Assessment of oxidative stress in autism spectrum disorder using reactive oxygen metabolites and biological antioxidant potential.

There are several studies on oxidative stress of Autism Spectrum Disorder (ASD), but in these cases there is no study to measure oxidative stress and antioxidant capacity at the same time or studies considering childhood development. Therefore, this study comprehensively assessed the level of oxidative stress in ASD children by simultaneously measuring reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP). The subjects were Japanese, 77 typical development (TD) children, 98 ASD children, samples were plasma. The subjects were divided into age groups: toddlers/preschool age (2-6 years) and school age (7-15 years), to compare the relationships among the d-ROMs levels and BAP/d-ROMs ratios. Furthermore, the correlations between the Parent-interview ASD Rating Scales (PARS) scores and the measured values were analyzed. The levels of d-ROMs were significantly higher in the ASD (7-15 years) than in TD (7-15 years). The PARS scores were significantly higher in the ASD and were significantly correlated with d-ROMs levels. These results suggested that d-ROMs and BAP/d-ROMs ratios could be objective, measured indicators that could be used in clinical practice to assess stress in ASD children.

Introduction

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is characterized by the core symptoms of lack of social communication skills and restricted behaviors and interests, and its prevalence is on the increase [. Some children with ASD have secondary symptoms caused by the accumulation of psychological stress, such as insomnia, depression and anxiety [, and a high proportion of them show aggressive behaviors, self-mutilation behaviors and pica [. On the other hand, factors associated with encephalopathy in ASD, such as autoimmune disorders, inflammatory changes in the brain and mitochondrial dysfunction, have been reported, and oxidative stress is assumed to be enhanced in these conditions [.

At present, ASD is diagnosed and evaluated according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth edition (DSM-5) criteria, but there are no objective/biological test criteria. Therefore, recently, various biomarkers have been explored. In particular, many oxidative stress markers have been reported [, including nitric oxide synthase (NOS), xanthine oxidase (XO), glutathione S-transferase (GST), paraoxonase-1 (PON-1), glutathione, methionine and cysteine, which have been suggested to be associated with neurodevelopmental disorders and oxidative stress [. However, these markers cannot be measured quickly in clinical settings because of the need for specialized test equipment, or cannot be used comprehensively to assess stress in vivo.

As with other conditions, it is beneficial for patients that tests which affect the assessment of ASD are quick and easy. Therefore, we paid attention to oxidative stress markers, namely, reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP). There has been no report on the relationship between ASD and d-ROMs test. However, the d-ROMs test is a method in which serum hydroperoxides generates alkoxy and peroxy radicals by the Fenton reaction with iron ions, and quantitatively measures them. There are several reports on the relationship between oxidative stress evaluation by ASD and Fenton reaction [, and d-ROMs seems to be a method applicable to oxidative stress evaluation as well. The BAP test is a test for examining the ability to reduce ferric ions (Fe3+) to ferrous ions (Fe2+). There are reports investigating the relationship between inflammation markers and iron reduction in ASD, suggesting that it can be applied to the evaluation of oxidative stress in ASD [. These can be measured quickly and efficiently in about 5 minutes for each test [ and are used as assessment indicators in various diseases [. The purpose of this study was to investigate whether d-ROMs and BAP could be objective indicators to assess oxidative stress in untreated ASD children.

Methods

Subjects

The subjects were Japanese children aged 2 to 15 years, including 77 typical development (TD) children (39 males and 38 females) and 98 children with untreated ASD (73 males and 25 females). “TD” children included children who were medically judged to be mentally and physically healthy by pediatric specialists; those who 1) had underlying disease, 2) were receiving therapeutic or prophylactic medication, and 3) had suffered from any disease within the previous one month were excluded from the healthy group of children. The “ASD” children included children who had been medically diagnosed by pediatric neurologists as having ASD based on the DSM-5 criteria and clinical symptoms; children who 1) had underlying disease other than the developmental disorders, 2) had received therapeutic intervention for ASD, and 3) were receiving any drugs were excluded from the ASD group of children. As comorbidities, insomnia 20(20.4%), depression 8(8.2%) and anxiety 37(37.8%) were observed.

