Article

Lipid Peroxidation via Regulating the Metabolism ofDocosahexaenoic Acid and Arachidonic Acid in Autistic
Behavioral Symptoms
Kunio Yui 1,2, *, George Imataka 3
1
2
3

*

Citation: Yui, K.; Imataka, G.;
Shiohama, T. Lipid Peroxidation via
Regulating the Metabolism ofDocosahexaenoic Acid andArachidonic Acid in Autistic

and Tadashi Shiohama 1

Department of Pediatrics, Graduate School of Medicine, Chiba University, Chiba 260-8677, Japan
Department of Urology, Fujita Health University School of Medicine, Toyoake 470-1192, Japan
Department of Pediatrics, Dokkyo Medical University, Tochigi 321-0293, Japan
Correspondence: yui16@bell.ocn.ne.jp

Abstract: The association between the lipid peroxidation product malondialdehyde (MDA)-modified
low-density lipoprotein (MDA-LDL) and the pathophysiology of autism spectrum disorder (ASD)
is unclear. This association was studied in 17 children with ASD and seven age-matched controls
regarding autistic behaviors. Behavioral symptoms were assessed using the Aberrant Behavior
Checklist (ABC). To compensate for the small sample size, adaptive Lasso was used to increase thelikelihood of accurate prediction, and a coefficient of variation was calculated for suitable variableselection. Plasma MDA-LDL levels were significantly increased, and plasma SOD levels weresignificantly decreased in addition to significantly increased plasma docosahexaenoic acid (DHA)
levels and significantly decreased plasma arachidonic acid (ARA) levels in the 17 subjects with ASD as
compared with those of the seven healthy controls. The total ABC scores were significantly higher inthe ASD group than in the control group. The results of multiple linear regression and adaptive Lassoanalyses revealed an association between increased plasma DHA levels and decreased plasma ARAlevels, which were significantly associated with total ABC score and increased plasma MDA-LDL
levels. Therefore, an imbalance between plasma DHA and ARA levels induces ferroptosis via lipid
peroxidation. Decreased levels of α-linolenic acid and γ-linolenic acid may be connected to the total
ABC scores with regard to lipid peroxidation.
Keywords: lipid peroxidation; ferroptosis; malondialdehyde-modified low-density lipoprotein;
autistic behavior; do cosahexaenoic acid; arachidonic acidBehavioral Symptoms. Curr. Issues
Mol. Biol. 2023, 45, 9149–9164.
https://doi.org/10.3390/
cimb45110574
Academic Editor: Gadi Turgeman
Received: 20 September 2023
Revised: 27 October 2023
Accepted: 6 November 2023
Published: 15 November 2023

Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Lipids, including polyunsaturated fatty acids (PUFAs), are the main component of cell
membranes and play an important role in maintaining the structural integrity of cells [1].
Lipid peroxidation is defined as a process in which oxidant-related free radicals attack
polyunsaturated fatty acids (PUFAs), and it has shown its important role in cell biology
and human health [1]. PUFAs produce malondialdehyde (MDA) and 4-hydroxy-2-nonenal(4-HNE) during lipid peroxidation chain reactions (Ay) [2]. MDA is used as a biomarker oflipid peroxidation [2]. Lipid peroxidation plays an important role in the pathophysiologyof neurodevelopmental disorders such as autism spectrum disorder (ASD) [3]. The plasma
MDA levels were significantly higher in 20 children with ASD than those in 20 age-matched
controls [4], and the blood MDA levels in 45 autistic children (aged 3–11 years) were higher
than those in 42 age-matched controls [5].
Potentially oxidizable PUFA species in plasma membrane samples are more abundant,
and there is a need to counterbalance the antioxidant activity [6]. This can easily induce
ferroptosis, leading to irreparable membrane damage and cell death [7,8]. In general,oxidants and antioxidants are driven by the activity of multiple redox-active enzymes
that produce lipid oxidation products [9], with increased MDA and decreased SOD [10].

Curr. Issues Mol. Biol. 2023, 45, 9149–9164. https://doi.org/10.3390/cimb45110574

https://www.mdpi.com/journal/cimb

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Ferroptosis is mediated by PUFA-related lipid peroxidation [11] via polyunsaturated phosphatidylethanolamines or complexes of 15-Lipoxygenase (15LOX) [12].
DHA triggers ferroptosis via the upregulation of nuclear receptor coactivator 4(NCOA4) [8], while arachidonic acid (ARA) facilitates ferroptosis via the elongation ofvery long-chain fatty acid protein 5 (ELOVL5) and fatty acid desaturase-1 (FADS1) [13] orarachidonic acid 15-lipoxygenase [14]. Therefore, ferroptosis might be an important factor
in lipid peroxidation in relation to DHA and ARA.
As another component of the lipid profile, low-density lipoprotein (LDL) has animportant role in brain development. Of reference, significantly higher blood LDL levels
were reported in 22 adults with Asperger’s syndrome (mean age, 40.8 ± 10.8 years) than inthose of 22 age-matched controls, suggesting abnormal cholesterol metabolism [15]. Thus,
blood LDL levels also contribute to the pathophysiology of ASD.
MDA undergoes a chemical reaction with acetaldehyde to form malondialdehydemodified low-density lipoprotein (MDA-LDL) [15–17]. MDA-LDL is an important biomarker
of lipid peroxidation. However, only a few studies have examined the role of MDA-LDL in
the pathophysiology of ASD. This study examines the pathophysiological role of MDALDA in behavioral symptoms in ASD.
The antioxidant protein superoxide dismutase (SOD) is associated with MDA; forexample, in a study, the lipid peroxidation and SOD levels were found to be significantly
higher in 30 autistic children than they were in 30 unaffected control children [17]. However,
the activity of SOD in plasma and erythrocytes is variable. For example, the serum SOD
levels were increased in 20 children with ASD compared with those in 25 age-matched
controls, suggesting the decreased activity of the antioxidant defense mechanism [4], while
the serum SOD levels were significantly lower in autistic children aged < 6 years thanthey were in the age-matched controls. Additionally, the MDA level was significantly
higher in these children than it was in the controls [4]. These findings suggest that children
with ASD are vulnerable to oxidative stress, resulting in increased lipid peroxidation anddeficient antioxidant defense mechanisms in younger children [4]. Furthermore, a close
relationship between MDA and SOD was reported in 90 autistic children who were treatedwith coenzyme Q10 [18]. The association between MDA and SOD in the pathophysiology
of ASD remains unclear. Additionally, dietary dihomo-γ-linolenic acid (DGLA; omega-6)
triggers ferroptosis in the germ cells of the model organism [19]. Similarly, omega-6
PUFA adrenic acid is a lipid peroxidation product [20]. α-linolenic acid (ALA) affects
lipid peroxidation due to increased oxidative stress [21]. The present study revealed thecontribution of DGLA and ALA to lipid peroxidation.
Taking these considerations into account, there are two important aims of this study:(a) determining the role of DHA- and ARA-induced ferroptosis via lipid peroxidation in
autistic behavioral symptoms and (b) clearly reporting the relationship between the plasma
levels of MDA-LDL and SOD in individuals with autistic behaviors.
In this study, we employed the adaptive Lasso technique to ensure accurate prediction.
Adaptive Lasso parameters can assist with estimation and variable selection in highdimensional sparse models. The simulation findings and real data examples indicate theimproved performance of the proposed penalized method [22]. The adaptive Lasso method
is a popular statistical approach based on weighted norm penalties in weights derived
from an initial estimate of a model parameter [23], and adaptive Lasso welcomes negative
variable effects while specifying adaptive weights to penalize the coefficients in a different
manner [23].
2. Materials and Methods
2.1. Subjects
Seventeen individuals with mild-to-moderate ASD and seven age-matched and healthy
male and female control individuals were randomly included in this study. Such an imbalance in case/control individuals was recognized in a series of case–control studies, such asa case versus control study including 18 symptomatic acute intermittent porphyria patients

