Journal of Translational Medicine and Developmental Disorders, 2017, Vol. 3, No. 1, 1-10
Available online at http://pubs.sciepub.com/jtmdd/3/1/1©Science and Education Publishing
DOI:10.12691/jtmdd-3-1-1

Imbalance between Urinary Hexanoyl-lysine and Total
Antioxidant Capacity Levels and Plasma Superoxide
Dismutase Levels in Individuals with
Autism Spectrum Disorder
Kunio Yui1,*, Nasoyuki Tanuma2, George Imataka3, Yohei Kawasaki4
1
Department of Cognitive-Behavioral Medicine, Kyoto University Graduate School of Meicine
Department of Pediatrics, Tokyo Metropolitan Fuchu Medical Center for the Disabled, Tokyo 183-8553, Japan3
Department of Pediatrics, Dokkyo Medical University, 880 Kitakobayashi, Mibu, 321-0293, Tochigi, Japan
4
Department of Pharmacoepidemiology, Graduate School of Meicine, University of Kyoto, Kyoto 606-8501 Japan*Corresponding author: yui16@bell.ocn.ne.jp
2

Abstract Bachgraund and aim: The imbalance between oxidants and antioxidants contributes to the pathophysiology
of ASD. Methods: We measured the urinary levels of an oxidative stress biomarker, hexanoyl-lysine (HEL), thetotal antioxidant capacity (TAC) and the DNA methylation biomarker 8-hydroxy-2′-deoxyguanosine (8-OHdG), andthe plasma levels of a major antioxidant enzyme, superoxide dismutase (SOD). We examined the relationshipbetween these four biomarkers and social responsiveness in 20 individuals with ASD and in 11 healthy controls.
Results: The sex and age distributions (11.4 ± 5.1 years vs. 13.9 ± 6.6 years) were not significantly different
between the groups. Social responsiveness was assessed using the Social Responsiveness Scale (SRS). Dietary TAC
from chocolate, biscuits and cookies, jam and marmalade was significantly higher in the ASD group than in thecontrol group, although the nutrient intake was not significantly different between the two groups. Urinary TAC
levels were significantly lower, but the urinary HEL levels were significantly higher in the ASD group compared tothe control group. Urinary 8-OHdG levels and plasma SOD levels were not significantly difference between thegroups. SRS scores were significantly higher in the ASD group than in the control group. Plasma SOD levels
significantly correlated with the urinary TAC levels. Stepwise regression analysis revealed that urinary TAC andHEL levels may predict differences in the biomarkers and SRS scores between the groups. Discussion andConclusion: A critical imbalance between the urinary HEL and TAC levels may contribute to impaired social
responsiveness in individuals with ASD without the DNA methylation. Plasma SOD levels may affect the impaired
antioxidant capacity.
Keywords: urinary levels of total antioxidant power, urinary levels of hel and 8-ohdg levels, social responsivenessscale, autism spectrum disorder, endogenous antioxidant capacity
Cite This Article: Kunio Yui, Nasoyuki Tanuma, George Imataka, and Yohei Kawasaki, “Imbalance between
Urinary Hexanoyl-lysine and Total Antioxidant Capacity Levels and Plasma Superoxide Dismutase Levels in
Individuals with Autism Spectrum Disorder.” Journal of Translational Medicine and Developmental Disorders,vol. 3, no. 1 (2017): 1-10. doi: 10.12691/jtmdd-3-1-1.

1. Introduction
The prevalence of ASD has markedly increased inrecent years [1]. ASD symptoms usually appear during
early development and generally lead to serious lifelong
limitations in function; thus, both genetic andenvironmental factors may contribute to its development[2,3]. The shift in the balance between oxidants andantioxidants in favor of oxidants is termed “oxidative
stress” [4]. Regulation of reduction/oxidation (redox) stateis important for cell viability, proliferation and organfunction [4], however, imbalance of oxidant/antioxidantbalance such as overproduction of ROS became toxic toneurons, inducing DNA methylation [5] and various
tissues damage [6]. Because the brain is highly vulnerable
to oxidative stress during early development [6], oxidative
stress-induced neuronal damage may occur in genetically
predisposed individuals [7]. Thus, deficits in antioxidant
capacity may important in the etiology of ASD. The total
antioxidant capacity (TAC) in urine and plasma is aparameter used to characterize the antioxidant status of the
body [8,9].
Recent studies in ASD have shown significantly
reduced urinary levels of TAC, indicating increased
vulnerability to oxidative damage [6], and significantly
higher urinary levels of 8-hydroxy-2′-deoxyguanosine(8-OHdG) [10]. further, the urinary levels of oxidative
stress markers such as hexanoyl-lysine (HEL) were found2

