Coenzyme Q10 supplementation reduces oxidative stress and decreases antioxidant enzyme activity in children with autism spectrum disorders

Coenzyme Q10 supplementation reduces oxidative stress and decreases antioxidant enzyme activity in children with autism spectrum disorders

Accepted Manuscript Coenzyme Q10 supplementation reduces oxidative stress and decreases antioxidant enzyme activity in children with Autism spectrum ...

NAN Sizes 0 Downloads 2 Views

Accepted Manuscript

Coenzyme Q10 supplementation reduces oxidative stress and decreases antioxidant enzyme activity in children with Autism spectrum disorders Elham Mousavinejad , Mohammad Ali Ghaffari , Forough Riahi , Maryam Hajmohammadi , Zeinab Tiznobeyk , Masoumeh Mousavinejad PII: DOI: Reference:

S0165-1781(17)31786-9 10.1016/j.psychres.2018.03.061 PSY 11293

To appear in:

Psychiatry Research

Received date: Revised date: Accepted date:

27 September 2017 2 March 2018 22 March 2018

Please cite this article as: Elham Mousavinejad , Mohammad Ali Ghaffari , Forough Riahi , Maryam Hajmohammadi , Zeinab Tiznobeyk , Masoumeh Mousavinejad , Coenzyme Q10 supplementation reduces oxidative stress and decreases antioxidant enzyme activity in children with Autism spectrum disorders, Psychiatry Research (2018), doi: 10.1016/j.psychres.2018.03.061

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Highlights  

AC

CE

PT

ED

M

AN US

CR IP T



This study has demonstrated that CoQ10 supplementation can improve the symptoms in autistic children. This study aims to address the recent advances in supplementary field and to clarify the possible role of CoQ10 in ASDs. Oxidative stress factors are directly related to the severity of ASDs.

1

ACCEPTED MANUSCRIPT

Coenzyme Q10 supplementation reduces oxidative stress and decreases antioxidant enzyme activity in children with Autism spectrum disorders

Running title: CoQ10 supplementation in the autistic children.

Hajmohammadi ,d Zeinab Tiznobeyk,e Masoumeh Mousavinejad,f a

CR IP T

Authors: Elham Mousavinejad,a,* Mohammad Ali Ghaffari,a,b , Forough Riahi,c Maryam

Department of Biochemistry, School of Medical Sciences, Ahvaz, Jundishapur University of medical Sciences, Ahvaz, Iran. b

c

AN US

Cellular and Molecular Research Center, Ahvaz, Jundishapour University of Medical Sciences, Ahvaz, Iran. Department of psychiatry, Medical School, Ahvaz, Jundishapour University of Medical Sciences, Ahvaz,

Iran. d

Department of Community Nutrition, School of Nutritional Sciences and Dietetics, Tehran, University

of Medical Sciences, Tehran, Iran. f

M

e

Biostatistics, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

Sheffield, UK. *Corresponding author:

ED

Centre for Stem Cell Biology (CSCB), Department of Biomedical Science, the University of Sheffield,

PT

Elham Mousavinejad, Department of Biochemistry, School of Medical Sciences, Ahvaz Jundishapur University of medical Sciences, Ahvaz, Iran.

CE

University of Medical Sciences, Golestan Blv, Ahvaz, Iran. Email: [email protected], [email protected]

AC

Tel: 09166151796, Fax: +98-61-33335291.

Abstract:

Antioxidants and oxidative stress can participate in pathobiochemical mechanisms of autism spectrum disorders (ASDs). The aim was to identify the effects of early CoQ10 supplementation on oxidative stress in children with ASDs. Ninety children with ASDs were included in this study, based on DSM-IV criteria

2

ACCEPTED MANUSCRIPT

and using Childhood Autism Rating Scale (CARS) scores. Concentrations of CoQ10, MDA, total antioxidant status (TAS) assay, and antioxidant enzymes (superoxide dismutase or SOD and glutathione peroxidase or GPx) activity were determined in serum before and after 100 days of supportive therapy with CoQ10 at daily doses of 30 and 60 mg. Data on children’s behavior were collected from parents and 2 30

mg

CR IP T

babysitters. CoQ10 supportive therapy was determined after three months with daily dose

improved oxidative stress in the children with ASDs. A relation was seen between serum MDA (r2 = 0.668) and TAS (r2 = 0.007), and antioxidant enzymes (SOD [r2 =0.01] and GPx [r2 =0.001]) activity and CARS score. Based on the results, high doses of CoQ10 can improve gastrointestinal problems (P=0.004)

AN US

and sleep disorders (P=0.005) in children with ASDs with an increase in the CoQ10 of the serum. We concluded that the serum concentration of CoQ10 and oxidative stress could be used as relevant biomarkers in helping the improvement of ASDs.

M

Keywords: Coenzyme Q10; Supplementation; Oxidative stress; Autistic spectrum disorder; Antioxidant.

1. Introduction

ED

ASDs are neurodevelopmental disorders resulting from pervasive abnormalities in social interaction and communication, repetitive behaviors, and restricted interests (Constantino et al., 2016). The etiology of

PT

autism stems from genetic, neurological, and environmental factors (Sealey et al., 2016). Studies suggest that ASDs may result from interactions between genetic, environmental, and immunological factors with

CE

oxidative stress as a mechanism linking these risk factors (Steullet et al., 2017).