In our previous study, younger children had higher oxidative stress levels [. And it has been reported that the stress level of children differs due to changes in the environment between preschool and school life [. Since age and living environment may affect stress, both TD and ASD children were divided into two groups, toddlers/preschool age children (aged 2 to 6 years) and school age children (aged 7 to 15 years). 2 years children were tracked and confirmed as ASD again at the age of 3.

shows the “characteristics” of the subjects. The mean age was 7.08±2.87 years in the ASD group of children and 8.49±3.75 years in the TD group of children. In both groups, the minimum age was 2 years and the maximum age was 15 years. The proportion of males was as high as 74.5% in the ASD group, while the male to female ratio was 1:1 in the TD group. Age did not differ between the TD group and the ASD group when tested by the chi-square distribution.

Lactate and pyruvate was measured in ASD participants, and calculated the lactate/pyruvate ratio. As a result, no patients had a ratio>20, and there were no symptoms or complications suspected of mitochondrial disease.

Indicator of oxidative stress (d-ROMs test)

For "oxidative stress evaluation", the level of excess free radicals produced in the body must be accurately measured. However, free radicals are difficult to measure in vivo because of their short lifetime and high reactivity. Thus, hydroperoxides, stable chemicals produced by oxidation of proteins, amino acids, peptides, glucosides, lipids, nucleotides and other molecules, were measured. Hydroperoxides produce free radicals (alkoxy radical, peroxy radical) in the presence of metal ions; therefore, “amount of hydroperoxides in the blood = amount of free radicals in the body.” U.CARR is used for the unit, and 1U.CARR is equivalent to the H2O2 of 0.08 mg/dL. In a study by Alberti et al., They investigated the adequacy of the d-ROMs measurement and demonstrated that there is a correlation between the d-ROMs test data and the Electron Spin Resonance (ESR) assay data [.

Indicator of antioxidant potential (BAP test)

The BAP test measures the ability to reduce Fe3+ to Fe2+ and is evaluated as the ability to stop the peroxide chain reaction caused by free radicals. The principle of measurement is to first add human serum to ferric chloride (FeCl3) and thiocyanate derivative (uncolored) to create a colored complex of ferric chloride with the thiocyanate derivative. Next, adding “molecule of blood plasma barrier with reducing/electron giving/antioxidant activity against ferric ions (BP (e-))”, ferrous chloride (FeCl2), thiocyanate derivative (uncolored) and oxidized form of BP (e-) generated. The BAP test evaluates the amount of Fe3+ reduced to Fe2+ in human serum by optical measurement at the bleaching level. Based on these facts, we defined “the amount of Fe ions reduced by the sample = antioxidant power”. The unit is μmol / L [.

The BAP test has been reported in metabolic syndrome [. In addition, various studies conducted in combination with d-ROMs have been reported to evaluate both oxidation and reduction. e.g. in conditions including diabetes, nonalcoholic steatohepatitis, epilepsy, carotid atherosclerosis [.

Measurement methods

Samples were collected from the subjects between December 2016 and September 2018. The blood samples were subjected to centrifugal separation at 1469g for 10 minutes to obtain plasma samples. The plasma levels of d-ROMs and BAP were measured using the free radical analyzer “FREE CARRIO DUO” (Diacron International, Grosscto, Italy).

Other examination items

The subjects’ scores on the Parent-interview ASD Rating Scales (PARS) [ were examined. PARS-TR is an evaluation scale that interviews parents about the developmental and behavioral symptoms of ASD, and evaluates the presence or absence and degree. The evaluation items consist of 57 items in 6 areas, including interpersonal, communication, commitment, banding, difficulty, and irritability. The level of each item are evaluated on a three-point scale (0, 1, 2), and a higher total score suggests ASD.

To determine whether intellectual functioning has affected the results of this study, an intelligence test was conducted in the ASD participants to examine the IQ (Intelligence Quotient). And IQ<70 was defined as intellectual disability, and it was analyzed whether intellectual disability affected d-ROMs, BAP and BAP/d-ROMs.