Curr. Issues Mol. Biol. 2023, 45

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and 33 healthy controls were performed in a recent study evaluating metabolic changes inacute intermittent porphyria patients via targeted metabolomics [24].
The information clearly presents the research methods, and the random recruitment
process was conducted according to the IRB submission requirements. All 17 individuals
with ASD were subjected to a medical examination at our medical clinic (Fujimoto Clinic,
Kobe City, Japan), taking place between January 2020 and June 2021. Seven healthy
individuals in the same region were included in this study and subjected to a medical
examination at our medical clinic during the same period.
Each of the 17 individuals received a clinical diagnosis of ASD. The 17 patients with
ASD included 11 males and 6 females (mean age: 11.2 ± 5.7 years). The 7 healthy controls
included 4 males and 3 females (mean age: 10.0 ± 4.1 years) (Table 1). The diagnosis of
ASD was conducted based on the Diagnostic and Statistical Manual of Mental Disorders,
Fifth Edition (DSM-5). The Autism Diagnostic Interview-Revised (ADI-R) instrument
was used to confirm the ASD diagnoses. The ADI-R interviews were conducted by oneof the authors (K.Y.), who is an expert in diagnosing ASD using the Japanese versionof the ADI-R. The ADI-R is a semi-structured interview conducted with the respective
patients’ parents. Seventeen individuals with mild-to-moderate ASD had core symptomsof the DSM-5 diagnostic criteria for ASD without any abnormal neurological symptoms.
These 17 individuals with mild-to-moderate ASD and the 8 healthy controls were matchedwith respect to feeding habits, age, and the full-scale intelligent quotient (Table 1). No
participants had any abnormalities in the results of their physical examinations or laboratory
findings. Their intelligence quotients were assessed using the Wechsler Intelligence Scale
for Children and Adolescents of 6–16 years of age (WISC-V) or the respective scale for adults(Wechsler Adult Intelligence Scale (WAIC-R)). We used the Wechsler Intelligence Scale forChildren, Third Edition (WISC-III), to include subjects with normal cognitive functions
and a full-scale IQ of ≤70. We excluded subjects who have comorbid psychiatric illnesses
using the Structured Clinical Interview for DSM-5. Individuals were excluded from thisstudy if they had epileptic seizures or obsessive-compulsive disorder or were diagnosed
with any additional psychiatric or neurological conditions. None of the individuals with
ASD took dietary supplements. The healthy control subjects were recruited locally using
an advertisement. All the control individuals underwent a comprehensive assessment of
their medical histories to exclude individuals with neurological or other medical disorders.
Table 1. Subject characteristics and plasma levels of PUFAS, MDA-LDL, and antioxidant proteins,and ABC total scores.

Age (years)
Sex (male/female)
Scores of Autism Diagnostic
Interview-revised
Domain A (Social)
Domain B (Communication)
Domain C (stereotyped)

ASD(n = 17)

Controls(n = 7)

U

p Value

11.2 ± 5.7
13/17

10.0 ± 4.1
4/17

45.00
χ2 = 0.46

0.49
0.50

13.6 ± 4.3
2.1 ± 2.3
17.6 ± 5.6

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Table 1. Cont.

Plasma biomarker levels
Cp (mg/dL)
Tf (mg/dL)
SOD (mg/dL)
MDA-LDLDHA
ARA
DHA/ARAAdA
DGLA
Total scores of Aberrant
Behavior Checklist

ASD(n = 17)

Controls(n = 7)

U

p Value

28.29 ± 7.21
275.71 ± 43.16
2.51 ± 0.47
90.94 ± 17.24
3.53 ± 0.98
6.34 ±1.110.59 ± 0.17
0.27 ± 0.25
1.27 ± 0.33

24.29 ± 7.25
262.29 ± 25.75
5.69 ± 4.64
71.29 ± 17.87
2.52 ± 0.90
6.68 ±1.67
0.37 ± 0.08
0.27 ± 0.07
1.60 ± 0.22

44.50
47.00
18.50
24.50
28.50
49.00
11.50
28.50
25.50

0.35
0.46
0.01 *
0.02 *
0.047 *
0.53
0.01 *
0.047 *
0.03 *

49.41 ± 25.57

0.71 ± 1.25

0.00

p < 0.001 **Cp, ceruloplasmin; Tf, transferrin; SOD, superoxide dismutase; MDA-LDL, malondialdehyde modified lowdensity lipoprotein; DHA, docosahexaenoic acid; ARA, arachidonic acid; DGLA, dihomo-γ-linolenic acid: AdA,
adrenic acid; ABC, Aberrant Behavior Checklist. Values are represented as mean ± SD (Mann–Whitney U test).
* p < 0.05. ** p < 0.001, versus normal controls.

2.2. Precautions for Mitigating the Effects of Small Sample Sizes
The small sample size in this study may limit the interpretation of the results. Therefore, we employed two precautions. First, the most appropriate method is needed witha small sample size [23]. Adaptive Lasso may select the desirable covariates to build anunbiased and statistically efficient propensity score estimator [22]. Therefore, we usedadaptive Lasso. Second, standard deviations (SDs) are useful for expressing variability.
Therefore, data reliability was evaluated using the coefficient of variation (CV, %), which isdefined as the SD/the mean value to measure the relative variation in a random variable.
The CV was employed to determine between- and within-subject reliability [25], and this isoften used to measure relative variation in small sample studies.
Moreover, a large sample may not be helpful in other situations because of the higher
possibility of errors and reduced validity. Analytical studies may provide more truthful
results with a small sample because intensive efforts can be made to control all the confounders; small samples can be studied to reach a valid conclusion in certain situations [26].
According to a former clinical study, despite the small sample size (12 patients), statistically significant improvement was obtained by an intensive neuro-rehabilitation surface
program with electromyography [27].
2.3. Assessment of Social Behaviors
Social behaviors were assessed using the Social Responsiveness Scale (SRS), which
is used to distinguish ASD from other psychiatric disorders [28]. The SRS is a 65-item
questionnaire completed by the respective parents of the subject to quantitatively assess
autistic traits [28]. The SRS was used to assess the severity of the ASD symptoms [29].
2.4. Controlling for Dietary Intake and Assessment of Nutrient Intake
The PUFA composition of blood reflects dietary intake. In this study, all the participants received the “Japanese Food Guide” (Ministry of Health, Labour and Welfare, andMinistry of Agriculture, Forestry and Fishers, Japanese Food Guide, 2012), which is used tomonitor the daily intake of nutrients and food based on the “Overview of Dietary Reference
Intake for Japanese (2010)” (Ministry of Health, Labour, and Welfare, 2010). All the subjects
were provided a diet based on the sample diet meal plan and menu (KAWASAKI FOODMODEL), which was edited according to the “Japanese Food Guide” (Ministry of Health,Labour and Welfare, and Ministry of Agriculture, Forestry and Fishers, Japanese Food
Guide, 2012). Moreover, to measure daily food and nutrient intake, a semi-constructive
questionnaire (DHQ) was performed in Japanese using a junior high school version of theCurr. Issues Mol. Biol. 2023, 45

9153

DHQ15 (DHQ Support Center, http://www.ebnjapan.org/, accessed on 1 January 2020).
The DHQ15 consists of 72 questions on the frequency of intake of 150 food and beverage
items and cooking methods. The DHQ15 was administered for two weeks before this study.
Because of the complicated assessment of DHQ15, only a subsample of 7 individuals with
ASD and 7 controls participated in this assessment. Because of difficulty with nutrientsin foods, the nutrient items and portion sizes in the questionnaire were derived primarily from the data in the Overview of Dietary Reference Intake for Japanese (Ministry of
Health, Labour, and Welfare. Overview of dietary reference Intake for Japanese. 2015).
The completed DHQ15 data sheets were checked by a psychiatrist (K.Y.). When inaccurate
information was recognized, the psychiatrist (K.Y.) confirmed the data using a phone. The
validity of the DHQ15 was verified. The daily intake of nutrients was calculated using a
DHQ system program (DHQ support center, Tokyo, Japan).
2.5. Measurement of Plasma PUFA, Cp, SOD, and Tf Levels
2.5.1. Blood-Sampling Procedures
Fasting exerts beneficial effects in mice and humans, including protection fromchemotherapy toxicity [30]. After a 3 h fasted interval, whole blood samples were collectedby venipuncture into EDTA tubes and then placed on ice. With respect to the fasting interval,
the extent of return to the fasting state 2 h after a glucose challenge among 1879 normoglycemic individuals is associated with a lower risk of incident prediabetes/type 2 diabetes
in the Coronary Artery Risk Development [31]. Moreover, nonfasting triglyceride levels
may replace fasting levels in assessing cardiovascular disease risk once standard reference
values are developed [32]. Therefore, a fasting time of 3 h after breakfast is reasonable forapplication for blood sampling in the present study. The blood samples were frozen at −80◦ C until the plasma levels of the variables were analyzed at a clinical laboratory (SRL Inc.,Tokyo, Japan).
Since the present study aimed to identify the association of lipid peroxidation withferroptosis, we did not measure eicosanoids such as prostaglandin (PG), thromboxane(TX), and leukotrienes (LT) in the present study. The association between the levels ofpro-inflammatory ARA and pro-inflammatory eicosanoids has been reported [33]; however,
we did not study the association between eicosanoids and peroxidation in the present study.
In response to the association between cytokine and lipid peroxidation, a few referencesindicated the association between proinflammatory cytokines and lipid peroxidation inpatients with severe dengue disease [34].
2.5.2. Plasma Levels of PUFAs
Blood samples were drawn from the 24 participants after at least 3 h of fasting. The
serum specimens were separated, frozen, and stored at −80 ◦ C until assaying. The plasma
fatty acid levels were measured using the gas chromatography method (SRL, Tokyo).
Twenty-four PUFAs, including the EPA, DHA, and AA concentrations, were measured.
The intra- and inter-assay coefficients of ARA 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, while 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%). The plasma levels are expressed as the mean ± SD weight (%) of thetotal PUFAs.
2.5.3. Plasma Levels of SOD
The plasma SOD levels were estimated via the cytochrome c method using an SOD
Assay Kit (Takara Bio Inc., Kusatsu, Japan) in a clinical analytical laboratory (SRL Inc.,Tokyo, Japan). The assay sensitivity was 0.3 U/mL. The intra- and inter-assay coefficientswere 2.11 and 2.10 U/mL, respectively. (Table 1).