Journal of Translational Medicine and Developmental Disorders

to be elevated in children with ASD, and higher HEL
values correlated with the hyperactivity component of
the Childhood Autism Rating Scale [11]. Thus, urinaryoxidative stress-related biomarkers, including HEL, whichis reportedly a more sensitive biomarker of oxidative
stress [12], TAC and biomarkers of DNA methylation may
provide useful information about oxidative stress-related
brain damage. However, few studies have measured
urinary levels of HEL, TAP and 8-OHdG.
Increased ROS with a concomitant decrease in antioxidant
capacity may induce more serious damage in diseases
such as leukemia [13,14]. However, few reliable studies
have shown increased HEL to be concomitant with decreased
antioxidant capacity in ASD.
Interestingly, a recent study revealed that decreased
serum levels of SODs, which are major antioxidant enzymes[15] and contribute to human neurodegenerative disorders[16], may also be implicated in the pathophysiology of
ASD [17]. Previous studies have also reported thataltered plasma levels of enzymatic antioxidants such as
SOD, ceruloplasmin (Cp), and transferrin (Tf) play animportant role in ASD [18-25]. In spite of the important
pathophysiological contribution of SOD in ASD, the roleof the plasma SOD levels in relation to the urinary levelsof oxidative stress-related biomarkers remains unclear.
These considerations taken together, we pursued threeavenues of research in this work. First, we evaluated thelink between the urinary levels of HEL, TAC and 8-OHdG
and social responsiveness in individuals with ASD. Next,
we assessed the role of plasma SOD levels in relation tothese urinary biomarkers. Finally, we determined therelationship between HEL and TAC in urine ofindividuals with ASD. Because previous clinical studies
have found that urinary levels of TAC are affected by diet
[26,27], we also assessed dietary nutrient intake andestimated dietary TAP.

2. Subjects and Methods
2.1. Subjects
This study included a total of 31 young, physically
healthy individuals in Hanshin area (Kobe and Osaka
prefectures) participated in this study. They were recruitedfrom the medical care facilities of the Research Institute ofPervasive Developmental Disorders of Ashiya University
based on their presenting for a medical consultation
between October 2013 and September2015. ASD was
diagnosed based on the DSM-5 criteria [28] and wasconfirmed by the Autism Diagnostic Interview-Revised(ADI-R) [29]. Two psychiatrists specializing in ASD anddevelopmental disorders diagnosed all 20 individuals with
ASD. Among the 31 participants, 20 had an independent
clinical diagnosis of ASD (13 males and 7 females, mean
age: 11.1 ± 5.2 years old, age range: 6-22 years old), andthe remaining 11 were normal healthy controls (7 males
and 4 females, mean age: 13.9 ± 6.6 years old, age range:
5-21 years old). These individuals with ASD had the core
symptoms of the DSM-5 diagnostic criteria for ASD
without any abnormal neurological symptoms (e.g.,
seizure or movement disorders). The sex and age
distributions were not significantly different between

the two groups. The 20 individuals with ASD and the 11
individuals in the control group were matched on homeenvironment, feeding habits and full intelligent quotient(IQ) scores (Table 1). The 11 normal controls wereconsidered 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 clinical laboratory examinations (hematology andplasma chemistry, including plasma fatty acids,cholesterol and triglycerides) were performed on all 31
participants. These 31 participants did not have any
abnormalities in their physical examinations andlaboratory findings. The IQs of the individuals were
estimated using the Wechsler Intelligence Scale forchildren and adolescents aged 6-16 years old [30] or therespective scale for adults (WAIS-R) (Table 1). Comorbid
psychiatric disorders were evaluated based on the
Structured Clinical Interview for DSM-IV Axis I
Disorders (SCID). None of the ASD or control individuals
had any history of neurological conditions, including
seizure, movement disorders, head injury, AttentionDeficit Hyperactivity Disorder or learning disorders. The
additional inclusion criteria were as follows: (a) the
absence of any other medical or comorbid psychiatricdisorders; (b) a baseline verbal or full IQ greater than 70
as calculated by the [30] or the respective scale for adults[31 (WAIS-R) because subjects with high-functioning
pervasive developmental disorders were required to have a
total IQ of at least 70 [32]; and (c) no treatment withantidepressants, anxiolytic medications or neuroleptics
within the three months prior to the study (the treatment of
ADHD symptoms with stimulants was allowed during thisstudy, provided that the patient’s dosage was stable for atleast 3 months before and during the study).
All procedures performed in this study were accordancewith the ethical standards of the Fujimoto Medical Clinic
in Kobe City, Japan with the 1964 Declaration of Helsinki
and its later amendments or comparable ethical standards.
This ethics committee is registered with Pharmaceuticals
and Medical Devices Agency of Japan to register the IRB
information (http://www.info.pmda.go.jp/). Most of theparticipants in this study were young people under thelegal age of 20 years; thus, we obtained parental
permission and applied information on the behalf of these
individuals (e.g., recognizing whether each participant’surinary TAP and plasma SOD levels were within thestandard values according to SRL, Inc, Tokyo, Japan). The
standard values of TAP levels were within standard ranges
established by the Department of Pediatrics, Tokyo
Metropolitan Fuchu Medical Center for the Disabled,
Tokyo, Japan (3700 - 4000 μM uric acid equivalents in 8
normal healthy subjects aged 6-37 years) [33], and thoseof plasma SOD levels were within standard ranges
established by SRL, Inc., Tokyo, Japan (1.8-3.2 U/ml in
164 healthy subjects) [34]. The studies have been
approved by the appropriate institutional and/or national
research ethics committee and have been performed inaccordance with the ethical standards as laid down in the1964 Declaration of Helsinki and its later amendments orcomparable ethical standards. Written informed consent
was obtained from the participants and/or their parents.
This study was registered at https:// dbcentre3.jmacct.med.
or.jp/jmactr (the Clinical Trials Registry, Japan Medical