AC

The nervous tissue is especially susceptible to oxidant threat because of its high rate of aerobic metabolism and readily oxidizable molecules similar to catecholamine (Ferreira et al., 2016). Neurodegenerative and neurodevelopmental diseases are linked to multiple biological processes such as depletion or insufficient synthesis of neurotransmitters, oxidative stress, and abnormal ubiquitination (Saravi et al., 2016; Hardingham et al., 2016; Liang et al., 2014). Oxidative stress is known to play a role in some neurological conditions like Parkinson’s disease, Alzheimer’s disease (Paul et al., 2017),

3

ACCEPTED MANUSCRIPT

schizophrenia (Ojala et al., 2017), and bipolar disorder (Nucifora et al., 2017; Vasconcelos-Moreno et al., 2017). Autism spectrum disorder or ASD, another important brain disorder, is related to central nervous system (CNS) inflammation and characterized by learning and social disabilities with no definite pathogenesis (Di Marco et al., 2016). Oxidative stress controls the activities of receptor and non-receptor

CR IP T

types of proteins like tyrosine kinases and protein kinase C, as well as transcription factors like NF-κβ, which are found to be dysregulated in ASDs (LaFlamme et al., 2014). Multiple biochemical and molecular features can be observed in the neurodevelopmental brain of ASDs including oxidative stress, reactivate astrocytes, microglia (Xanthos et al., 2014, Harada et al., 2016), neuronal loss, and

AN US

development of proinflammatory cytokines (Masi et al., 2015). Consistent with previous studies increased oxidative stress biomarkers included reducing endogenous antioxidant capacity, specifically the total GSH levels, altered GPx, SOD activities were found in autistic individuals compared to controls (Seminotti et al., 2016, González-Fraguela., 2013). A high dose of N-acetylcysteine (NAC) is included to

M

enhance production of glutathione (Schmitt et al., 2015). NAC has been tried as a treatment for a number of psychiatric diseases (Minarini et al., 2017). Additional indications of oxidative stress in ASDs are

ED

derived from the evidence of impaired energy metabolism. Reduced synthesis of adenosine triphosphate (ATP) and higher lactate and pyruvate levels may suggest mitochondrial dysfunction in ASDs. Increased

PT

reactive oxygen species (ROS) metabolism induced by dysfunctional mitochondria may elicit chronic

CE

oxidative stress (Hollis et al., 2017). The amount of SOD present in cellular and extra-cellular is crucial for the prevention of diseases linked to oxidative stress (Hininger-Favier et al., 2016).

AC

CoQ10 (also called ubiquinone) is a lipid-soluble benzoquinone. CoQ10 is recognized as an intracellular antioxidant that protects membrane phospholipids, mitochondrial membrane proteins, and low-density lipoproteins from free radical-induced oxidative damage (Littarru et al., 2017). Coenzyme Q10 is added to support mitochondrial functions such as shuttling electrons, serving as a potent antioxidant, and working as an electron transport chain to generate ATP (Cornelius et al., 2017). Children with autism have mitochondrial dysfunction (Elham et al., 2016), In psychiatric medication, CoQ10 is added to 4

ACCEPTED MANUSCRIPT

support mitochondrial functions to improve the functional brain (Bhardwaj et al., 2016). ASDs use a restricted diet relative to health children, either due to sensory sensitivities or due to therapeutic measures. Recent studies show that vitamin therapy and other nutritional supplements are generally consumed to treat ASDs (Sathe et al., 2017, Horvath et al., 2017).

CR IP T

In this study, we further investigated the hypothesis that insufficient serum concentration of CoQ10 may play role in ASDs (Khemakhem et al., 2017). This study followed a relation between serum CoQ10 concentration and severity of ASDs, which may contribute to the higher susceptibility of some

M

2. Materials and Methods

AN US

individuals to ASDs, especially Iranians.

ED

2.1. Study Design and Participants (Table 1)

This study was designed as a randomized, parallel, placebo-controlled study (Henschel et al., 2010). The

PT

sample size was calculated using an alpha of 0·05. Ninety children (mean age: 7.91±2.55 years; range: 3– 12 years) suffering from mild to severe ASDs were enrolled in the study; healthy children (N= 90; mean

CE

age: 7.61±2.55 years; range: 3–12 years) were also selected and diagnosed by a pediatric endocrinologist. Children with ASDs were diagnosed according to the Diagnostic and Statistical Manual of Mental

AC

Disorders, Fourth Edition, Text Revision (DSM-IVTR), American Psychiatric Association; Autism Diagnostic Observation Schedule (ADOS); and using CARS, a test combining parent reports and direct observation by a professional (Föcker et al., 2017). This study excluded children (healthy children and ASDs children) with a history of fragile X disorders, tuberous sclerosis, phenylketonuria (PKU), LeschNyhan syndrome, fetal alcohol syndrome, or a history of maternal illicit drug use (Feng et al., 2017). Additionally, this study excluded any children (healthy children and ASDs children) with a history of 5

ACCEPTED MANUSCRIPT

chelation therapy, and vitamin or mineral supplements (Nath, 2017). All children (healthy children and ASDs children) with a history of seizure disorders, severe head injuries, psychotic disorders, and any other major acute or chronic physical or mental illnesses were excluded from the groups. Written informed consent was obtained from the children’s parents (healthy children and ASDs children). This

Jundishapour University of Medical Sciences, Ahvaz, Iran.

AN US

2.2. CoQ10 Administration and Sample Collection (Figure 1)

CR IP T

study was conducted with the approval of the Human Subjects Institutional Review Board of

Patients received liquid soft gels CoQ10 supplementation once and twice daily for 100 days. CoQ10 (ubiquinone)

was

obtained

from

Nature

Made

company

(http://www.naturemade.com/supplements/coq10/coq1030-mg) and placebo capsules (starch) were available preparations from a pharmacologist. Placebo capsules were made to resemble CoQ10 capsules

M

and were kept in a similar bottle. Participants were adjusted in groups according to their age, sex, and

ED

body mass index or BMI (Table 1). All subjects were Iranian, born and living in the state of Khuzestan. We enrolled 90 patients with ASDs in this study but 12 subjects declined to participate during the course

PT

of the study. The remaining 78 patients were randomly assigned to one of two groups – a placebo group (N: 26; one capsule daily) or a coenzyme Q10 group (N: 52; 30 and 60 mg/d) (Miles et al., 2006 and

CE

Adams et al., 2011; Gvozdjáková et al., 2014). Intervention was administered for 100 days. To monitor compliance, participants were screened throughout the study via telephone for individualized dosing

AC

titration and potential adverse effects. The research team checked vacant capsule bags every 14 days to confirm use of the capsule. This was done by the study nurse with supervision from the study psychiatrist, both of whom were blind to group assignment. A placebo group for group Q10 (60 mg/day) of children with ASDs was not included in the study for ethical reasons (Gvozdjáková et al., 2014). After overnight fasting, venous blood was drawn from the antecubital vein, first at the time of study enrollment and then, after 100 days of oral CoQ10 (ubiquinone) treatment. 6

ACCEPTED MANUSCRIPT

2.3. Anthropometric Measurements (Table 1) The weight, height, and BMI of all participants were measured. BMI is the body weight (kg)/height squared (m−2), and the standard deviation (SD) scores for BMI (BMI-SDS) were calculated (Júlíusson et al., 2017).

