0.17
0.02
−0.21

0.002⁎⁎
0.002⁎⁎
0.23
0.88
0.14

0.42
0.36
−0.13
0.05
−0.28

0.002⁎⁎
0.009⁎⁎
0.36
0.73
0.045⁎

0.45
0.47
−0.16
−0.04
−0.22

0.001⁎⁎
0.000⁎⁎
0.24
0.77
0.13

Cp, ceruloplasmin; Tf, transferrin; SOD, superoxide dismutase; r, Spearman correlation coefﬁcient.
*p b 0.05, **p b 0.01, ***p b 0.001 were considered statistical signiﬁcance.

210

K. Yui et al. / Life Sciences 145 (2016) 205–212

Table 5The intake of nutrients in the random subsamples of 18 of the 28 individuals with ASD and
10 of the 21 normal controls.
ASD (n = 18)

Control (n = 10) U

Age (years)
11.8 ± 4.1
13.3 ± 5.1
High (cm)142.9 ± 23.0
151.6 ± 1 9.1
Wight (kg)40.0 ± 23.0
45.6 ± 15.6
Energy (kcal)
2153.2 ± 558.2 2421.0 ± 503.3
Fat (g/day)
72.0 ± 27.9
87.3 ± 22.2
Unsaturated fatty acid (g/day)
14.7 ± 4.2
18.4 ± 4.5
Saturated fatty acid (g/day)
24.4 ± 14.0
28.0 ± 10.2
Omega-3 PUFAs (g/day)
2.6 ± 0.9
3.1 ± 0.5
Omega-6 PUFAs (g/day)
12.3 ± 3.7
15.6 ± 4.2
EPA (mg/day)
226.8 ± 169.6
197.8 ± 103.7
DPA (mg/day)
73.3 ± 47.9
72.3 ± 26.5
DHA (mg/day)
400.8 ± 241.0
380.8 ± 115.8
AA (mg/day)
168.5 ± 15.9
209.1 ± 75.0
Iron (mg/day)
9.3 ± 2.99.5 ± 3.0
Copper (g/day)
1.3 ± 0.4
1.3 ± 0.4
Protein (g/day)
80.3 ± 24.7
90.5 ± 24.1
Animal protein (mg/day)
32.6 ± 8.6
32.3 ± 12.5
Cholesterol (mg/day)
124.9 ± 186.4
95.6 ± 169.6
Carbohydrates (g/day)
292.3 ± 60.0
309.1 ± 59.7
Vitamin B6 (mg/day)
1.4 ± 0.5
1.4 ± 0.4
Vitamin C (mg/day)
158.5 ± 79.2
121.4 ± 82.9
Vitamin D (mg/day)
10.0 ± 5.4
9.1 ± 2.4

24.0
22.5
20.5
20.0
18.0
15.0
21.0
19.0
16.0
27.5
25.5
26.5
19.5
25.0
28.0
20.0
27.0
22.0
25.0
25.0
19.0
27.5

p value
0.69
0.54
0.38
0.40
0.28
0.15
0.46
0.33
0.19
0.96
0.78
0.87
0.34
0.78
1.00
0.40
0.96
0.54
0.78
0.78
0.34
0.96

Values are mean ± SD.

Thus, the lowered plasma Cp levels in association with the increased
plasma DHA/AA and EPA/AA ratios may contribute to behavioral symptoms in the 28 individuals with ASD.
Interestingly, the plasma levels of arachidic acid were signiﬁcantly
higher in the ASD group than in the control group. Hydrogenation inhibits the release of prostacyclin, which is a prostanoid metabolized
from endogenous AA [70]. This occurs by inhibiting the conversion of
LA to AA or shunting the free AA released by the phospholipase A2 action to be metabolized by another pathway, leaving less AA available
for phospholipase A2 synthesis [71,72]. In addition, hydrogenated fatsincrease the production of PGE2-related tumor necrosis factor α (TNFα) [73]. Collectively, increased plasma arachidic acid might reﬂect
increased AA hydrogenation, which might inhibit the production of
AA-related eicosanoids involving Cp.
The plasma adrenic levels were signiﬁcantly lower in the ASD group.
Adrenic acid is a quantitatively signiﬁcant metabolic fate for AA [74], isformed by AA chain elongation or the elongation and desaturation of

Table 6
Results of the multiple linear regression.
Model

DHA/AACp
Tf
SOD
ABC total score
Group (1 = ASD,
2 = control)
EPA/AACp
Tf
SOD
ABC total score
Group (1 = ASD,
2 = control)

Model

Model

Coefﬁcients

R2

p-valueB

Beta
coefﬁcientsp value

0.34

0.003⁎⁎
0.013 ± 0.006−0.001 ± 0.001−0.003 ± 0.010−0.001 ± 0.001−0.268 ± 0.098

0.295−0.274−0.133−0.463−0.6210.046
0.047#
0.299
0.645
0.009##

0.006 ± 0.004−0.001 ± 0.000−0.005 ± 0.007
0.000 ± 0.001−0.122 ± 0.068

0.226−0.251−0.0910.071−0.430

0.142
0.083
0.499
0.759
0.080

0.265

0.018⁎

R2 = R-squared values; Cp, ceruloplasmin; Tf, transferrin; SOD, superoxide dismutase;B = unstandardized coefﬁcients; ABC, Aberrant Behavior Checklist.
*p b 0.05, **p b 0.01, signiﬁcance of R square. #p b 0.05, signiﬁcant; ##p b 0.01, signiﬁcant
contribution.