Journal of Translational Medicine and Developmental Disorders

Association, 2010/6/14; renewal, 2015/12/31; ID: JMA-IIA
00162).

2.2. Assessment of Dietary Nutrient Intake
Urinary levels of TAC may be affected by dietary foodintake may [26,27]. Berries, fruits, nuts, chocolate,vegetables [35], and cafe and soybean oil had the highest
antioxidant values [36]. Thus, participants who habituallyingested foods and snacks containing high antioxidant
values (berries, fruits, nuts, chocolate, vegetables, coffeeand soybean oil) were excluded from the study.
To assess the daily food 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 10 individuals with ASD
and 10 normal controls by the order of their submission to
medical consultation during January 2013 and June 2015.
The food and beverage items and portion sizes in thequestionnaire were derived primarily from the data in the
Overview of Dietary Reference Intake for Japanese [37].
If there was any missing or unclear information recordedon the DHG 15 by the parents or subjects, the researcher(KY) questioned them by phone or e-mail, andinformation was corrected in the DHQ 15. The estimatedintake of nutrients was calculated using a dedicatedprogram for the DHQ system (DHQ Support Center,
Tokyo, Japan) [38]. The validity of theDHQ15 has been
already verified [39].

3

The urinary levels of HEL were measured in duplicate
using a competitive ELISA kit (Japan International
Cooperation Agency-JICA, Shizuoka, Japan) [43].
For measurement of the urinary levels of 8-OHdG,
urine samples were centrifuged and the supernatant after
proper dilution was used in duplicate for assessment witha competitive enzyme-linked immunosorbent assay kit (8OHdG check ELISA kit, JalCA, Japan Institute for theControl of Aging, Shizuoka, Japan). These results werethen corrected to the urinary concentration of creatinine,
and the urinary 8-OHdG/creatinine levels were used insubsequent analyses [44].
For measurement of the urinary levels of urinary total
antioxidant capacity, the urinary antioxidant capacity wasdetermined by competitive enzyme-linked Immunosorbent
assay (ELISA) [44]. This assay provides accurate
determination of the total antioxidant activity in a sample
that results from the combined activities of theconstituents (Oxford Biomedical Research).

2.5. Plasma Levels of SOD
Plasma SOD levels were estimated from the rate ofdecrease in nitrite produced by hydroxylamine and thesuperoxide anions based on the nitrite method, using a
Versa max instrument (Molecular Devices Co, Tokyo,
Japan). Human plasma was assayed using an SOD Assay
Kit (Takara Bio, Tokyo) according to the cytochrome cmethod, and 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 and 2.10
U/ml, respectively.