Curr. Issues Mol. Biol. 2023, 459154

2.5.4. Plasma Levels of CP
To estimate the plasma CP levels, a Bering BN II Nephelometer (Siemens Healthcare
Diagnostics K.K., Issaquah, WA, USA) was used. The assay sensitivity was 3.0 mg/dL.
2.5.5. Plasma Levels of TF
To assess the plasma TF levels, a standard turbidimetric assay and an automatedbiochemical analyzer (JCA-BM8000 series, JEOL Ltd., Tokyo, Japan) were used.
2.6. Plasma Levels of MDA-LDL
An enzyme-linked immunosorbent assay (ELISA) was used to measure the plasma
levels of MDA-LDL; the measurement was performed in a clinical analytical laboratory(SRL Inc., Tokyo, Japan). The detection limit was 6.3 U/L, and the intra- and inter-assay
coefficients were <5.6% and <9.4%, respectively.
2.7. Sex Differences in Plasma Variables and the Total SRS Scores
To compare the plasma variables and the total SRS scores between the male and female
individuals, we conducted the Mann–Whitney U test.
2.8. Statistical Analyses
The relationship between the plasma variables and SRS scores in the two groups
was confirmed via multiple linear regression analysis (Table 2). To identify the mosteffective variables in small sample data, adaptive Lasso was employed. Adaptive Lasso
has a good statistical performance in solving independent variables and enhancing modelinterpretation and prediction accuracy. Adaptive Lasso is useful for consistent variableselection and identifying important variables [22] in small samples [23]. All of the statisticalanalyses were performed using SPSS version 27.0.
Table 2. Results of the multiple linear regression.

Model
DHA
ALA
DGLA
MDA-LDLSOD
ABC total scores

Model
R2

Model
Value

0.997

p < 0.001 **Group
1 = ASD, 2 = controls
DHA/ARAALA
DGLA
MDA-LDLSOD
ABC total scoresGroup
1 = ASD, 2 = control

0.982

CoefficientsB

Beta
Coefficientsp Value

0.849 0.245−0.210 0.029−0.057 0.124−0.0076 0.020
0.002 0.002

0.164−0.421−0.025−0.203
0.067

0.018
0.002
0.667
0.012
0.253

−0.057 0.124−0.0250.667−0.342 0.124−0.122 0.058
0.002 0.001
0.008 0.050
0.022 0.024−0.395−0.230.270
0.023
0.348

0.041
0.090
0.096
0.895
0.183

0.007 0.055

0.019

0.899

0.004 **

R2 , R-squared values; B, Unstandardized coefficients; ALA, α-linolenic acid; DGLA, dihomo-γ-linolenic acid;
SOD, superoxide dismutase; MDA-LDL, malondialdehyde-modified low-density lipoprotein; ABC, Aberrant
Behavior Checklist. ** p < 0.001.

Curr. Issues Mol. Biol. 2023, 459155

3. Results
3.1. Study Population
Age did not significantly differ between the two groups of participants. The 17 individuals with ASD showed restricted and stereotypical behavior patterns (n = 10) or irritability
and crying (n = 7). Their mean total ABC score was 49.41 ± 24.58 (Table 1). A previous
study reported a total ABC score of 60.14 in 29 patients with ASD aged 13–27 years [35],
Therefore, the behavioral symptoms of the 17 children in this study were moderate.
3.2. Plasma Levels of Lipid-Peroxidation-Related Biomarkers
In the ASD group, plasma MDA-LDL levels were significantly higher, and plasma
SOD levels were significantly lower in comparison with the control group. The plasma
DHA level and the plasma DHA/ARA ratio were significantly higher, and the plasma
level of adrenic acid (AdA), and omega 6 PUFA, was significantly lower than in the control
group (Table 1) (Figures 1 and 2).

Figure 1. The association between the DHA and DHA/ARAS ratio and other plasma variables andthe ABC.

Curr. Issues Mol. Biol. 2023, 3, FOR PEER REVIEW
Curr. Issues Mol. Biol. 2023, 45

89156

Figure 2. The results of adaptive Lasso.
Figure 2. The results of adaptive Lasso.

Plasma levels of the omega-3 PUFA DHA and the ratio of plasma DHA/ARA were
Plasma levelsof thewhereas
omega-3plasma
PUFA levels
DHA andthe ratiofamilyof plasma
DHA/ARA weresignificantly
higher,of omega-6
dihomo-γ-linolenicacid
significantly
higher,whereasplasmalevelsof omega-6
familydihomo-γ-linolenic(DGLA) andadrenic
acid (AdA)
weresignificantly
lower
in theASD group (darkacid
green)(DGLA)
and(AdA)
significantly
than intheadrenic
controlacid
group(lightwere
green)(Table 1). lower in the ASD group (dark green)
than in the control group (light green) (Table 1).
3.3. Predictor Variables: Multiple Linear Regression Analysis
3.3. Predictor
Variables:
Multiple
Linear analysis
Regression
Analysis
The multiple
linearregression
revealed
that the plasma DHA levels (R2 = 0.997,
2p<0.001)and plasma
ratio (R revealed= 0.982, pthat= 0.004)
were significantly
associatedThe
multiplelinear DHA/ARA
regression analysis
the plasma
DHA levels
(R2 =
with
the
total
ABC
scores,withadjustmentsinplasmavariables,
and
the
total
SRS
2
0.997, p < 0.001) and plasma DHA/ARA ratio (R = 0.982, p = 0.004) were significantly scoresasin the with
two subject
groupsThesefindings revealedthatplasma DHA
levelssociated
the total
ABC (Table
scores,3).
withadjustments
in plasma
variables,
and thetotalandratios groups
may predictvariablesin thetwo groups.
Via theuse ofSRSplasmascores DHA/ARA
in the two subject(Tablethese3). These
findings
revealed
that plasma
DHAtheplasmalevelsof
α-linolenic
acid
(ALA)
as
the
dependent
variable,theselevelsshowedlevels and plasma DHA/ARA ratios may predict these variables in the two groups. Via
contribution
by the plasma
DHAaslevels(unstandardized
coefficients,
thetheusesignificantof the plasma
levels ofmade
α-linolenicacid (ALA)
the dependent
variable,
these
B
=
0.849
±
0.245
β
=
0.1641,
p
=
0.02)
and
the
plasmaratiosof
DHA/ARA(unstandardized
levels showed the significant contribution made by the plasma DHA levels (unstandardcoefficients,
0.342±±0.245
0.124β β= =−0.395,= 0.041)2) (Figure
ized
coeﬃcients,B B= =−0.849
0.1641,p =p0.02)
and (Table
the plasma
ratios1).
of Therefore,
DHA/ARAthe
plasmaDHA
levelsandplasmaDHA/ARA
ratio
distinguished
the
ASD
group
from
(unstandardized coeﬃcients, B = −0.342 ± 0.124 β = −0.395, p = 0.041) (Table 2) (Figure1).thecontrol group.
Therefore, the plasma DHA levels and plasma DHA/ARA ratio distinguished the ASD
group from the control group.
Table 3. Results of adaptive Lasso.

Intercept

Table 3. Results of adaptive Lasso.
p Value
Estimate
SE

Intercept

EstimateSE

p Value

DHAscores
ABC total
DHA/ARADHA
α-linoleic acid
DHA/ARAMDA-LDLα-linoleic
DHA acid
DHA/ARAMDA-LDLα-linoleic acid
DHA
DHA/ARAα-linoleic acid136.50136.50 −74.88−75.4854.89
54.8946.97
26.21

0.012
0.111
0.012
0.004

ABC total scores−74.88−75.48 −105.88
98.81

46.97
26.2150.74

0.111
0.004 0.038

32.21

0.002

95%

Confidence Interval

Lower Bounds Confidence
Upper Bounds
95％Interval
Lower Bounds
Upper Bounds
29.11−166.94−126.8429.11−166.94−126.84 −205.32
35.67

243.9017.18
243.90−24.12

17.18−24.12−6.44

161.95−205.32 −22.52−6.4487.28
35.67
161.95−22.5287.28
We employed the adaptive Lasso approach to increase the predictive accuracy and3.4.interpretability
Results of Adaptive
Lassoof thestatistical model for small samples [20,21]. The plasma levels of DHA(standardcoefficient,136.50; 95%
CI,approach
29.10 to 243.90;
p = 0.017)
and the plasma
We employed
the adaptive
Lasso
to increase
the predictive
accuracyratios
and ofARA(coefficient,−104.84;95%CI,
193.69to25.98
p=0.000)
andALA(coefficient,−interpretability of the statistical model for small samples [20,21]. The plasma levels 75.48)
ofwere
selectedcoeﬃcient,
for the ABC
total scores(TableDHA(standard
136.50;95% CI,
29.103)to(Figure243.90; 2).
p = 0.017) and the plasma ratios−105.8831.38
50.7428.01 0.038 0.247
98.81
32.21
0.002
of Adaptive Lasso
31.383.4. Results 28.01
0.247

of ARA(coeﬃcient,CI, 193.69 to 25.98 p = 0.000) and ALA (coeﬃcient, −75.48)
3.5. The
Profiles of−104.84;
Nutrient95%
Intakewere selected for the ABC total scores (Table 3) (Figure 2).
The intake of nutrients in the random subsamples of 7 of the 17 individuals and 5of the 7 normal controls were analyzed, and there were no significant differences in the
nutrient intake profile between both subsamples (Table 4).