CR IP T

2.4. Blood Samples Fasting blood samples were collected in the morning between 8 am and 10 am. The serum was separated by centrifugation at 1300 g for 10 minutes at +4ºC and divided into 150 µl of aliquots, which were immediately stored at -20º C; Heparin tubes were centrifuged for 20 minutes at 2500 ×g, after which plasma was collected quickly, frozen, and kept at −20ºC until the time of analysis at the medical school

AN US

laboratory, Jundishapour University of Medical Sciences, Ahvaz, Iran. The storage time was less than one month. 2.5. Biochemical Analyses For all biochemical analyses:

M

Intra-assay precision (precision within an assay): CV %< 8%

Inter-assay precision (precision between assays): CV %< 10%

ED

2.5.1. Total Antioxidant Status Assay

TAS was measured in the serum samples using the antioxidant assay kit (Cayman Chemical). This

PT

technique was based on ABTS (1, 2-Azino-di-[3-ethylbenzthiazoline] sulphonate); a dark blue green radical was reduced to a colorless ABTS by antioxidants in the samples. The change in absorbance was

CE

directly proportional to antioxidant levels in the samples. The assay was calibrated with a trolox

AC

equivalent analogue.

2.5.2. Determination of MDA and CoQ10 Concentrations MDA was measured in the serum sample using the antioxidant assay kit (Cayman Chemical). Oxidative stress in the cellular environment results in the formation of highly reactive and unstable lipid hydroperoxides. Decomposition of unstable peroxides derived from polyunsaturated fatty acids results in the formation of MDA, which can be quantified calorimetrically following its controlled reaction with 7

ACCEPTED MANUSCRIPT

thiobarbituric acid. There is a well-established method for screening and monitoring lipid peroxidation for measuring these thiobarbituric acid reactive substances (TBARS). CoQ10 serum was measured using the ELISA kit (My Bio Source). 2.5.3. Glutathione Peroxidase Activity Colorimetric Assay

CR IP T

Glutathione peroxidase was measured in heparinized whole blood using a kit (Cayman Chemical). Cayman Chemical’s glutathione peroxidase assay kit measured GPx activity indirectly through a coupled reaction with glutathione reductase (GR). Oxidized glutathione (GSSG) was produced upon reduction of an organic hydroperoxide by GPx, and was recycled to its reduced state by GR and NADPH. The

AN US

oxidation of NADPH to NADP+ was accompanied by a decrease in absorbance at 340 nm. The rate of decrease in A340 was directly proportional to GPx activity in the sample. 2.5.4. Superoxide Dismutase Activity Colorimetric Assay

Superoxide dismutase (SOD) activity colorimetric assay kit (Cayman Chemical) is a sensitive kit using

M

WST-1 that produces a water-soluble formazan dye upon reduction with superoxide anion. The rate of

ED

reduction with a superoxide anion is linearly related to xanthine oxidase (XO) activity and is inhibited by SOD. Therefore, inhibition activity of SOD can be determined by a colorimetric method. SOD is one of

PT

the most important antioxidative enzymes. It catalyzes the dismutation of the superoxide anion into

CE

hydrogen peroxide and molecular oxygen.

AC

2.6. Ethics Approval and Consent This study was approved by the regional Ethics Committee of the Medical University of Medical Sciences, Ahvaz, Iran. Written informed consent was obtained from the children’s parents.

2.7. Statistical Analysis The sample size was calculated using an α of 0·05 and the power of analysis was 80%. Data analysis was performed using SPSS (version 19). The values were expressed as mean ± SD (standard deviation). Normal distribution and homogeneity of the variance of data were tested by the Kolmogorov-Smirnov 8

ACCEPTED MANUSCRIPT

and Levene tests, respectively. For those variables that did not meet these premises, the Mann-Whitney U nonparametric test was used for comparison between patients and control individuals. Furthermore, the values obtained for each study participant diagnosed with ASDs were evaluated for their correlation with the severity of disorders as derived from the CARS scoring conducted on each study participant using the unweighted least squares test statistics. For all statistical tests, a two-tailed p value ≤ .05 was considered statistically significant. Accuracy of diagnostic stress oxidative measurement tests was assessed via curve

data) variables was evaluated using the chi-square test. 3. Results

CR IP T

analysis of the receiver operating characteristic (ROC). The association between qualitative (categorical

No significant differences were observed between these groups (ASDs, health) with respect to sex, age, weight, and BMI (Table 1). Data indicated that levels of measured factors in autistic children were not

AN US

significantly increased and decreased (p>0.05) compared to their matched control after 30 mg/day of CoQ10 and placebo (Tables 2a and 2c). Serum CoQ10 concentration was higher in the Q10-60 group (p=0.003) as compared to the placebo and Q10-30 groups at Week 4 (p=0.39 and p=0.63). 3.1. Status of Oxidative Stress and Antioxidant Enzyme Activity after Intervention (Table 2)

M

It was observed that study subjects with ASDs had significantly decreased GPx and SOD activity after intervention with 60 mg/day of CoQ10 (p=0.001). Study subjects with ASDs had significantly decreased

PT

ED

levels of serum MDA (p=0.01) and increased TAS (p=0.02) after intervention with 60 mg/day of CoQ10.