linoleic acid and can be converted to AA by β-oxidation [75]. The
lowered plasma levels of adrenic acid may therefore reﬂect the lowered
plasma AA levels.
Most studies of ASD have reported elevated serum Cp levels [37] orlower serum Cp levels [36] compared to the age-matched controls.
Thus, previously reported ﬁndings on blood Cp levels in ASD wereinconsistent [36,37]. Moreover, there is no data on the mechanisms
underlying the alteration of Cp in ASD. The present study is the ﬁrst to
reveal that increased plasma DHA/AA and EPA/AA ratios, which is indicative of increased plasma DHA and EPA levels and decreased plasma AA
levels, may be the underlying mechanisms of the reduced plasma Cplevels. Moreover, this is the ﬁrst report that increased AA hydrogenation
may have contributed to decreased plasma Cp levels.
In the ASD group, the increased plasma levels of the omega-3 PUFA
fractions, such as EPA, DPA and DHA, may reﬂect an increased biosynthesis of these omega-3 PUFAs, while the reduced plasma levels of AA
and adrenic acid may reﬂect the reduced biosynthesis of AA and AAformed adrenic acid. In human cells, PUFAs are converted into theirelongated and desaturated forms by elongase and desaturase enzymes[76], particularly delta-5 and delta-5 desaturases. The activity of these
enzymes is primarily regulated at the transcriptional level [76,77].
Therefore, alterations in fatty acid biosynthesis may depend on thepresence of transcriptional factors. In addition, the existence of DHA
synthesis from EPA through a Sprecht-independent pathway in somemammals has been reported [78]. Further studies of the altered biosynthesis of omega-3 and omega-6 PUFAs and saturated fatty acids inpsychiatric disorders are needed.
Eicosanoids, which are signaling mediators, are primarily derived
from the enzymatic oxygenation of AA [79–81]. Thus, the increased
plasma DHA/AA and EPA/AA ratios may be related to the downregulation of AA-related eicosanoid signaling mediators, as indicatedby the lowered plasma Cp levels in the ASD group.
Previous studies have shown altered PUFA compositions and havenot mentioned the competitive interaction between omega-6 andomega-6 PUFAs in autistic children aged 3–17 years old [6], 3–
15 years old [7] and 4–12 years old [82], as well as those in children
younger than 5 years of age [83]. In this study, an imbalance between
the plasma levels of omega-3 PUFAs and the plasma AA levels wasfound in ASD individuals with an average age of 13.5. At higher ages,
omega-3 PUFAs may protect against neurodegeneration [84]. By
protecting the brain from oxidative stress, omega-3 PUFAs minimize
brain damage and deterioration that occurs with aging [85]. Therefore,
the difference in the results between these previous studies and thepresent study might be due to an age-dependent metabolic mechanism.
Our study had some limitations: a) the concentrations of eicosanoid
family members (e.g., cyclooxygenase [86]) were not measured, and we
examined only three biomarkers of copper and iron signaling mediators. As AA-derived eicosanoid signaling mediators include many
prostaglandin families [38] and these mediators are affected by multiple
factors [39], studying AA-derived eicosanoids may be very complex.
In this study, we examined plasma copper and iron signaling mediators. The regulation of iron and copper homeostasis is essential forlife and may be related to the pathophysiology of several neurodegenerative disorders [87]. Therefore, we measured Cp, Tf and SOD
levels. Further studies should measure plasma levels of PUFA-related
eicosanoid family members and other useful biomarkers, such as 20hydroxyeicosatetraenoic acid [88], the plasma levels of 18 eicosanoids[89], or the urinary eicosanoid levels (e.g., tetranor-Prostaglandin E metabolite [90]). b) Even though the high number of male participants
with ASD may affect the study results, this disorder is most prevalent
in males, with a male to female ratio of 4 to 1 [91]. In this study, ASD
and normal control groups were age- and gender-matched. Further
studies should evaluate the effect of gender on the composition of
PUFAs. c) The small sample size in this study affects our ability to generalize our results to entire ASD populations. Future studies should investigate the increased biosynthesis of PUFAs using large sample sizes.

K. Yui et al. / Life Sciences 145 (2016) 205–212

In conclusion, high plasma DHA/AA and EPA/AA ratios, which wereindicative of increased plasma DHA and EPA levels and decreased
plasma AA levels, were correlated with decreased plasma levels of Cp,
which is a neuroprotectant involved in the pathogenesis of neuronal
disorders. It is plausible that increased AA hydrogenation might havecontributed to reduced plasma Cp levels. It is plausible that increased
plasma DHA/AA and EPA/AA ratios may be risk factors in the development of behavioral symptoms in young individuals with ASD.
Conﬂict of interest statement
All authors must disclose any ﬁnancial and personal relationships with other people
or organizations that could inappropriately inﬂuence our work.

Acknowledgments
We appreciated the assistance of Dr. Yuji Kobayashi (Information
and Planning Department, SRL, Inc., Tokyo, Japan) with the assessmentof the plasma levels of PUFAs, Cp, SOD and Tf. Dr. Kunio Yui received a
Grant-in-Aid for Scientiﬁc Research on Innovative Areas (Grant No
21200017) (2010–2012) and a Grant-in-Aid for Scientiﬁc Research(C) (2014–2016) from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
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