2.6. Assessment of Social Impairment
2.3. Estimation of Dietary TAC

The social responsiveness scale (SRS) can be used to
Urinary levels of TAC have been reported to be
distinguish clinically significant ASD from otheraffected by dietary food intake. Grape juice (200 ml) [38]psychiatric disorders based on varying levels of socialand cocoa powder (40 g) [39] increased urinary TAC.
impairment [45]. The SRS is a 65-item questionnaire
Thus, a dietary TAC value was assigned to each food item
completed by subjects’ parents for a quantitativein the DHQ15. The 31 participants in this study wereassessment of autistic traits across a wide spectrum ofchildren or young adolescents with age range: 6-22 years
children aged 4–18 or 19 and more years (adult version).
old, their nutrients were less varied and were of smaller
The total SRS raw scores range from 0 to195, with theamounts than those of adults. We therefore calculated
highest scores corresponding to significant social
dietary TAC value of the remaining 34 foods in the
impairment, as observed in individuals with severe ASD
DHQ15 according to a previous study [35,42]. TAC(Di Martino et al, 2009). The SRS is able to reliably
g ofdistinguishfood for other psychiatric conditions from ASD [46],values were expressed as mmol Fe2+/100
ferric. reducing ability of plasma (FRAP) [42] orand assess the severity of autistic trait [47].
mmol/100g oxygen radical absorbance capacity (ORAC)[35,42]. Finally, The TAC values of 18 foods were
2.7. Statistical Analyses
calculated as FRAP or those of 4 foods were calculated as
Because the data were not normally distributed, theORAC.
non-parametric Mann-Whitney U test for multiple
comparisons was used to determine the significant
2.4. Urinary Assay of HEL, 8-OHdG anddifferences in the urine levels of HEL, 8-OHdG and TAC
TAC Levels
and plasma SOD levels as well as the five subscale andtotal scores on the SRS, and the intake of nutrients
Urines were collected as a spot sample and immediately
between the random subsamples of 10 individuals withstored at-80C until analysis. After the dissolving process,
ASD and 10 of the normal controls. Moreover, stepwise
the urines were centrifuged to remove all insoluble
regression analysis was used to confirm the relationshipmaterials. The specialists at the Department of Pediatrics,
between the urinary oxidative stress-related biomarkers
Tokyo Metropolitan Fuchu Medical Center for the(levels of HEL, 8-OHdG and TAC) and plasma SOD
Disabled (Tokyo, Japan) measured the urinary levels oflevels, and the other main variables, adjusting for the two
HEL, 8-OHdG and TAC.

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Journal of Translational Medicine and Developmental Disorders

subject groups, and the SRS five subscale and total scores(Table 2). We conducted statistical analyses using SPSS
version 18.0 (IBM Tokyo, 2009).

Urinary levels of 8-OHdG and plasma levels of SOD were
not significantly different between the groups (Table 1).

3.3. Predictor Variables

3. Results
3.1. Study Population
Clinical features of in the 20 individuals with ASD
were characterized by failure of normal back-and-forth
conversation (n = 8), deficits in socio-emotional
reciprocity (n = 9) and deficits in understanding
relationships (n = 3). The mean total SRS score for ourpatients was 111.0 ± 33.1 (Table 1). Previous studies
reported total SRS scores of 120.21 for children andadolescents with moderate-to-severe ASD [48].
Considering that an SRS total score between 60 and 80 is
associated with a mild form of ASD [49], our patients
suffered moderate or severe social impairment from ASD.
Ages did not differ significantly between the ASD andcontrol groups (p = 0.30).

3.2. Urinary Levels of Oxidative StressMarkers
There was a significant increase in urinary HEL levels(p = 0.018) and a significant decrease in urinary TAC
levels (p = 0.049) in the 20 individuals with ASD
compared to those observed in the 11 normal controls.

Table 2 summarizes the results of our stepwise multiple
regression analysis. Urinary levels of TAC contributed
significantly to the variables after adjusting for the two
subject groups, urinary HEL levels, 8-OHdG levels,
plasma SOD levels, and the five subscale and total ABC
scores (Step 1, R2 = 0.216, P = 0.008; step 2, R2 = 0.411,
p = 0.001). Using Group used as a dependent variableshowed a significance of contribution to the urinary TAC
levels (step 1, unstandardized coefficients, B = 1152.564± 407.338, β = 0.5465, and p = 0.008; step 2,
unstandardized coefficients, B = 3027.688 ± 713.591,
β = 1.222, and p = 0.000) and to the SRS cognition scores(unstandardized coefficients, B = 83.864 ± 27.572,
β = 0.876, and p = 0.005).
Furthermore, the procedure using urinary HEL levels as
the independent variable terminated at step 2, explaining
22.1 % of the variance. The SRS subscale and total scores
being used as the dependent variables, the statisticalsignificance of contribution of the SRS subscale ofawareness to the urinary HL levels (unstandardized
coefficients, B = 2.312 ± 0.807, β = 0.470, and p = 0.008).
These findings indicate that urinary levels of TAC andHEL were predictable measures for differences in thebiomarkers and the SRS scores between the ASD groupand the control group (Table 2).