CE

3.2. Effects and Side Effects of CoQ10 on Psychological Functions and Behavior of Children with ASDs (Tables 3 and 4)

AC

In rare cases, especially in the first days of CoQ10 supplementation, opposite effects were observed including increased destructive behavior, hyperactivity, and manifestations of anger. However, supplementation was not stopped (Table 3). After three months of Q10-60 treatment, concentrations of CoQ10 were in the range of 6.71 ng/ml. Significant improvement in autistic symptomology was observed after three months of CoQ10 supplementary therapy in children, prevailingly at over 6.71 ng/ml of CoQ10 serum concentration (p=0.003) (Table 2b). A Global Impressions parental questionnaire found that the

9

ACCEPTED MANUSCRIPT

supplement group reported statistically significant improvements in sleep (65.38%) and gastrointestinal problems (61.53%) as compared to the control group (p=0.005 and p=0.004) (Table 4). 3.3. Correlation between Serum Oxidative Stress and Severity of ASDs (Figure 2)

CR IP T

The scatter plot showed that the relationship between the two variables is positive or negative. According to these diagrams, oxidative stress factors are directly related to the severity of autism (CARS). Moreoverthe, relationship is positive and stronger between MDA and CARS (r=0.668; p>0.05).

3.4. Comparison of ROC Curves for Oxidative Stress in Children with ASDs and Healthy Children (Figure 3 and Table 5)

AN US

ROC curve should become the gold standard for identification of parameters that are specific and sensitive enough to support diagnosis of autism; its utility in prognosis, evaluation of therapeutic interventions, and risk assessment await further studies. In Table 5, a ROC analysis was performed to assess the diagnostic value of serum oxidative stress and CoQ10 levels for children with ASDs from all

M

examined cases. This figure showed that serum levels of oxidative stress cannot be used as differentiating

ED

biomarkers in children with ASDs because AUC is close to 0.5. Since an AUC rate close to 1 indicates a brilliant predictive marker, a curve that lies close to the diagonal (AUC = 0.5) has no diagnostic value. An

CE

biomarker.

PT

AUC rate close to 1 is always accompanied by satisfactory rates of specificity and sensitivity of the

AC

4. Discussion

In a previous study, the level of serum coenzyme Q10 was low at the baseline in healthy subjects (Elham, 2017). The serum coenzyme Q10 concentration can be lowered under chelation therapy (Lee et al., 2016) but patients being treated with chelating agents were excluded. Our study showed that after three months of 60 mg/day of CoQ10 treatment, concentrations of CoQ10 were in the range of 6.71±1.52 ng/ml. Subjects with ASDs had significantly increased concentrations of CoQ10 (p= 0.003) after intervention with 60 10

ACCEPTED MANUSCRIPT

mg/day of coenzyme Q10 (Table 2b). As a result, it is believed that supplementation of CoQ10 in patients with ASDs at a higher dosage may provide sustainable antioxidation and better absorption. An increase in the concentration of CoQ10 might affect the mitochondrial respiratory function (Hargreaves, 2014). CoQ10 has a better synergistic effect than other antioxidant vitamins like Vitamins E, A, and C (Kobori et al.,

CR IP T

2014). Previous data indicated decreased levels of TAS in serum and plasma of children with ASDs (Ranjan et al., 2015; Kondolot et al., 2016). Significantly increased TAS and decreased MDA levels in children with ASDs were observed after three months of CoQ10 supplementary therapy, prevailingly in the range of

AN US

6.71±1.52 ng/ml of CoQ10 serum concentration (Table 2b). In previous studies, MDA levels in patients with autism increased plasma concentrations as compared to control (Frustaci et al., 2012; Ghanizadeh et al., 2012; Kondolot et al., 2016). Several factors can contribute to the MDA serum level in patients with ASDs such as metabolic changes, age, and mitochondrial dysfunction of brain and skeletal muscles. In

M

this study, obese children with autism and children with diabetes, epilepsy, or comorbid diseases were not included, while unification was done according to subject and control groups. Coenzyme Q10 supplement

ED

at a dose of 60 mg as compared to a dose of 30 mg significantly reduced lipid peroxidation in this study. Finally, a significant direct correlation was observed between MDA serum level and severity of autism

PT

using the CARS score (r=0.667, p=0.005) (Figure 2d). In 2013, M. M. Essa et al. reported no significant correlation (p=0.75, r=0.000) between the CARS score and serum TAS level (M. M. Essa et al., 2013).

CE

Our data is consistent with this study showing no correlation between severity of autism and TAS

AC

(p=0.55, r=0.007) (Figure 2c). Results generally showed some significant irregularities in biomarkers of oxidative stress in participants diagnosed with ASDs. Moreover, there was a significant inverse correlation between GPx and SOD activity, and severity of ASDs measured through CARS scoring. GPx and SOD activity in ASDs may be caused by an imbalance between the generation of ROS by endogenous or exogenous pro-oxidants and the defense mechanism against ROS by antioxidants (Chauhan et al., 2013). Treatment of oxidative stress

11

ACCEPTED MANUSCRIPT

with antioxidants and other dietary additions may ameliorate mitochondrial dysfunction in individuals with ASDs (Castrén et al., 2014; Gu et al., 2014). Additionally, it seemed clear that coenzyme Q10 had a protective effect against oxidative stress, which may be ascribed to its antioxidant role (Tarry-Adkins et al., 2015). CoQ10 supplement at a dose of 60 mg as compared to a dose of 30 mg showed significant

CR IP T

decreased oxidative stress implying that its effect is dose-dependent supplement coenzyme Q10. In this study, it was observed that study subjects with ASDs had significantly decreased GPx and SOD activity after intervention with 60 mg/day as compared to 30 mg/day of CoQ10 and placebo group (Table 2). That is to say, due to the reduction of oxidative stress factors, the activity of these enzymes also decreased; On

AN US

the other hand, in some studies indicated coenzyme Q10 supplements could decrease oxidative stress and increase antioxidant enzyme activity (Lee et al., 2012; Sanoobar et al., 2013). In 2013, M. M. Essa et al. reported a significant positive correlation between severity of the disease and SOD activity (p<0.01, r=0.588), and a weak correlation between severity of the disease and GPx activity

M

(p<0.05, r=0.303) (M. M. Essa et al., 2013). Mitochondrial dysfunction and antioxidant defense systems are included in the pathobiochemical mechanism of ASDs (Kovacic et al., 2017). Studies have

ED

documented increased antioxidant enzymatic activities (Yui et al., 2017; Smaga, 2015). Other studies

PT

have shown reduced levels of blood antioxidant enzymes in children with ASDs (Frye and James, 2014). Till today, diagnosis of ASDs is based exclusively on clinical observation of changed behavior and can be