Curr. Issues Mol. Biol. 2023, 45

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Table 4. The intake of nutrients in the random subsamples of 7 of the 17 individuals and 5 of the
7 normal controls.
ASD (n = 7)

Control (n = 5)U

p ValueAge (years)

11.4 ± 4.3

11.4 ± 3.2

14.0

0.87

Fat (g/day)

72.2 ± 30.1

87.4 ± 25.8

12.0

0.43

Unsaturated fatty acid (g/day)

14.8 ± 4.4

18.7 ± 5.5

9.0

0.20

Omega-3 PUFAs (g/day)

2.6 ± 0.8

3.1 ± 0.5

9.0

0.20

Omega-6 PUFAs (g/day)

12.1 ± 3.9

15.9 ± 5.1

11.0

0.34

EPA (mg/day)

181.2 ± 118.7

176.2 ± 73.6

15.5

0.76

DHA (mg/day)

332.6 ± 170.4

345.0 ± 83.31

15.5

0.76

ARA (mg/day)

168.1 ± 17.1

221.0 ± 87.7

11.5

0.34

Protein (g/day)

78.1 ± 25.8

89.2 ± 25.8

13.0

0.53

Animal protein (mg/day)

32.0 ± 9.1

30.4 ± 14.7

14.0

0.64

Cholesterol (mg/day)

139.1± 186.4

31.9 ± 10.3

17.0

1.00

286.2 ± 62.1

304.21 ± 72.4

14.0

0.64

Carbohydrates (g/day)

EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; ARA, arachidonic acid. Values are mean ± SD.

3.6. Sex Difference
There were no gender differences in plasma levels of the main variables (Table 5).
Table 5. Sex differences in plasma variables and the total SRS scores.
MDA-LDL

DHA

ARA

SOD

Total ABC Score

Male (n = 12)

92.55 ± 14.69

3.43 ± 1.07

6.43 ± 1.12

2.76 ± 0.53

41.81 ± 29.03

Female (n = 5)

88.93 ± 29.88

3.05 ± 0.71

6.32 ± 1.77

4.21 ± 3.54

19.78 ± 29.68

U

112.00100.50139.00

99.00

45.50

p values

0.34

0.17

0.99

0.16

0.07

DHA, docosahexaenoic acid; ARA, arachidonic acid; SOD, superoxide dismutase; ABC, Aberrant Behavior
Checklist.

4. Discussion
Two measures were taken in this study because of the small sample size. The mean CVs
for the plasma PUFAs were 0.451 (45.17%) and 0.277 (27.7%) in the ASD and control groups,
respectively. With respect to the CVs, the plasma levels of the anti-cancer drug palbociclib
were variable, with a CV from 38.8% to 48.5% [36]. Furthermore, CV in cardiothoracic
ratio on chest radiograph in pediatric heart disease was 32.5% Gro [37]. Thus, the CVs
in the present study were comparable to these previously reported CVs, suggesting theappropriateness of our variable selection.
In the present study, in the ASD group, the plasma MDA-LDL levels were significantly
higher, and the plasma SOD levels were significantly lower in comparison with thoseof the control group. The plasma DHA level and the plasma DHA/ARA ratio weresignificantly higher in the 17 subjects with ASD than they were in the control group(Table 1). Additionally, the omega-3 PUFA family AdA and omega-6 PUFA family GLA
levels in the plasma were more significantly decreased in the ASD group as comparedwith those of the control group. Notably, the results of multiple linear regression analysis
and the adaptive Lasso method revealed that increased DHA and decreased ARA plasma
levels were appropriate variables that distinguished the ASD group from the normal
control group. Importantly, higher plasma MDA-LDL levels in the ASD group indicatedferroptosis [9,10]. PUFAs are highly susceptible to lipid peroxidation under oxidative
stress [7]. The lipid peroxidation of cellular membranes caused by the disruption of theantioxidant defense system induces ferroptosis, which is a type of regulated cell death.

Curr. Issues Mol. Biol. 2023, 45

9158

Ferroptosis is a recently proposed novel mode of cell death. The inhibition of ferroptosis
may provide the possibility to prevent and improve impaired neurocognitive disorders [38].
DHA and EPA prevent seizure and depression-like behavior by inhibiting ferroptosis
and neuroinflammation [39]. DHA is sensitized to ferroptosis via an increase in ROS
accumulation and lipid peroxidation (Shan) [40]. Omega-6 PUFAs have relatively greater
susceptibility to lipid peroxidation and can participate in ferroptosis [7]. In a study, thedownregulation of arachidonate 5-lipoxygenase (ALOX5) inhibited ferroptosis, while theoverexpression of ALOX5 promoted ferroptosis [40]. ARA associated with endoplasmic
reticulum phospholipids induces ferroptosis due to the interaction with T-cell-generated
interferon, contributing to death by ferroptosis [7]. Omega-3 PUFAs promote intracellularantioxidant synthesis and reduce the formation of hydroperoxides, inducing ferroptosis [41]. DHA is sensitized to ferroptosis via an increase in ROS accumulation and lipid
peroxidation [7].
Omega-6 PUFAs have relatively greater susceptibility to lipid peroxidation and canparticipate in ferroptosis [7]. In a study, the downregulation of arachidonate 5-lipoxygenase(ALOX5) inhibited ferroptosis, while the overexpression of ALOX5 promoted ferroptosis [7].
ARA associated with endoplasmic reticulum phospholipids induces ferroptosis due to theinteraction with T-cell-generated interferon, contributing to death by ferroptosis [7].
Furthermore, ARA transport may be an important contributor to ferroptosis sensitivity(Dier). These previously reported findings support the present findings in that increased
DHA levels and decreased ARA levels in plasma may induce ferroptosis in the ASD group.
The present study included decreased plasma levels of ALA and DGL in the ASD
groups. ALA can induce ferroptosis via an isomer, α-eleostearic acid [42]. Based on theaccumulated evidence, the ACSL4-catalyzed biosynthesis of arachidonoyl-CoA contributes
to the execution of ferroptosis via triggering phospholipid peroxidation [12]. DGL-related
ferroptosis is a complex area of research, but there have been studies of GLA metabolism
and its relationship with inflammatory processes, including ferroptosis [43]. DGLA induces
ferroptosis-mediated neurodegeneration in dopaminergic neurons, representing a newclass of lipid metabolite that induces neurodegeneration via ferroptosis [44].
Regarding the effects of the combination of DHA and ARA on lipid peroxidation, aprevious study found that both DHA and ARA are susceptible to peroxidation by oxygen free radicals, producing 4-hydroxynonenal (4-HNE) from ARA via 12-hydroperoxyeicosatetraenoate and 4-hydroxyhexenal (4-HHE) and from DHA via the deacylation–
reacylation cycle [45]. Thus, these results regarding the contribution of both plasma DHA
and ARA levels to lipid peroxidation are reasonable. DHA promotes synaptic connectivity
and the prospective therapeutic capacity for neurodegenerative diseases [46]. DHA’s antioxidant activity plays an important role in signaling and lipid mediator production [47].
Furthermore, DHA has an important role in synaptogenesis and the synaptic expressionof synapsin, which exerts beneficial effects on neuronal plasticity [48] and modulates theoxidant/antioxidant balance by increasing the SOD level and decreasing the MDA-LDL
level [49]. Therefore, increased plasma DHA levels may play an important role in synaptic
function and in regulating redox homeostasis, thus supporting our findings. However, theintake of high doses of DHA can promote lipid peroxidation [47], possibly due to cellular
damage caused by oxidation [50]. Therefore, the incorporation of DHA may enhancepeople’s susceptibility to lipid peroxidation and disrupt the antioxidant system. Theseprevious findings may indicate sensitivity to lipid peroxidation via DHA, as shown inthis study.
The release of ARA induces lipid peroxidation (4-HNE) via cytosolic phospholipase
A2 α (cPLA2 α), making ARA an important lipid signal in activated immune cells [51], while
ARA is metabolized to prostaglandin E2 (PGE2), and both abnormal PGE2 signaling and
Wnt signaling contribute to ASD [52]. Therefore, ARA plays an important role in lipid
peroxidation in relation to the development of ASD. In addition, with respect to differencesin the activities of lipid peroxidation between DHA and ARA, the activity of DHA may be
due to an increase in DHA-related 4-HHE due to the Nrf2/antioxidant response element