Table 1. Subject characteristics and urinary levels of HEL, 8-OHdg and TAP, and the SRS scores in the 20 individuals with ASD and 11
normal controls
ASD(n=20)

Controls(n= 11)11.1±5.2
13/7

U

p value13.9±6.6

84.00

0.30

7/4

χ2=0.00

1.00

100.9±25.91

109.38±18.45

32.00

0.52

13.6±6.3―

Domain B (communication)

8.0±4.5―

Domain C (stereotyped)

11.2±6.0―

HEL (pmol/mg Cre)77.10± 30.56

51.76 ± 26.16

53.00

0.018*

8-OHdG (ng/mL Cre)11.31± 5.50

19.51± 32.16

105.500.86

2986.18 ± 999.25

4169.71±1285.03

62.000.

049*

3.86 ± 3.12

3.64 ± 2.82

103.000.79

12.80 ± 4.4

12.73 ± 3.70

8.50

0.000***
0.000***

Variables
Age (Year)
Mean±S.D.
Sex (male/female)
Full IQ
Scores of Autism Diagnostic
Interview-Revised (ADI-R)
Domain A (social)

Urinary levels

TAP (μMUric Acid equivalents)
Plasma SOD levels (U/ml)
Subscale scores of the SRS
Irritability
Social withdrawal

25.80 ± 7.13

3.10±4.7

40.00

Stereotypy

41.25 ±11.05

4.82±7.17

1.50

0.000***

Hyperactivity

21.60±5.28

4.73±5.49

2.50

0.000***

Inappropriate speech

26.35±27.00

2.18±3.97

0.50

0.000***

Total

114.94±31.34

17.36± 23.40

3.00

0.000***

HEL, hexanolyl-lysinr; 8-OHdG, 8-hydoxy-2’-deoxyguanosine, TPA, Total Antioxidant Power; SRS,
Social Responsiveness Scale
Values are mean ± SD
* p < 0.05, *** p < 0.001compared to normal controls.

Journal of Translational Medicine and Developmental Disorders

5

Table 2. Results of the stepwise regression analysis
Model

Model

Model Coefficients

R2

P-value

0.219

0.008*

0.416

0.001*BBeta

p value

1183.529 ± 415.231

0.468

0.008*

109.753±725.554

1.229

0.000**

86.149± 28.034

0.881

0.005*

2.244± 0.803

0.460

0.009*TAP
Step 1
Group (1 = ASD, 2 = control)
Step 2
Group (1 = ASD, 2 = control)
SRS cognition scores
HEL
SRS awareness scores

0.212

0.009*

2

TAP = total antioxidant power; R = R-squared values; B =unstandardized coefficients; ASD = autism spectrum disorder; SRS =Social ResponsivenessScale* p < 0.05, ** p <0.001, significant contribution.
Table 3. The intake of nutrients in the random subsamples of 10 individuals with ASD and random subsample of 10 normal controls
ASD (n = 10)11.4± 3.9

Control (n = 10)10.1±3.9

U
35.0

p value
0.45

High (cm)142.1±21.7

141.1±20.2

42.5

0.84

Wight (kg)39.5 ± 21.6

39.2± 18.1

45.5

1.00

Energy (kcal)

2166.0±523.6

2231.8 ±617.5

40.0

0.72

Fat (g/day)

71.6± 26.2

78.8±23.3

38.0

0.60

Protein (g/day)Animal protein (mg/day)

79.2± 23.3
32.4±8.0

80.8± 27.0
33.8±12.0

43.0
41.0

0.91
0.78

Cholesterol (mg/day)

138.4±179.0

127.8 ±134.6

37.0

0.55

Carbohydrates (g/day)

297.8±58.6

292.2±86.8

45.0

1.00

Vitamin B2 (mg/day)

1.8± 0.7

1.7± 0.8

44.0

0.97

Vitamin B6 (mg/day)

1.4± 0.5

1.3± 0.4

42.5

0.84

Vitamin B12 (mg/day)
Vitamin C (mg/day)

7.3± 4.0
1678.0± 79.4

7.0±2.3
119.4± 74.5

44.5
28.0

0.97
0.18

Omega-3 (mg/day)

216.5± 161.6

192.7±90.8

36.0

0.50

Omega-6 (mg/day)

384.5 ±230.7

357.7± 121.3

35.0

0.44

Age (years)

Values are mean ± SD.
Table 4. Dietary TAP in the random subsamples of 10 individuals with ASD and 10 normal controls
ASD (n = 10)