CE

made only around two years of age since clinical diagnosis is hard and ambiguous in younger children (Estes et al., 2015). Therefore, valid biomarkers are needed that will allow improving and forestalling

AC

diagnosis. The importance of the availability of strong biomarkers in research on ASDs cannot be underestimated (Abruzzo et al., 2015). Even the discovery of biological networks underlying pathophysiology of ASDs can be improved by their identification, as well as by personalized treatments and development of new methods to cure or, at least, improve the symptoms of the disease. In this study, Figures 3 and table 5 analyzed oxidative stress data in order to identify a panel of peripheral markers associated with ASDs by focusing on information that used ROC analysis for optimally assessing both 12

ACCEPTED MANUSCRIPT

specificity and sensitivity of a putative marker. According to our results (Figures 3 and Table 5), our study factors could not make available predictive information on the clinical outcome of ASDs. Our study had three limitations. First, in regard to effect of sample size, the number of participants was small and the study group was limited. Second, the study was designed using just 30 and 60 mg of

CR IP T

coenzyme Q10 supplements per day for three months; larger and longer interventions are needed to establish the beneficial effect of a high dosage of coenzyme Q10 supplementation in patients with ASDs. Finally, our assessment was limited to children. We suggested that the study be extended to adults in various age groups.

AN US

4.1. Conclusions

CoQ10 supplements at a dose of 60 mg can reduce MDA, decrease antioxidant enzyme activity, increase TAS, and improve sleep and gastrointestinal problems in children with ASDs. We believe a higher dose of coenzyme Q10 supplements (>60 mg/day) may provide rapid and sustainable antioxidation in children with ASDs. However, additional study is needed to show whether a high dose of coenzyme Q10

M

correlates with clinical benefits. Additionally, potential treatment protocols evaluate to attenuate oxidative stress observed in the present study. In future, CoQ10 can be used as a promising therapeutic

ED

agent for ASD disorders due to its antioxidative activity. We suggested that further analyses be done in an expanded cohort to evaluate additional biomarkers for oxidative stress. Perhaps, overall supplementation

Acknowledgment

PT

with CoQ10 can offer promising alternatives to current therapies for neurodevelopmental disorders.

This research was funded by a grant from the Cellular and Molecular Research Center (CMRC) at

CE

Jundishapur University of Medical Sciences with the following code: CMRC-106. Special thanks to all

AC

members of CMRC and to families with autistic children for their help and participation.

Conflict of Interest The authors have no financial relationship with the laboratory used in this study.

Table 1: Demographic and clinical characteristics of autistic children and healthy children. P < 0.05. 13

ACCEPTED MANUSCRIPT

Children with ASDs (N† = 90)

Characteristics

Healthy children (N† = 90)

t-value

p-value‡

7.91(2.55) * 7.61(2.55) -0.93 boys 66† 66† Sex N† girls 24† 24† Weight (kg) * 26.77 (8.33) * 27.87(8.43) -0.36 Height (cm) 126.8 (15.74) 127.1(15.74) -0.35 BMI (kg/m2) 16.16 (1.36) 16.15 (1.43) -0.15 BMI-SDS 0.34 (0.17) 0.24 (0.15) -0.11 BMI: body mass index, BMI-SDS: body mass index-standard deviation scores, N are numbers. * Data are mean (standard deviation). † Data are numbers. ‡ All p-values from by two-tailed Student's t- test.

0.42

0.76 0.74 0.80 0.67

CR IP T

Age (years)

MDA(µM)

0.19 (0.17)

TAS(mM)

0.09 (0.047)

SOD activity (U/ml)

0.011(0.003)

GPx activity (U/ml)

141(14.2)

CoQ10 (ng/ml)

3.91(1.84)

PT

(a).

ASDs Before 60mg/dayQ10

CE

Serum Levels

ASDs After 30mg/dayQ10

AC

MDA(µM) TAS(mM) SOD activity(U/ml) GPx activity(U/ml) CoQ10(ng/ml) (b).

Serum Levels

0.39 (0.19) 0.07 (0.02) 0.013 (0.004) 85.6 (31.9) 4.57 (1.74)

ASDs Before placebo

t-value

p-value

0.15 (0.12)

1.06

0.29

0.11 (0.037)

-1.6

0.11

0.01 (0.003)

1.23

0.22

96.8 (37.20)

1.52

0.14

4.1(2.00)

-0.48

0.63

M

ASDs Before 30mg/dayQ10

ED

Serum Levels

AN US

Table 2. Concentration of plasma coenzyme Q10, lipid peroxidation, and antioxidant enzyme activity after intervention. Data are mean (standard deviation). P < 0.05.

ASDs After 60mg/dayQ10

t- value p- value

0.21 (0.17) 0.14 (0.04) 0.007 (0.002) 62.1 (16.30) 6.71 (1.52)

3.74 -17.82 5.7 3.9 -4.07

ASDs After placebo 14

t-value

0.01 0.02 0.001 0.001 0.003

p-value

ACCEPTED MANUSCRIPT

0.27 (0.11)

0.28 (0.03)

0.24

0.8

TAS (mM)

0.16 (0.08)

0.16 (0.05)

0.08

0.93

SOD activity (U/ml)

0.01 (0.003)

0.01 (0.003)

-0.96

0.34

GPx activity (U/ml)

119.40 (27.7)

113.6 (10.50)

0.22

0.82

CoQ10 (ng/ml)

3.20 (1.66)

3.6 (1.62)

-0.86

0.39

(c). 1

CR IP T

MDA (µM)

ASDs =Autism Spectrum disorders. Data are mean (standard deviation). 3 MDA = malondialdehyde. 4 TAS = Total antioxidant status. 5 SOD = Activity Superoxide Dismutase 6 GPx = Glutathione Peroxidase Activity. 7 CoQ10 = Coenzyme Q10. Data are mean (standard deviation). N are numbers.