Curr. Issues Mol. Biol. 2023, 459159

system, whereas ARA-related 4-HNE activity depends mainly on the stimulation of cellswith lipopolysaccharides related to the increase in cPLA2 [45]. The metabolic link between
DHA and the 4-HHE pathway may be useful in therapeutic strategies against oxidative
damage due to cerebral ischemia and other brain injuries [40]. Furthermore, upon exceeding
the buffering capacity of triglyceride storage into lipid droplets, both omega-3 and omega-6
PUFAs induce peroxidation, leading to cytotoxic effects in the presence of diacylglycerol
acyltransferase inhibitors (DGATis) [41]. Therefore, the present finding that both DHA andARA contribute to lipid peroxidation may be reasonable. The decreased plasma levels of
ALA may be involved in both DHA and ARA lipid peroxidation.
The DHA/ARA balance is important in the cognitive and behavioral developmentof infants [53]. Our previous study revealed that an increased plasma omega-3/omega-6
ratio due to decreased plasma ARA levels results in a decrease in plasma levels of signaling
proteins, such as ceruloplasmin, and these mechanisms contribute to the pathophysiology
of ASD [54]. An omega-6/omega-3 PUFA ratio of 4:1 is advocated because of competition
for the same enzymes during desaturation and elongation and the protective and stabilizing
capacity of the neuronal membrane (Yahuda) [55]. Values of 17 mg/100 kcal for DHA
and 34 or 25 mg/100 kcal for ARA and (DHA/ARA ratio, 17/25, 2.3/1) advocate for thedevelopment of the nervous system of infants [53]. Importantly, the reported beneficial
ratio of DHA/ARA is at least 2:1.5 [53]. Therefore, an imbalance between plasma DHA
and ARA levels might induce autistic behavioral symptoms and altered plasma MDA-LDL
levels. The average DHA/ARA ratio in the ASD group in the present study was 0.59 (1:1.9),
which might have been non-beneficial.
DHA is synthesized from ALA via ELOVL Fatty Acid Elongase 2 (ELOVL2), a ratelimiting enzyme of DHA synthesis in cells [56]. Therefore, the increase in plasma DHA
levels in the present study may reflect a comparable increase in the activity of ELOVL2.
Moreover, since the metabolism of omega-3 PUFA are synthesized by delta-12 and delta-15
desaturase enzymes [57], this enzyme may be lowered, resulting in low DAH levels.
The plasma levels of omega-6 PUFA family members DGLA and adrenic acid (AdA)induced oxidative stress blocked serotonin synthesis and serotoninergic neuron activity [58].
In the present study, the AdA level was significantly lower in the ASD group than it wasin the control group. AdA upregulates the production of intracellular oxidative-stressrelated ROS. Therefore, reduced plasma AdA levels may indicate weak anti-oxidative
stress activity and may contribute to autistic behaviors in ASD individuals, as shown in thepresent findings. The plasma levels of omega-6 PUFA family member DGLA can be further
converted to prostaglandin E1, which possesses anti-inflammatory properties [59]. The
anti-inflammatory effects of DGLA downregulate the synthesis of lipid mediators of inflammation, thereby contributing to the improvement in clinical symptoms of inflammatory
disorders such as ASD [60]. DGLA can be further converted to prostaglandin E1, which
possesses anti-inflammatory properties [60]. Taken together, reduced plasma DGLA in thepresent study may weaken the anti-inflammatory activity. Overall, reduced plasma AdA
and DGLA levels may weaken antioxidant and anti-inflammatory activities, contributing
to autistic social behaviors.
MDA is an endogenous genotoxic product of enzymatic and oxygen-radical-induced
lipid peroxidation [45]. DHA secondarily elicits MDA production [61]. Therefore, plasma
MDA-LDL, as the end-product of lipid peroxidation, might have had a small, indirect effecton the behavioral symptoms of the ASD individuals in our study.
In the present study, the plasma SOD levels were significantly lower in the ASD groupthan they were in the control group. SOD controls oxidative damage and regulates signaling
activities [62]. The loss of SOD2 activity may, therefore, result in various pathologicalphenotypes in the central nervous system [63]. We previously reported that reduced
plasma SOD levels may be related to the decreased endogenous antioxidant capacity [63].
Therefore, decreased plasma SOD levels may contribute to the neuronal deficit related tobehavioral symptoms in autistic individuals.

Curr. Issues Mol. Biol. 2023, 45

9160

Several limitations associated with the present study warrant mention. First, 4-HNE,
as the main phospholipid product, is considered a second messenger of ROS, which is an
important factor in stress- and age-associated diseases [64]. 4-HNE plays an important rolein not only physiological and protective functions as a signaling molecule but also plays acytotoxic role in inhibiting gene expression and promoting cell death [2]. Therefore, weused the plasma levels of MDA-LDL as a biomarker of lipid peroxidation. Second, thesmall sample size limits the generalization of these results to the entire ASD population.
Importantly, we applied two useful measures to ameliorate the difficulty in drawing
significant conclusions. A previous review article indicated similar results to our findings
concerning the association between MDA and SOD [4,64]. Furthermore, many previous
studies have indicated that DHA/ARA ratios are important for lipid peroxidation [65–67].
Third, the ratio of control subjects to cases was small (2.3:1), and the average DHA/ARA
ratio in the ASD group in the present study was 0.59 (2:3.79). A previous case–control
genomic study found that ratios of 3:1 or 4:1 induced more significant results comparedwith 1:1 or 2:1 [68]. This ratio of the experimental group to the control group was 2:1 in apreviously randomized clinical trial of a new drug [69]. Importantly, the most beneficial
ratio of DHA/ARA was reported to be at least 2:1.5 [53]. The average DHA/ARA ratio in
the ASD group in the present study was 0.59 (2:3.79), which might have been non-beneficial.
ALA may protect against mercurial-induced abnormal social interactions and stereotypical behaviors in mice [70]. Similarly, ALA has a protective role against valproic-acidinduced autism-like features [71]. Thus, decreased ALA and GLA levels may be associatedwith the total ABC scores.
DHA-ARA balance is important in cognitive and behavioral development in infants [53]. Our previous study revealed that an increased plasma omega-3/omega-6
ratio reflects decreased plasma ARA levels, resulting in decreases in the plasma levels of
signaling proteins such as ceruloplasmin and that this ratio may contribute to the pathophysiology of ASD [63]. An omega-6/omega-3 PUFA ratio of 4:1 is advocated because ofcompetition for the same enzymes during desaturation and elongation and the protective
and stabilizing capacity of the neuronal [68]. ARA:DHA ratio greater than 1:1 is associatedwith improved cognitive outcomes [61]. Therefore, an imbalance between plasma DHA
and ARA levels might contribute to autistic behavioral symptoms. Importantly, while thereported beneficial ratio of DHA/ARA is at least 2:1.5 [53], the average DHA/ARA ratio in
the ASD group in the present study was 0.59 (2:3.79), which might have been unbeneficial.
DHA is synthesized from ALA in the body via ELOVL fatty acid elongase 2 (ELOVL2),
a rate-limiting enzyme of DHA synthesis in cells [72]. Therefore, our study suggests forthe first time that increased plasma DHA levels may reflect a comparable increase in theactivity of ELOVL2 and an insufficient supply of ALA and then induce an unbeneficial
DHA/RA ratio.
The plasma levels of omega-6 PUFA family member DGLA were significantly lower
in the ASD group than in the control group. The anti-inflammatory effects of dihomo-γlinolenic acid (DGLA) downregulate the synthesis of lipid mediators of inflammation [73],thereby contributing to the improvement in the clinical symptoms of inflammatory disorders such as ASD [63].
Several limitations associated with the present study warrant mention. First, 4-HNE,
as the main phospholipid product, is considered a second messenger of ROS [45]. At thesame time, MDA is an endogenous genotoxic product of enzymatic and oxygen-radicalinduced lipid peroxidation [74]. Since MDA is a biomarker of the lipid peroxidation ofn-3 and n-6 PUFAs [2], we used plasma levels of MDA-LDL but not 4-HNE. Second, thesmall sample size limits the generalization of these results to the entire ASD population.
Importantly, we applied two measures to ameliorate the difficulty in drawing significant
conclusions. A previous review article indicated similar results concerning the association
between MDA and SOD [4,17]. Furthermore, many previous studies have indicated that
DHA/ARA ratios are important for lipid peroxidation [53–55]. Third, the ratio of control
to case subjects was small (2.3:1), and the average DHA/ARA ratio in the ASD group inCurr. Issues Mol. Biol. 2023, 45