Statistical analysis

The relationship between gender and each test value was analyzed by the Mann–Whitney U test. The subjects were divided into TD 2–6 (TD children aged 2–6 years), ASD 2–6 (ASD children aged 2–6 years), TD 7–15 (TD children aged 7–15 years), and ASD 7–15 (ASD children aged 7–15 years), and the relationship between each age group and each test value was analyzed by the Kruskal-Wallis test. Correlation coefficients (r) and p values between the PARS scores and levels of d-ROMs and BAP were also calculated and compared among the groups. The relationship between intellectual function and d-ROMs, BAP and BAP/d-ROMs was analyzed by the Mann-Whitney U test. The statistical analysis software IBM SPSS Statistics version 21 was used.

Ethical considerations

This study was conducted with the approval of the Ethics Committee of the Japanese Red Cross Tokushima Hinomine Rehabilitation Center for People with Disabilities. A written explanation was given to the subjects and their families, and written informed consent was obtained from the subjects, as much as possible. They were explained that they could refuse to participate or withdraw from the study at any time without any negative consequences.

Results

compares the gender differences among each measured value and age groups. There were no significant gender differences in the plasma levels of d-ROMs and BAP/d-ROMs ratio.

shows the relationship between the age groups and plasma d-ROMs levels in the TD and ASD groups of children. Among the TD children, the plasma d-ROMs levels were significantly higher in TD 2–6 than in TD 7–15 (p<0.001). ASD 7–15 had a significantly higher plasma d-ROMs level than TD 7–15 (p<0.001). However, between TD 2–6 and ASD 2–6 (p = 0.072) and between ASD 2–6 and ASD 7–15 (p = 0.171), there was no significant difference.

Comparison of each measured level and age group.

TD 2–6 = 2–6 years TD children, ASD 2–6 = 2–6 years ASD children, TD 7–15 = 7–15 years TD children, ASD 7–15 = 7–15 years ASD children. (A) d-ROMs levels. TD 2–6, the d-ROMs levels were significantly higher than that in TD 7–15 (p<0.001). ASD 7–15, the d-ROMs levels were significantly higher than that in TD 7–15 (p<0.001). The mean and standard deviation of the levels ​​in each group. 2–6 TD = 405.1±58.1, 2–6 ASD = 433.1±67.8, 7–15 TD = 295.0±26.8, 7–15 ASD = 412.1±64.0. (B) BAP/d-ROMs ratio. TD 7–15, the BAP/d-ROMs ratio was significantly higher than that in TD 2–6 and ASD 7–15 (p<0.001).The mean and standard deviation of the levels in each group. 2–6 TD = 5.571±1.12, 2–6 ASD = 6.337±1.06, 7–15 TD = 8.044±0.98, 7–15 ASD = 6.319±1.21.

shows the relationship between the age groups and the BAP/d-ROMs ratio in the TD and ASD groups of children. The BAP/d-ROMs ratio was significantly higher in TD 7–15 than in TD 2–6 or ASD 7–15 (p<0.001). However, between TD 2–6 and ASD 2–6 (p = 0.063) and between ASD 2–6 and ASD 7–15 (p = 0.770), there was no significant difference.

Comparison of the PARS scores between the healthy and ASD groups of children revealed that the scores were significantly higher in the ASD group of children (p<0.001). shows the relationship between the PARS-TR scores and plasma d-ROMs levels in all subjects. The plasma d-ROMs levels increased as the PARS scores increased, with a significant correlation ( group aged 2–6 years r = 0.343, p = 0.045; group aged 7–15 years r = 0.679, p<0.001). BAP was similarly analyzed and the results are shown in .There was no significant correlation between BAP levels and PARS scores in both the 2–6 years group and the 7–15 years group. ( group aged 2–6 years r = 0.211, p = 0.078; group aged 7–15 years r = 0.166, p = 0.107).

Relationship between PARS scores and d-ROMs levels in all subjects.

〇 = TD children, ● = ASD children. (A) 2–6 years toddlers/preschool age children. The relationship between d-ROMs levels and PARS scores in all subjects aged 2–6 years was analyzed. As a result, a significant positive correlation was observed between the d-ROMs levels and the PARS scores. (r = 0.343, p = 0.045). (B) 7–15 years school age children. The relationship between d-ROMs levels and PARS scores in all subjects aged 7–15 years was analyzed. As a result, a significant positive correlation was observed between the d-ROMs levels and the PARS scores. (r = 0.791, p<0.001).