AN US

2

Table 3: Side effects of CoQ10 and placebo on psychological functions and behavior in children with

Placebo group

destructive hyperactivity

CoQ10 60 mg/day

(N=26)

group (N=26)

group (N=26)

% 15.38

% 3.84

% 11.53

% 11.53

% 7.69

% 15.38

% 23.07

% 15.38

% 7.69

CE

Aggression

CoQ10 30 mg/day

PT

Side effects

ED

M

ASDs. Data are expressed as percent (%).

AC

Table 4: Effects of CoQ10 on psychological functions and behavior in children with ASDs. Data are expressed as percent (%). Characteristic Groups

gastrointestinal problems N†† (%)

Placebo group (N=26) Improving

5 (19.23)

Deterioration

2 (7.69)

15

P-value†

ACCEPTED MANUSCRIPT

CoQ10 30 mg/day (N=26)* Improving

5 (19.23)

Deterioration

3 (11.53)

0.63

CoQ10 60 mg/day (N=26)** 16 (61.53)

Deterioration

2 (7.69)

Characteristic

Sleep disorders

Groups

N (%)

AN US

Placebo group (N=26)

0.004

CR IP T

Improving

Improving

P-value†

4 (15.38)

Deterioration

1 (3.84)

CoQ10 30 mg/day (N=26)*

6 (23.07)

M

Improving Deterioration

ED

CoQ10 60 mg/day (N=26)** Improving

2 (7.69)

17 (65.38)

0.005

3 (11.53)

CE

PT

Deterioration

0.36

Characteristic

N (%)

AC

Groups

Verbal communication

Placebo group (N=26) Improving

0 (0.0)

Deterioration

2 (7.69)

CoQ10 30 mg/day (N=26)*

16

P-value†

ACCEPTED MANUSCRIPT

Improving

2 (7.69)

Deterioration

3 (11.53)

0.245

CoQ10 60 mg/day (N=26)** 4 (15.38)

Deterioration

5 (19.23)

Characteristic

0.668

Playing with friend

Groups

N (%)

Placebo group (N=26) 1 (3.84)

P-value†

AN US

Improving

CR IP T

Improving

Deterioration

2 (7.69)

CoQ10 30 mg/day (N=26)* Improving

2 (7.69)

2 (7.69)

M

Deterioration

0.5

CoQ10 60 mg/day (N=26)**

ED

Improving Deterioration 1

5 (19.23)

0.419

2 (7.69)

PT

CoQ10 = Coenzyme Q10. †P-value is for Chi-Squar test (Fisher′s Exact test). ††N are numbers.

* Group coenzyme Q10 (30 mg/day) compared with the placebo group.

CE

** Group coenzyme Q10 (60 mg/day) compared with the group CoQ10 (30 mg/day). Table 5: Comparison of receiver operating characteristic (ROC) curves for Co-Q, MDA, Gpx, SOD, and TAS in autistic and healthy children. P < 0.05.

AC

Area Under the Curve

Test Result Variable(s)

Area

Std. Errora

17

Asymptotic Sig.b

Asymptotic 95% Confidence Interval Lowe r Uppe r Boun d Boun d

ACCEPTED MANUSCRIPT

coQ

0.364

0.042

0.002

0.282

0.446

MDA

0.121

0.030

0.000

0.063

0.179

Gpx

0.240

0.037

0.000

0.167

0.313

SOD

0.308

0.039

0.000

0.231

0.385

TAS

0.078

0.019

0.000

0.041

0.115

CR IP T

The test result variable(s): CoQ, MDA, Gpx, SOD has at least one tie between the positive actual state group and the negative actual state group. Statistics may be biased. a. Under the nonparametric assumption b. Null hypothesis: true area = 0.5

AC

CE

PT

ED

M

AN US

Fig. 1. Flow diagram.

Figure 2. A summary of the correlation between lipid peroxidation, and antioxidant enzyme activity and ASDs severity. P < 0.05.

18

AN US

CR IP T

ACCEPTED MANUSCRIPT

(c).

(d).

SOD activity

ED

M

Figure 3: Interactive dot diagram comparing levels of lipid peroxidation, and antioxidant enzyme activity in autistic and healthy children. P < 0.05.

MDA

PT

1

0.8 0.6

CE

ASDs Health

AC

(a).

0.4 0.2

ASDs

0

(b).

19

Health

(c).

CR IP T

ACCEPTED MANUSCRIPT

(d).

AN US

References:

Abruzzo, P.M., Ghezzo, A., Bolotta, A., et al., 2015. Perspective biological markers for autism spectrum disorders: advantages of the use of receiver operating characteristic curves in evaluating marker sensitivity and specificity. Dis. Markers, 2015. http://dx.doi.org/10.1155/2015/329607.

M

Adams, J.B., Audhya, T., McDonough-Means, S., et al., 2011. Effect of a vitamin/mineral supplement on children and adults with autism. BMC pediatr., 11(1), 111.

ED

Bhardwaj, M., Kumar, A., 2016. Neuroprotective mechanism of Coenzyme Q10 (CoQ10) against PTZ induced kindling and associated cognitive dysfunction: Possible role of microglia inhibition. Pharmacol Rep., 68(6), 1301-1311.

PT

Castrén, M.L., Westermarck, T., Atroshi, F., 2014. Oxidative Stress and Dietary Interventions in Autism: Exploring the Role of Zinc, Antioxidant Enzymes and Other Micronutrients in the Neurobiology of Autism. Pharmacology and Nutritional Intervention in the Treatment of Disease. InTech. http://dx.doi.org/10.5772/57512.

CE

Constantino, J.N. and Charman, T., 2016. Diagnosis of autism spectrum disorder: reconciling the syndrome, its diverse origins, and variation in expression. The Lanc. Neurology, 15(3), 279-291.

AC

Cornelius, N., Wardman, J.H., Hargreaves, I.P., et al., 2017. Evidence of oxidative stress and mitochondrial dysfunction in spinocerebellar ataxia type 2 (SCA2) patient fibroblasts: Effect of coenzyme Q10 supplementation on these parameters. Mitochondrion, 34, 103-114. Di Marco, B., M Bonaccorso, C., Aloisi, E., et al., 2016. Neuro-inflammatory mechanisms in developmental disorders associated with intellectual disability and autism spectrum disorder: a neuroimmune perspective. CNS Neurol. Disord. Drug Targets, 15(4), 448-463.