Control (n = 10)U

p value

Green tea

7.13± 5.7

8.72±4.17

42.5

0.58

Fruit juice

2.79± 4.28

1.89±4.19

31.5

0.17

FRAP
Non alcohol beverages

Vegetables
Cabbage

0.26 ±0.26

0.16±0.13

39.5

0.44

Tomatoes
Lettuce0.08± 0.08
0.09 ± 0.05

0.10± 0.09
0.07± 0.06

43.0
41.0

0.63
0.53

Oranges0.65 ± 0.78

0.41± 0.51

39.5

0.44

Strawberries

1.10± 1.78

0.44± 0.48

43.0

0.63

Chocolate
Jam and marmalade

0.94± 0.80
0.15± 0.13

0.29± 0.33
0.00 ± 0.00

20.0
45.0

0.02*
0.007*

Coockies and biscuits

0.06± 0.05

0.02± 0.02

22.0

0.04*

Pancakes

0.03± 0.03

0.02± 0.02

41.0

0.53

Breakfast cereals

0.63± 0.46

0.42±0.35

40.5

0.48

Egg
Meat and meet products

0.03± 0.01
0.17 ± 0.09

0.02±0.01
0.20±0.20

35.0
43.5

0.28
0.63

Poultry and poultry products

0.06± 0.05

0.07±0.07

42.5

0.58

FruitsSugar confectioneri

ORAC

FRAP = ferric reducing ability of plasma; ORAC = oxygen radical absorbance capacity
Values are mean ± SD
*p < 0.05.

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Journal of Translational Medicine and Developmental Disorders

3.4. Association between Urinary HEL, TAC
and 8-OHdG Levels, and Plasma SOD
Levels and Their Relations to the SRS
ScoresThere were significant correlations between urinary
TAC levels, and plasma SOD levels (r = 0.46, p =0.04)
and SRS subscale scores of cognition (r = 0.47. p = 0.04)
in the ASD group. Urinary HEL levels were significantly
correlated with the SRS subscales of awareness (r = 0.60.
p = 0.005) and mannerisms (r = 0.69, p = 0.001). Plasma
SOD levels significantly correlated with SRS subscalescores of communication (r = 0.58, p = 0.007) and total
scores (r = 0.65, p = 0.02).

3.5. Assessment of Nutrient Intake
There were no significant differences in weight, high,energy intake, or in the intake of protein (p = 1.00),
cholesterol (p = 0.49), vitamin B2 (p = 0.97), vitamin B12(p = 0.97), vitamin C (p = 0.18), omega-6 (p = 0.44), ironP = 0.55), copper (p = 0.55), omega-3 (p = 0.50) andomega-6 PUFAs (p = 0.44) between the ASD and control
groups (Table 3).

3.6. Dietary Total Antioxidant PowerThe ASD group consumed significantly more dietary
TAC in the form of chocolate (p = 0.02), cookies andbiscuits (p = 0.04), and jam and marmalade (p = 0.007)
compared to the control group. However, there was no
significant correlation between dietary TAC and urinarylevels of TAP (r = 0.02–0.57, p = 0.95–0.053) in ASD
groups (Table 4).

4. Discussion
The present study first measured a set of urinary levels
of HEL, TAC and 8-OHdG, and revealed significantly
decreased levels of TAC and significantly increased levels
of HEL in urine of the 20 individuals with ASD comparedwith the 11 healthy controls. It was also shown that there
were no significant differences in urinary 8-OHdG levels
between the groups.
Stepwise multiple regression analysis identified
significant correlations between the urinary levels of both
TAC and HEL and the adjusting variables (the two subject
groups, the levels of other urinary biomarkers and the total
ABC scores) in the whole population. Thus, the urinarylevels of TAC and HEL were appropriate models for
distinguishing the ASD group from the control group andsignificantly predicted the adjusting variables. Thesefindings suggest that the urinary levels of TAC and HEL
may contribute to social impairment in individuals withASD.
A previous study revealed that the urinary levels of
TAC were significantly lower without a concomitant
increase in urinary catalase activity and total thiol
molecules, which are the main indicators of antioxidant
capacity [50] in 29 children with ASD aged 6–12 years
than in 24 age-matched healthy controls [6]. Further, 34
adolescent individuals with Asperger syndrome (mean age