9161

the present study was 0.59. A previous case–control genomic study found that ratios of
3:1 or 4:1 induced more significant results compared with 1:1 or 2:1 [53]. This ratio of theexperimental group to the control group was 2:1 in a previous randomized clinical trial of a
new drug [53]. Importantly, the beneficial ratio of DHA/ARA was reported to be at least
2:1.5 [53]. The average DHA/ARA ratio in the ASD group in the present study was 0.59
(2:3.79), which might have been non-beneficial.
5. Conclusions
These multiple linear regression analysis and adaptive Lasso analysis results revealed
that the concomitance of increased plasma DHA levels and decreased plasma ARA levels may induce increased plasma MDA-LDL levels. This imbalance between DHA and
ARA levels in plasma induced ferroptosis via lipid peroxidation in patients with autistic
behavioral symptoms. The disturbed metabolism of decreased levels of ALA subsequently
induced decreased levels of GLA and may have induced oxidation with regard to lipid
peroxidation. This biomedical mechanism may be due to the rate-limiting enzyme of DHA
synthesis, cell elongase 2 (ELOVL2). These neurological impairments may contribute toautistic behavioral symptoms.
Author Contributions: Conceptualisation, K.Y.; methodology, K.Y. and G.I.; formal analysis, K.Y.;
investigation, K.Y. and T.S.; writing—original draft preparation, K.Y.; writing— review and editing,
G.I. and T.S.; visualisation, T.S.; supervision, K.Y.; funding acquisition, T.S. All authors have read andagreed to the published version of the manuscript.
Funding: T.S. received The Practical Research Project for Rare/Intractable Diseases of the Japan
Agency for Medical Research and Development (AMED) Number: JP23ek0109521.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Corresponding author (Kunio Yui) may response to requires.
Acknowledgments: The authors are grateful to Tomoyo Hayashi for her contribution of research
fees.
Conflicts of Interest: The authors declare no conflict of interest.

References
1.
2.
3.

4.
5.
6.
7.
8.
9.
10.

Gaschler, M.M.; Stockwell, B.R. Lipid peroxidation in cell death. Biochem. Biophys. Res. Commun. 2017, 482, 419–425. [CrossRef][PubMed]Ayala, A.; Muñoz, M.F.; Argüelles, S. Lipid Peroxidation: Production, Metabolism, and Signaling Mechanisms of Malondialdehyde
and 4-Hydroxy-2-Nonenal. Oxid. Med. Cell. Longev. 2014, 2014, 360438. [CrossRef] [PubMed]
Cortelazzo, A.; De Felice, C.; Guerranti, R.; Signorini, C.; Leoncini, S.; Zollo, G.; Leoncini, R.; Timperio, A.M.; Zolla, L.; Ciccoli, L.;et al. Expression and Oxidative Modifications of Plasma Proteins in Autism Spectrum Disorders: Interplay Between Inflammatory
Response and Lipid Peroxidation. Proteomics Clin. Appl. 2016, 10, 1103–1112. [CrossRef]
Meguid, N.A.; Dardir, A.A.; Abdel-Raouf, E.R.; Hashish, A. Evaluation of Oxidative Stress in Autism: Defective Antioxidant
Enzymes and Increased Lipid Peroxidation. Biol. Trace Elem. Res. 2011, 143, 58–65. [CrossRef]
González-Fraguela, M.E.; Hung, M.D.; Vera, H.; Maragoto, C.; Noris, E.; Blanco, L.; Galvizu, R.; Robinson, M. Oxidative StressMarkers in Children with Autism Spectrum Disorders. BJMMR 2013, 3, 307–317. [CrossRef] [PubMed]
Perez, M.A.; Magtanong, L.; Dixon, S.J.; Watts, J.L. Dietary Lipids Induce Ferroptosis in Caenorhabditiselegans and Human Cancer
Cells. Dev. Cell. 2020, 54, 447–454.e4. [CrossRef]
Mortensen, M.S.; Ruiz, J.; Watts, J.L. Polyunsaturated fatty acids drive lipid peroxidation during ferroptosis. Cells 2023, 12, 804.
[CrossRef] [PubMed]
Zhang, Z.; Wang, X.; Wang, Z.; Zhang, Z.; Cao, Y.; Wei, Z.; Shao, J.; Chen, A.; Zhang, F.; Zheng, S. Dihydroartemisinin alleviates
hepatic fibrosis through inducing ferroptosis in hepatic stellate cells. Biofactors 2021, 47, 801–818. [CrossRef]
Tang, D.; Chen, X.; Kang, R.; Kroemer, G. Ferroptosis: Molecular mechanisms and health implications. Cell Res. 2021, 31, 107–125.
[CrossRef]
Mu, Y.; Sun, J.; Li, Z.; Zhang, W.; Liu, Z.; Li, C.; Peng, C.; Cui, G.; Shao, H.; Du, Z. Activation of pyroptosis and ferroptosis is
involved in the hepatotoxicity induced by polystyrene microplastics in mice. Chemosphere 2022, 291 Pt 2, 132944. [CrossRef]

Curr. Issues Mol. Biol. 2023, 45

11.
12.

13.
14.
15.

16.

17.
18.

19.
20.

21.

22.
23.
24.
25.
26.
27.
28.
29.

30.

31.

32.
33.
34.

35.