Elham, M., 2017. Coenzyme-Q10 deficiency and Stress oxidative in Children with Autism Spectrum Disorders; A poster presentation on the 17th international conference neurology and neuroscience. J.

20

ACCEPTED MANUSCRIPT

Neurol. Neurorehabil. Res, 2(3), 54. Elham, M., Mohammad-Ali, G., Mohammad-Reza, A., et al., 2016. Mitochondrial Dysfunction in Autistic Children and Oral Coenzyme Q10 Supplementation Treatment. Autism-Open Access, 6(4). doi:10.4172/2165-7890.1000189.

CR IP T

Essa, M.M., Braidy, N., Waly, M.I., et al., 2013. Impaired antioxidant status and reduced energy metabolism in autistic children. Res. Autism Spectr. Disord, 7(5), 557-565. Estes, A., Zwaigenbaum, L., Gu, H., et al., 2015. Behavioral, cognitive, and adaptive development in infants with autism spectrum disorder in the first 2 years of life. J. Neurodev. Disord., 7(1), 24. Feng, J., Shan, L., Du, L., et al., 2017. Clinical improvement following vitamin D3 supplementation in autism spectrum disorder. Nutr. Neurosci., 20(5), 284-290.

AN US

Ferreira, D.J.S., Sellitti, D.F., Lagranha, C.J., 2016. Protein undernutrition during development and oxidative impairment in the central nervous system (CNS): potential factors in the occurrence of metabolic syndrome and CNS disease. J. Dev. Orig. Health Dis., 7(5), 513-524. Föcker, M., Antel, J., Ring, S., et al., 2017. Vitamin D and mental health in children and adolescents. Eur. Child Adolesc. Psychiatry, 1-24.

M

Frustaci, A., Neri, M., Cesario, A., et al., 2012. Oxidative stress-related biomarkers in autism: systematic review and meta-analyses. Free Radic. Biol. Med,52(10), 2128-2141.

ED

Frye, R.E., James, S.J., 2014. Metabolic pathology of autism in relation to redox metabolism. Biomarkers, 8(3), 321-330. Ghanizadeh, A., Akhondzadeh, S., Hormozi, M., et al., 2012. Glutathione-related factors and oxidative stress in autism, a review. Curr. Med. Chem., 19(23), 4000-4005.

PT

González-Fraguela, M.E., Hung, M.L.D., Vera, H., et al., 2013. Oxidative stress markers in children with autism spectrum disorders. Br. J. Med. Med. Res., 3(2), 307.

CE

Gu, F., Chauhan, V., Chauhan, A., 2013. Impaired synthesis and antioxidant defense of glutathione in the cerebellum of autistic subjects: alterations in the activities and protein expression of glutathione-related enzymes. Free Radic. Biol. Med., 65, 488-496.

AC

Gu, F., Chauhan, V., Chauhan, A., 2014. Oxidative stress and mitochondrial dysfunction in ASDs. Frontiers in autism research: New horizons for diagnosis and treatment, 407-428. Gvozdjáková, A., Kucharská, J., Ostatníková, D., et al., 2014. Ubiquinol improves symptoms in children with autism. Oxid. Med. Cell. Longev., 2014. Harada, K., Kamiya, T., Tsuboi, T., 2016. Gliotransmitter release from astrocytes: functional, developmental and pathological implications in the brain. Front. Neurosci., 9, 499. Hardingham, G.E., Do, K.Q., 2016. Linking early-life NMDAR hypofunction and oxidative stress in schizophrenia pathogenesis. Nat. Rev. Neurosci., 17(2), 125-134. 21

ACCEPTED MANUSCRIPT

Hargreaves, I.P., 2014. Coenzyme Q 10 as a therapy for mitochondrial disease. Int. J. Biochem. Cell Biol. 49, 105-111. Henschel, A.D., Rothenberger, L.G., Boos, J., 2010. Randomized clinical trials in children-ethical and methodological issues. Curr. Pharm. Des., 16(22), 2407-2415.

CR IP T

Hininger-Favier, I., Osman, M., Roussel, A.M., et al., 2016. Positive effects of an oral supplementation by Glisodin, a gliadin-combined SOD-rich melon extract, in an animal model of dietary-induced oxidative stress. Phytothérapie,14(1), 29-34. Hollis, F., Kanellopoulos, A.K., Bagni, C., 2017. Mitochondrial dysfunction in Autism Spectrum Disorder: clinical features and perspectives. Curr. Opin. Neurobiol. 45, 178-187. Horvath, A., Łukasik, J., Szajewska, H., 2017. ω-3 Fatty Acid Supplementation Does Not Affect Autism Spectrum Disorder in Children: A Systematic Review and Meta-Analysis. J. Nutr.147(3), 367-376.

AN US

Júlíusson, P.B., Roelants, M., Benestad, B., et al., 2017. Severe obesity is a limitation for the use of body mass index standard deviation scores in children and adolescents. Acta Paediatr. Khemakhem, A.M., Frye, R.E., El-Ansary, A., et al., 2017. Novel biomarkers of metabolic dysfunction is autism spectrum disorder: potential for biological diagnostic markers. Metab. Brain. Dis., 1-15. Kim, A., Nam, Y.J., Shin, Y.K., et al., 2017. Rotundarpene inhibits TNF-α-induced activation of the Akt, mTOR, and NF-κB pathways, and the JNK and p38 associated with production of reactive oxygen species. Mol. Cell Biochem., 1-13.

ED

M

Kobori, Y., Ota, S., Sato, R., et al., 2014. Antioxidant cosupplementation therapy with vitamin C, vitamin E, and coenzyme Q10 in patients with oligoasthenozoospermia. Arch. Ital. Urol. Nefrol. Androl., 86(1), 1-4. Kondolot, M., Ozmert, E.N., Ascı, A., et al., 2016. Plasma phthalate and bisphenol a levels and oxidantantioxidant status in autistic children. Environ. Toxicol. Pharmacol., 43, 149-158.