12.89 ±2.58 years) showed significantly reduced plasmaand erythrocyte levels of TAP compared to 34 agematched controls, indicating a chronically low detoxifying
capacity in these ASD individuals [25]. Regarding urinary
HEL levels, a previous study reported that 24 children
with ASD aged 5 - 12 years showed elevated levels ofurinary HEL without concomitant alterations in theurinary levels of 8-OHdG or the erythrocyte levels of
SOD, which may result from a deficit in membrane
fluidity [11]. Further, a case-control study reported that 68
children with ASD aged 3–10 years showed significantly
increased levels of 8-OHdG in peripheral lymphocyte
DNA compared to 54 age-matched healthy controls [10],indicating a deficit in antioxidant and methylation
capacity that promote cellular damage and alter epigenetic
gene expression. As indicated in this earlier works,
pro-oxidant environmental stressors may act in conjunction
with a genetic predisposition toward the development of
ASD [10]. Previous studies evaluated the following
parameters: 1) urinary levels of TAP alone [25]; 2) urinarylevels of TAC in concomitance with urinary catalase
activity and total thiol molecules [6]; 3) urinary levels of
8-OHdG alone [10,51]; and 4) urinary levels of 8-OHdG
in concomitance with HEL [11]. According to recent
measurements, a set of oxidative stress-related biomarkers
such as urinary levels of HEL, TAC and 8-OHdG may
provide useful information on neuronal damage byoxidative stress [52,53]. The present study first examined
the urinary levels of this set of three oxidative stressrelated biomarkers in ASD.
The significant increase in urinary HEL levels and the
reduction in urinary TAC levels in individuals with ASD
demonstrated in this study indicated an imbalance
between oxidative stress and antioxidant capacity.
Moreover, stepwise regression analysis revealed that theurinary levels of TAC and HEL were reliable measures for
distinguishing the ASD group from the control group. A
previous review article suggested that declining ROS
levels are coupled with increased antioxidant enzyme
activities, depending on the formation of antioxidant
enzymes relative to the changes in the levels of free
radicals [54]. Moreover, growing evidence indicates thatthe imbalance between ROS production and TAC may
correlate with ASD [55,56]. Thus, the present study firstrevealed detailed and concrete findings on the imbalance
between oxidative stress and TAC in the urine ofindividuals with ASD. These findings may provide
additional and useful information on the significance ofimbalance between increased oxidative stress and impaired
urinary antioxidant systems in the pathophysiology ofindividuals with ASD. Although elevated levels of
8-OHdG in plasma or urine is a commonly usedbiomarker for assessing oxidative DNA damage during
inflammatory disease and pro-oxidant exposures [10],DNA methylation analysis of ASD reveals multiple
dysregulated biological pathways [57]. Thus, the ASD
group in this study may be a subset of ASD without DNA
methylation damage.
Stepwise regression analysis and Spearman correlation
coefficients revealed that urinary TAC levels weresignificantly correlated to plasma SOD levels and the SRS
subscale scores for cognition. Previous studies reported
significant decrease in plasma and urine SOD andJournal of Translational Medicine and Developmental Disorders

TAC levels in 47 sarcoma patients [58] and significant
increase in urine SOD and TAC levels after administrationof L-carnitine in 12 healthy subjects [59], indicatingconcomitant changes of plasma and urine SOD and TAC,
supporting our findings. A previous study reported asignificant positive correlation between plasma TAC
levels and cognitive function in 105 subjects with early
onset-first episode psychosis [60], and an association
between lowered plasma TAP levels and cognitive
dysfunction in 138 older adults with mild cognitive
dysfunction [61] and in 90 healthy care workers [62]. The
Spearman correlation coefficient also revealed a significant
association between plasma SOD levels and the SRS
subscale of communication. A previous study reported
significant correlations between blood levels of SOD andpsychosocial functioning such as “relation to friends” and“taking initiative and self-sufficiency” in 51 subjects withbipolar depression [63]. Social interaction-mediatedlifespan extension of SOD mutants has been found in
human [64]. Thus, SOD may be significantly correlated tosocial behavior, which supports our present findings.
Stepwise regression analysis and Spearman correlation
coefficients further revealed that the urinary HEL levels
were significantly correlated to the SRS subscale scoresfor awareness and mannerisms. Of reference, the plasma
levels of 3-nitrotyrosine (3-MT), which is an oxidative
stress marker [65], have previously been associated withlow scores in SRS subscales of awareness in 18 children
with ASD [66]. Sulforaphane, which has therapeuticpotential against oxidative stress, was shown to improve
SRS scores by 17% [48]. Thus, oxidative stress biomarkers
are associated with the SRS scores, which supports our
findings.
A previous assessment of daily nutrient intake using
DHQ 15 revealed no significant differences in the intakeof fat, protein, vitamin B2, vitamin B6, vitamin B12,
vitamin C, omega-6, or omega-3 PUFAs between random
subsamples of 10 individuals with ASD and those of 10
healthy controls. With respect to the relationship between
dietary intake and the urinary levels of TAP, intake ofvirgin olive oil [67] and tryptophan-enriched cereal [68]have been shown to increase urinary TAC. Conversely,refined potato starch resulted in decreased urinary levelsof TAC [69]. Therefore, the effect of dietary intake on
urinary TAC may be variable, and differences in dietaryintake between the groups may not affect urinary TAC.
The dietary TAC of chocolate, biscuits and cookies,
jam and marmalade was significantly higher in the ASD
group than in the control group. Cocoa products such as
chocolate are good sources of dietary antioxidants [70].
Cookies containing chocolate chips have been shown topossess the highest antioxidant capacity [71]. Biscuits
including 5 % cocoa increased antioxidant properties [72].
Jam and marmalade processing has been shown toincrease antioxidant capacity [73], and bilberry jams havebeen revealed as a good source of antioxidant compounds[74,75]. Thus, chocolate, cookies, biscuits, jam andmarmalade possess higher antioxidant power. However,urinary TAC levels in the ASD group were significantly
lower than those in the control group, indicating thatantioxidant capacity in the ASD group may be impaired.
Oxidative stress contributes to many pathologicalconditions. Aerobic organisms have integrated antioxidant