9162

Wang, Y.; Peng, X.; Zhang, M.; Jia, Y.; Yu, B.; Tian, J. Revisiting Tumors and the Cardiovascular System: Mechanistic Intersections
and Divergences in Ferroptosis. Med. Cell Longev. 2020, 2020, 9738143. [CrossRef] [PubMed]
Lee, J.Y.; Nam, M.; Son, H.Y.; Hyun, K.; Jang, S.Y.; Kim, J.W.; Kim, M.W.; Jung, Y.; Jang, E.; Yoon, S.J.; et al. Polyunsaturated fatty
acid biosynthesis pathway determines ferroptosis sensitivity in gastric cancer. Proc. Natl. Acad. Sci. USA 2020, 117, 32433–32442.
[CrossRef] [PubMed]
Liao, Y.; Cao, P.; Luo, L. Identification of Novel Arachidonic Acid 15-lipoxygenase inhibitors based on the bayesian classifiermodel and computer-aided high-throughput virtual screening. Pharmaceuticals 2022, 15, 1440. [CrossRef] [PubMed]
Dziobek, I.; Gold, S.M.; Wolf, O.T.; Convit, A. Hypercholesterolemia in Asperger syndrome: Independence from lifestyle,obsessive-compulsive behavior, and social anxiety. Psychiatry Res. 2007, 149, 21–24. [CrossRef]
Kreuzer, J.; White, A.L.; Knott, T.J.; Jien, M.L.; Mehrabian, M.; Scott, J.; Young, S.G.; Haberland, M.E. Amino terminus of
Apolipoprotein B Suffices to Produce Recognition of Malondialdehyde-Modified Low Density Lipoprotein by the Scavenger
Receptor of Human Monocyte-Macrophages. J. Lipid Res. 1997, 38, 324–342. [CrossRef]
Ogawa, K.; Tanaka, T.; Nagoshi, T.; Sekiyama, H.; Arase, S.; Minai, K.; Ogawa, T.; Yoshimura, M. Increase in the oxidised
low-density lipoprotein Level by smoking and the possible inhibitory effect of statin therapy in patients with cardiovasculardisease: A retrospective study. BMJ Open 2015, 5, e005455. [CrossRef]
Al-Gadani, Y.; El-Ansary, A.; Attas, O.; Al-Ayadhi, L. Metabolic biomarkers related to oxidative stress and antioxidant status in
Saudi autistic children. Clin. Biochem. 2009, 42, 1032–1040. [CrossRef]
Mousavinejad, E.; Ghaffari, M.A.; Riahi, F.; Hajmohammadi, M.; Tiznobeyk, Z.; Mousavinejad, M. Coenzyme Q10 Supplementation Reduces Oxidative Stress and Decreases Antioxidant Enzyme Activity in Children with Autism Spectrum Disorders.
Psychiatry Res. 2018, 265, 62–69. [CrossRef]
Perez, M.A.; Clostio, A.J.; Houston, I.R.; Ruiz, J.; Magtanong, L.; Dixon, S.J.; Watts, J.L. Ether lipid deficiency disrupts lipid
homeostasis leading to ferroptosis sensitivity. PLoS Genet. 2022, 18, e1010436. [CrossRef]
Sánchez-Illana, Á.; Shah, V.; Piñeiro-Ramos, J.D.; Di Fiore, J.M.; Quintás, G.; Raffay, T.M.; MacFarlane, P.M.; Martin, R.J.;
Kuligowski, J. Adrenic acid non-enzymatic peroxidation products in biofluids of m; oderate preterm infants. Free Radic Biol. Med.
2019, 142, 107–112. [CrossRef]
Fortin, É.; Blouin, R.; Lapointe, J.; Petit, H.V.; Palin, M.F. Linoleic acid, α-linolenic acid and enterolactone affect lipid oxidationand expression of lipid metabolism and antioxidant-related genes in hepatic tissue of dairy cows. Br. J. Nutr. 2017, 117, 1199–1211.
[CrossRef]
Wahid, A.; Khan, D.M.; Hussain, I. Robust Adaptive Lasso Method for Parameter’s Estimation and Variable Selection in
High-Dimensional Sparse Models. PLoS ONE 2017, 12, e0183518. [CrossRef] [PubMed]
Abdul-Razik Ismail, E. Behavior of lasso quantile regression with small sample sizes. J. Multidiscip. Eng. Sci. Technol. 2015,
2, 388–394.
Gomez-Gomez, A.; Aguilera, P.; Langohr, K.; Casals, G.; Pavon, C.; Marcos, J.; To-Figueras, J.; Pozo, O. Evaluation of metabolicchanges in acute intermittent porphyria patients by targeted metabolomics. Int. J. Mol. Sci. 2022, 23, 3219. [CrossRef] [PubMed]
Clubb, J.; Towlson, C.; Barrett, S. Measurement properties of external training load variables during standardised games in soccer:
Implications for training and monitoring strategies. PLoS ONE 2022, 17, e0262274. [CrossRef]
Indrayan, A.; Mishra, A. The importance of small samples in medical research. J. Postgrad. Med. 2021, 67, 219–223. [CrossRef]
Hackshaw, A. Small studies: Strengths and limitations. Eur. Respir. J. 2008, 32, 1141–1143. [CrossRef]
Constantino, J.H.; Gruber, C.P. Social Responsiveness Scale-Second Edition (SRS-2); Western Psychological Services: Torrance, CA,
USA, 2012.
Sipsock, D.; Tokadjian, H.; Righi, G.; Morrow, E.M.; Sheinkopf, S.J.; Rhode Island Consortium for Autism Research and Treatment(RI-CART). Autism severity aggregates with family psychiatric history in a community-based autism sample. Autism Res. 2021,
14, 2524–2532. [CrossRef]
Barradas, M.; Plaza, A.; Colmenarejo, G.; Lázaro, I.; Costa-Machado, L.F.; Martín-Hernández, R.; Micó, V.; López-Aceituno,
J.L.; Herranz, J.; Pantoja, C.; et al. Fatty acids homeostasis during fasting predicts protection from chemotherapy toxicity. Nat.
Commun. 2022, 13, 5677. [CrossRef]
Vivek, S.; Carnethon, M.R.; Prizment, A.; Carson, A.P.; Bancks, M.P.; Jacobs, D.R., Jr.; Thyagarajan, B. Association of the extent
of return to fasting state 2-hours after a glucose challenge with incident prediabetes and type 2 diabetes: The CARDIA study.
Diabetes Res. Clin. Pract. 2021, 180, 109004. [CrossRef]
Keirns, B.H.; Sciarrillo, C.M.; Koemel, N.A.; Emerson, S.R.J. Fasting, non-fasting and postprandial triglycerides for screening
cardiometabolic risk. Nutr. Sci. 2021, 10, e75. [CrossRef] [PubMed]
Regidor, P.A.; de la Rosa, X.; Müller, A.; Mayr, M.; Gonzalez Santos, F.; Gracia Banzo, R.; Rizo, J.M. PCOS: A Chronic Disease That
Fails to Produce Adequately Specialized Pro-Resolving Lipid Mediators (S Ms). Biomedicines 2022, 10, 456. [CrossRef]
Soundravally, R.; Hoti, S.L.; Patil, S.A.; Cleetus, C.C.; Zachariah, B.; Kadhiravan, T.; Narayanan, P.; Kumar, B.A. Association
between proinflammatory cytokines and lipid peroxidation in patients with severe dengue disease around defervescence. Int. J.
Infect. Dis. 2014, 18, 68–72. [CrossRef] [PubMed]
Singh, K.; Connors, S.L.; Macklin, E.A.; Smith, K.D.; Fahey, J.W.; Talalay, P.; Zimmerman, A.W. Sulforaphane treatment of autism
spectrum disorder (ASD). Proc. Natl. Acad. Sci. USA 2014, 111, 15550–15555. [CrossRef] [PubMed]

Curr. Issues Mol. Biol. 2023, 45

36.

37.

38.
39.

40.

41.

42.
43.
44.

45.

46.
47.
48.
49.
50.

51.
52.
53.
54.
55.
56.

57.
58.

59.

9163

Molenaar-Kuijsten, L.; Braal, C.L.; Groenland, S.L.; de Vries, N.; Rosing, H.; Beijnen, J.H.; Koolen, S.L.W.; Vulink, A.J.E.; vanDongen, M.G.J.; Mathijssen, R.H.J.; et al. Effects of the Moderate CYP3A4 Inhibitor Erythromycin on the Pharmacokinetics ofPalbociclib: A Randomized Crossover Trial in Patients with Breast Cancer. Clin. Pharmacol. Ther. 2023, 114, 933–1141. [CrossRef][PubMed]Grotenhuis, H.B.; Zhou, C.; Tomlinson, G.; Isaac, K.V.; Seed, M.; Grosse-Wortmann, L.; Yoo, S.J. Cardiothoracic ratio on chest
radiograph in pediatric heart disease: How does it correlate with heart volumes at magnetic resonance imaging? Pediatr. Radiol.
2015, 45, 1616–1623. [CrossRef]
Song, Y.; Wu, Z.; Xue, H.; Zhao, P. Ferroptosis is involved in regulating perioperative neurocognitive disorders: Emerging
perspectives. J. Neuroinflammation 2022, 19, 219. [CrossRef]
Wang, X.; Xiao, A.; Yang, Y.; Zhao, Y.; Wang, C.C.; Wang, Y.; Han, J.; Wang, Z.; Wen, M. DHA and EPA Prevent Seizure andDepression-Like Behavior by Inhibiting Ferroptosis and Neuroinflammation via Different Mode-of-Actions in a PentylenetetrazoleInduced Kindling Model in Mice. Mol. Nutr. Food Res. 2022, 66, e2200275. [CrossRef]
Shan, K.; Feng, N.; Zhu, D.; Qu, H.; Fu, G.; Li, J.; Cui, J.; Chen, H.; Wang, R.; Qi, Y.; et al. Free docosahexaenoic acid promotes
ferroptotic cell death via lipoxygenase dependent and independent pathways in cancer cells. Eur. J. Nutr. 2022, 61, 4059–4075.
[CrossRef]
Dierge, E.; Debock, E.; Guilbaud, C.; Corbet, C.; Mignolet, E.; Mignard, L.; Bastien, E.; Dessy, C.; Larondelle, Y.; Feron, O.
Peroxidation of n-3 and n-6 polyunsaturated fatty acids in the acidic tumor environment leads to ferroptosis-mediated anticancer
effects. Cell Metab. 2021, 33, 1701–1715. [CrossRef]
Do, Q.; Zhang, R.; Hooper, G.; Xu, L.J. Differential contributions of distinct free radical peroxidation mechanisms to the inductionof ferroptosis. JACS Au. 2023, 3, 1100–1117. [CrossRef]
Sergeant, S.; Rahbar, E.; Chilton, F.H. Gamma-linolenic acid, Dihommo-gamma linolenic, Eicosanoids and Inflammatory Processes.
Eur. J. Pharmacol. 2016, 785, 77–86. [CrossRef] [PubMed]
Sarparast, M.; Pourmand, E.; Hinman, J.; Vonarx, D.; Reason, T.; Zhang, F.; Paithankar, S.; Chen, B.; Borhan, B.; Watts, J.L.;
et al. Dihydroxy-metabolites of dihomo-γ-linolenic acid drive ferroptosis-mediated neurodegeneration. ACS. Cent. Sci. 2023, 9,
870–882. [CrossRef]
Yang, B.; Kevin, L.; Fritsche, K.L.; Beversdorf, D.Q.; Gu, Z.; Lee, J.C.; Folk, W.R.; Greenlief, C.M.; Sun, G.Y. Yin-Yang mechanisms
regulating lipid peroxidation of docosahexaenoic acid and arachidonic acid in the central nervous system. Front. Neurol. Actions
2019, 10, 642. [CrossRef]
Carbone, B.E.; Abouleish, M.; Watters, K.E.; Vogel, S.; Ribic, A.; Schroeder, O.H.; Bader, B.M.; Biederer, T. Synaptic connectivity
and cortical maturation are promoted by the ω-3 fatty acid docosahexaenoic acid. Cereb. Cortex 2020, 30, 226–240. [CrossRef]
Roig-Pérez, S.; Guardiola, F.; Moretó, M.; Ferrer, R. Lipid Peroxidation Induced by DHA Enrichment Modifies Paracellular
Permeability in Caco-2 Cells: Protective Role of Taurine. J. Lipid Res. 2004, 45, 1418–1428. [CrossRef] [PubMed]
Dyall, S.C. Long-Chain Omega-3 Fatty Acids and the Brain: A review of the independent and shared effects of EPA, DPA andDHA. Front. Aging Neurosci. 2015, 7, 52. [CrossRef] [PubMed]
Gupta, R.K.; Patel, A.K.; Kumari, R.; Chugh, S.; Shrivastav, C.; Mehra, S.; Sharma, A.N.; Asianm Pac, J. Interactions between
oxidative stress, lipid profile and antioxidants in breast cancer: A case control study. Cancer Prev. 2012, 13, 6295–6298. [CrossRef]
Miralles-Pérez, B.; Méndez, L.; Nogués, M.R.; Sánchez-Martos, V.; Fortuño-Mar, À.; Ramos-Romero, S.; Hereu, M.; Medina, I.;
Romeu, M. Effects of a fish oil rich in docosahexaenoic acid on cardiometabolic risk factors and oxidative stress in healthy rats.
Mar. Drugs 2021, 19, 555. [CrossRef]
Liu, G.Y.; Moon, S.H.; Jenkins, C.M.; Sims, H.F.; Guan, S.; Gross, R.W. Synthesis of oxidized phospholipids by sn-1 acyltransferaseusing 2–15-HETE Lysophospholipids. J. Biol. Chem. 2019, 294, 10146–10159. [CrossRef]
Wong, C.T.; Ahmad, E.; Li, H.; Crawford, D.A. Prostaglandin E2 alters Wnt-dependent migration and proliferation in neuroectodermal stem cells: Implications for autism spectrum disorders. Cell Commun. Signal. 2014, 12, 19. [CrossRef]
Colombo, J.; Jill Shaddy, D.J.; Kerling, E.H.; Gustafson, K.M.; Carlson, S.E. Docosahexaenoic Acid (DHA) and Arachidonic Acid
(ARA) Balance in Developmental Outcomes. Prostaglandins Leukot. Essent. Fatty Acids 2017, 121, 52–56. [CrossRef] [PubMed]
Yui, K.; Imataka, G.; Kawasak, Y.; Yamada, H. Increased ω-3 polyunsaturated fatty acid/arachidonic acid ratios and upregulationof signaling mediator in individuals with autism spectrum disorders. Life Sci. 2016, 145, 205–212. [CrossRef]
Yehuda, S. Omega-6/omega-3 Ratio and Brain Related Functions. World Rev. Nutr. Diet. 2003, 92, 37–56. [CrossRef]
Smink, W.; Gerrits, W.J.J.; Gloaguen, M.; Ruiter, A.; van Baal, J. Linoleic and α-linolenic acid as precursor and inhibitor for thesynthesis of long-chain polyunsaturated fatty acids in liver and brain of growing pigs. Animal 2012, 62, 262–270. [CrossRef][PubMed]Nguyen, Q.V.; Malau-Aduli, B.S.; Cavalieri, J.; Malau-Aduli, A.E.O.; Nichols, P.D. Enhancing Omega-3 Long-Chain Polyunsaturated Fatty Acid Content of Dairy-Derived Foods for Human Consumption. Nutrients 2019, 11, 743. [CrossRef] [PubMed]
Wu, C.H.; Hsu, W.L.; Tsai, C.C.; Chao, H.R.; Wu, C.Y.; Chen, Y.H.; Lai, Y.R.; Chen, C.H.; Tsai, M.H. 7,10,13,16-Docosatetraenoic
acid impairs neurobehavioral development by increasing reactive oxidative species production in Caenorhabditis elegans. Life
Sci. 2023, 319, 121500. [CrossRef]
Wang, X.; Lin, H.; Gu, Y. Multiple Roles of dihomo-γ-linolenic acid against proliferation diseases. Lipids Health Dis. 2012, 11, 25.
[CrossRef]