CE

PT

Kovacic, P., Somanathan, R., 2017. Mechanistic aspects of autism involving electron transfer, reactive oxygen species, oxidative stress, pollutants, antioxidants, cell signaling and genes. Novel Appro. in Drug Desig. and Devel., 1(3), 8. LaFlamme, B., 2014. NF-[kappa][beta] signaling disrupted in neurodevelopmental disorders. Nature Genet., 46(9), 933-933.

AC

Lee, B.J., Huang, Y.C., Chen, S.J., et al., 2012. Coenzyme Q10 supplementation reduces oxidative stress and increases antioxidant enzyme activity in patients with coronary artery disease. Nutrition, 28(3), 250255.

Lee, Y.K., Lau, Y.M., Ng, K.M., et al., 2016. Efficient attenuation of Friedreich's ataxia (FRDA) cardiomyopathy by modulation of iron homeostasis-human induced pluripotent stem cell (hiPSC) as a drug screening platform for FRDA. Int. J. Cardiol. 203, 964-971. Liang, C.C., Tanabe, L.M., Jou, S., et al., 2014. TorsinA hypofunction causes abnormal twisting movements and sensorimotor circuit neurodegeneration. Eur J Clin Invest., 124(7), 3080. 22

ACCEPTED MANUSCRIPT

Masi, A., Quintana, D.S., Glozier, N., et al., 2015. Cytokine aberrations in autism spectrum disorder: a systematic review and meta-analysis. Mol. Psychiatry., 20(4), 440-446. Miles, M.V., Patterson, B.J., Schapiro, M.B., et al., 2006. Coenzyme Q10 absorption and tolerance in children with Down syndrome: a dose-ranging trial. Pediatr. Neurol. , 35(1), 30-37. Minarini, A., Ferrari, S., Galletti, M., et al., 2017. N-acetylcysteine in the treatment of psychiatric disorders: current status and future prospects. Expert. Opin. Drug Metab. Toxicol., 13(3), 279-292.

CR IP T

Nath, D., 2017. Complementary and Alternative Medicine in the School-Age Child With Autism. J. Pediatr. Health Care, 31(3), 393-397. Nucifora, L.G., Tanaka, T., Hayes, L.N., et al., 2017. Reduction of plasma glutathione in psychosis associated with schizophrenia and bipolar disorder in translational psychiatry. Transl. Psychiatry,7(8), e1215.

AN US

Ojala, J.O., Sutinen, E.M., 2017. The Role of Interleukin-18, Oxidative Stress and Metabolic Syndrome in Alzheimer’s Disease. J. Clin. Med. Res., 6(5), 55. Paul, R., Choudhury, A., Kumar, S., et al., 2017. Cholesterol contributes to dopamine-neuronal loss in MPTP mouse model of Parkinson’s disease: Involvement of mitochondrial dysfunctions and oxidative stress. PLoS ONE, 12(2), e0171285. Ranjan, S., Nasser, J.A., 2015. Nutritional status of individuals with autism spectrum disorders: do we know enough?. ADV. NUTR.,6(4), 397-407.

ED

M

Sanoobar, M., Eghtesadi, S., Azimi, A., et al., 2013. Coenzyme Q10 supplementation reduces oxidative stress and increases antioxidant enzyme activity in patients with relapsing–remitting multiple sclerosis. Int. J. Dev. Neurosci., 123(11), 776-782.

PT

Saravi, S.S.S., Dehpour, A.R., 2016. Potential role of organochlorine pesticides in the pathogenesis of neurodevelopmental, neurodegenerative, and neurobehavioral disorders: A review. Life Sci., 145, 255264.

CE

Sathe, N., Andrews, J.C., McPheeters, M.L., et al., 2017. Nutritional and dietary interventions for autism spectrum disorder: a systematic review. Pediatrics, e20170346.

AC

Schmitt, B., Vicenzi, M., Garrel, C., et al., 2015. Effects of N-acetylcysteine, oral glutathione (GSH) and a novel sublingual form of GSH on oxidative stress markers: A comparative crossover study. Redox Biol., 6, 198-205. Sealey, L.A., Hughes, B.W., Sriskanda, A.N., et al., 2016. Environmental factors in the development of autism spectrum disorders. Environ. Int., 88, 288-298. Seminotti, B., Amaral, A.U., Ribeiro, R.T., et al., 2016. Oxidative stress, disrupted energy metabolism, and altered signaling pathways in glutaryl-CoA dehydrogenase knockout mice: potential implications of quinolinic acid toxicity in the neuropathology of glutaric acidemia type I. Mol. Neurobiol., 53(9), 64596475.

23

ACCEPTED MANUSCRIPT

Smaga, I., Niedzielska, E., Gawlik, M., et al., 2015. Oxidative stress as an etiological factor and a potential treatment target of psychiatric disorders. Part 2. Depression, anxiety, schizophrenia and autism. Pharmacol Rep., 67(3), 569-580. Steullet, P., Cabungcal, J.H., Coyle, J., et al., 2017. Oxidative stress-driven parvalbumin interneuron impairment as a common mechanism in models of schizophrenia. Mol. Psychiatry.

CR IP T

Tarry-Adkins, J.L., Fernandez-Twinn, D.S., Hargreaves, I.P., et al., 2015. Coenzyme Q10 prevents hepatic fibrosis, inflammation, and oxidative stress in a male rat model of poor maternal nutrition and accelerated postnatal growth. Am. J. Clin. Nutr., 103(2), 579-588. Vasconcelos-Moreno, M.P., Fries, G.R., Gubert, C., et al., 2017. Telomere Length, Oxidative Stress, Inflammation and BDNF Levels in Siblings of Patients with Bipolar Disorder: Implications for Accelerated Cellular Aging. Int. J. Neuropsychopharmacol., 20(6), 445-454.

AN US

Xanthos, D.N., Sandkühler, J., 2014. Neurogenic neuroinflammation: inflammatory CNS reactions in response to neuronal activity. Nature. Rev. Neurosci., 15(1), 43-53.

AC

CE

PT

ED

M

Yui, K., Tanuma, N., Yamada, H., et al., 2017. Decreased total antioxidant capacity has a larger effect size than increased oxidant levels in urine in individuals with autism spectrum disorder. Environ. Sci. Pollut. Res. Int., 24(10), 9635-9644.

24