7

systems, which include enzymatic and nonenzymatic
antioxidants that are usually effective in blocking theharmful effects of ROS [76]. Such systems include theendogenous antioxidant defense system [77] and intrinsic
antioxidant defenses [78]. Most previous studies on
antioxidant capacity in individuals with ASD havesuggested a vulnerability in antioxidant enzymes [24] or achronically low detoxifying capacity [25]. These
antioxidant defense systems may be a part of theendogenous antioxidant systems. Indeed, recent research
on antioxidant networks has demonstrated that antioxidant
enzymes such as SOD, glutathione peroxidase, andglutathione act as an antioxidant network within specificintracellular or extracellular components of the antioxidant
system [79]. Further, a recent work has suggested that the
autophagy-lysosomal activities of these antioxidant
enzymes may serve an essential function in preventing
neurodegenerative diseases by removing damaged as partof an essential cellular antioxidant pathway [80]. These
considerations taking together suggested that theendogenous intrinsic antioxidant system may be impaired
in individuals with ASD. However, the present study did
not examine the specific neurobiological roles of urinary
TAP and plasma SOD, and further studies will need todetermine which specific components may be affected in
ASD.
Compromised intrinsic antioxidant defense contributeto blood-brain barrier (BBB) disruption through oxidative
damage to cellular molecules [81]. When BBB disruption
occurs, brain-speciﬁc proteins circulating inside the brain,
are observed in the peripheral blood as an index for
estimating the extent of the increase in BBB permeability
and brain damage [81]. As described above, overproductionof ROS results in oxidative stress the imbalance between
ROS and antioxidant capacity (referred to oxidative stress)
became toxic to neurons, damaging DNA, proteins andlipids, inducing neurodegeneration [4] such as ASD [82].
Thus, the imbalance between the generation of ROS andTAC may disrupt BBB, and be related to impaired social
responsiveness through biochemical processes such asimbalance between increased urinary levels of HEL as
oxidants and urinary levels of TAC as antioxidant inindividuals with ASD.
This study has some limitations. Previous studies
examined urinary oxidative stress biomarkers such as
F2-isoprostanes and their association with the activity ofplasma enzymatic antioxidants such as SOD andglutathione peroxidase [83,84]. In this study, the urinarylevels of 2-isoprostanes and plasma levels of glutathione
were not measured; however, we examined a useful andinformative set of oxidative stress-related biomarkers anddiscovered novel important information on impaired
antioxidant capacity that was not revealed in previous
studies [6,11,25,85]. Second, ASD is most prevalent inmales, with a male to female ratio of 4 to 1 [86]. However,in this study, the ASD and control groups were age- andgender-matched. Finally, the small sample size affects ourability to generalize our results to the entire population ofindividuals with ASD.
In conclusion, the present study firstly found reduced
levels of TAC and increased levels of HEL in urine inindividuals with ASD with a lack of significant alterationsin urinary 8-OHdG levels. Thus, the imbalance between

8

Journal of Translational Medicine and Developmental Disorders

the generation of ROS and TAC was impaired inindividuals with ASD. In addition, as stepwise regression
analysis revealed that urinary TAC levels showed
preferentially contribute to the adjusting variables
compared to urinary HEL levels, urinary TAC levels may
be more appropriate model for distinguishing the ASD
group from the control groups. Thus, endogenous
antioxidant defense systems may be impaired in young
individuals with ASD. Plasma SOD levels may be
associated with urinary TAC levels and may be related tothe impaired antioxidant capacity. These defects in thebalance between oxidative stress and antioxidant capacity
in urine may be related to the pathophysiology of social
impairment in individuals with ASD.

[11] Ghezzo, A., Visconti, P., Abruzzo, PM., Bolotta, A., Ferreri, C.,

[12]

[13]
[14]

[15]

Acknowledgements[16]This study was supported by 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.

Conflict of Interest

[17]
[18]

[19]

The authors declare that they have no conflict of interest.

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