Curr. Issues Mol. Biol. 2023, 45

60.

61.
62.
63.
64.

65.

66.

67.
68.
69.

70.

71.

72.
73.
74.

9164

Novichkova, E.; Chumin, K.; Eretz-Kdosha, N.; Boussiba, S.; Gopas, J.; Cohen, G.; Khozin-Goldberg, I. DGLA from the Microalga
Lobosphaera Incsa P127 Modulates Inflammatory Response, Inhibits iNOS Expression and Alleviates NO Secretion in RAW264.7
Murine macrophages. Nutrients 2020, 12, 2892. [CrossRef] [PubMed]
Mahéo, K.; Vibet, S.; Steghens, J.P.; Dartigeas, C.; Lehman, M.; Bougnoux, P.; Goré, J. Differential sensitization of cancer cells todoxorubicin by DHA: A Role for Lipoperoxidation. Free Radic. Biol. Med. 2005, 39, 742–751. [CrossRef]
Wang, Y.; Branicky, R.; Noë, A.; Hekimi, S. Superoxide dismutases: Dual roles in controlling ROS damage and regulating ROS
signaling. J. Cell Biol. 2018, 17, 1915–1928. [CrossRef]
Yui, K.; Imataka, G.; Sasaki, H.; Shiroki, R. The role of lipid peroxidation in individuals with autism spectrum disorders. Metab.
Brain Dis. 2020, 35, 1101–1108. [CrossRef]
Jaganjac, M.; Milkovic, L.; Gegotek, A.; Cindric, M.; Zarkovic, K.; Skrzydlewska, E.; Zarkovic, N. The Relevance of Pathophysiological Alterations in Redox Signaling of 4-Hydroxynonenal for Pharmacological Therapies of Major Stress-Associated Diseases.
Free Radic. Biol. Med. 2020, 157, 128–153. [CrossRef] [PubMed]
Oborna, I.; Wojewodka, G.; De Sanctis, J.B.; Fingerova, H.; Svobodova, M.; Brezinova, J.; Hajduch, M.; Novotny, J.; Radova, L.;
Radzioch, D. Increased lipid peroxidation and abnormal fatty acid profiles in seminal and blood plasma of normozoospermic
males from Infertile couples. Hum. Reprod. 2010, 25, 308–316. [CrossRef] [PubMed]
Starčević, K.; Roškarić, P.; Šperanda, M.; Ðidara, M.; Kurilj, A.G.; Maurić, M.; Mašek, T. High Dietary n6/n3 Ratio Decreases
Eicosapentaenoic to Arachidonic Acid Ratios and Upregulates NF-κB/p50 Expression in Short-Term Low Dose Streptozotocin
and High-Fructose Rat Model of Diabetes. Prostaglandins Leukot. Essent. Fatty Acids 2019, 149, 1–7. [CrossRef]
Hadley, K.B.; Ryan, A.S.; Forsyth, S.; Gautier, S.; Salem, N., Jr. The Essentiality of Arachidonic Acid in Infant Development.
Nutrients 2016, 8, 216. [CrossRef]
Kang, M.; Choi, S.; Koh, I.S. The Effect of Increasing Control-to-Case Ratio on Statistical Power in a Simukated Case-Cintrl SNP
Association Study. Genomics Inform. 2009, 7, 148–151. [CrossRef]
Li, J.; Zhang, C.; Wu, Z.; Wang, G.; Zhao, H.; Li, J. The Mechanism and clinical outcome of patients with corona virus disease.
Trials 2019 Whose nucleic acid test has changed from negative to positive, and the therapeutic efficacy of Favipiravir: A structured
summary of a study protocol for a randomised controlled trial. Trials 2020, 21, 488. [CrossRef] [PubMed]
Namvarpour, Z.; Nasehi, M.; Amini, A.; Zarrindast, M.R. Protective role of alpha-lipoic acid in impairments of social andstereotyped behaviors induced by early postnatal administration of thimerosal in male rat. Neurotoxicol Teratol. 2018, 67, 1–9.
[CrossRef]
Yadav, S.; Tiwari, V.; Singh, M.; Yadav, R.K.; Roy, S.; Devi, U.; Gautam, S.; Rawat, J.K.; Ansari, M.N.; Saeedan, A.S.; et al.
Comparative Efficacy of Alpha-Linolenic Acid and Gamma-Linolenic Acid to Attenuate Valproic Acid-Induced Autism-Like
Features. J. Physiol. Biochem. 2017, 73, 187–198. [CrossRef]
Ding, Y.; Yang, J.; Ma, Y.; Yao, T.; Chen, X.; Ge, S.; Wang, L.; Fan, X. MYCN and PRC1 cooperatively repress docosahexaenoic acidsynthesis in neuroblastoma via ELOVL2. J. Exp. Clin. Cancer Res. 2019, 38, 498. [CrossRef]
Dooper, M.M.; van Riel, B.; Graus, Y.M.; M’Rabet, L. Dihomo-gamma-linolenic acid inhibits tumour necrosis factor-alphaproduction by human leucocytes independently of cyclooxygenase activity. Immunology 2003, 110, 348–357. [CrossRef] [PubMed]
Niedernhofer, L.J.; Daniels, J.S.; Rouzer, C.A.; Greene, R.E.; Lawrence, J.; Marnett, L.J. Malondialdehyde, a product of lipid
Peroxidation, is mutagenic in human cells. J. Biol. Chem. 2003, 278, 31426–31433. [CrossRef] [PubMed]

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