Relationship between PARS scores and BAP levels in all subjects.

〇 = TD children, ● = ASD children. (A) 2–6 years toddlers/preschool age children. The relationship between BAP levels and PARS scores in all subjects aged 2–6 years was analyzed. As a result, there was no significant correlation between BAP levels and PARS scores. (r = 0.211, p = 0.078). (B) 7–15 years school age children. The relationship between BAP levels and PARS scores in all subjects aged 7–15 years was analyzed. As a result, there was no significant correlation between BAP levels and PARS scores. (r = 0.166, p = 0.107).

shows the relationship between intellectual function among ASD participants and measured values (d-ROMs, BAP and BAP/d-ROMs). When comparing the group without intellectual disability with the group with intellectual disability, no significant difference was observed in any of the measurements. (d-ROMs p = 0.821, BAP p = 0.987, BAP/d-ROMs p = 0.749).

Discussion

d-ROMs levels (hydroperoxides levels) are substances that are converted to alkoxy and peroxy radicals in the presence of iron ions. Because it is not a reactive oxygen species or free radicals, it is a stable substance with low reactivity with oxygen. In addition, the measurement requires very small samples of blood (20μL of plasma), and numerical values are obtained within 5 minutes. Thus, this measurement is minimally invasive and provides results within a very short time [. Furthermore, plasma d-ROMs levels are also highly reproducible [ and, in combination with the BAP, oxidative stress can be assessed from the aspect of both “oxidation” and “antioxidation.”

Nagata et al. reported the absence of any gender differences in the plasma d-ROMs levels or BAP/d-ROMs ratio in TD adults [. The present study showed that there was no gender difference in children.

Comparison of the plasma d-ROMs levels between TD children and children with ASD aged 2–6 years revealed no significant differences. In both healthy and ASD groups of children aged 2–6 years, the higher the plasma d-ROMs level, the younger the age. Similar trends have been reported for other oxidative stress markers (acrolein-lysine, pentosidine, nitrite/nitrate and 8-hydroxy-2'-deoxyguanosine [8-OHdG]) [. These findings are speculated to be associated with rapid synaptic pruning and regeneration in childhood [. In addition, they may also be influenced by psychological stress due to anxiety and fear, because toddlers/preschool age children aged 2 to 6 years have more anxiety and fear toward blood collection than school age children aged 7 to 15 years.

In contrast, comparison between TD children and children with ASD aged 7–15 years revealed significantly higher d-ROMs levels in the children with ASD. In TD children, the plasma d-ROMs levels decreased with growth and reached the reference range in TD adults (approximately < 300 U.CARR) by the age range of 7–15 years. However, in the children with ASD, the plasma d-ROMs levels failed to decrease. In addition, the BAP/d-ROMs ratio (antioxidant capacity) was significantly lower in the children with ASD aged 7–15 years than in those with ASD aged 2–6 years. Based on these findings, we speculate that oxidative stress may not change after growth in children with ASD, because they have a poor ability to cope with stress. Furthermore, they consider after entering school life to be more stressful due to impaired social communication and interpersonal interactions that are characteristic of ASD [.

At the cellular level, abnormalities in mitochondria-related biomarkers, such as bisphenol A and GST, have been reported in patients with ASD [. Free radical scavenging often depends on enzymes, and major radical-scavenging enzymes, such as glutathione peroxidase and catalase, scavenge radicals in and around the mitochondria [, suggesting that increased plasma d-ROMs levels may be caused by mitochondrial dysfunction.

When mitochondrial dysfunction occurs due to oxidative stress, the core symptoms of ASD occur/worsen, causing stress, which induces further mitochondrial dysfunction; thus, it is speculated that a negative chain reaction is established. Therefore, periodic measurements of oxidative stress markers could be useful to assess stress in children with ASD. Rossignol DA and Frye RE focused on immune dysregulation or inflammation, oxidative stress, mitochondrial dysfunction and environmental toxicant exposures as the four major causes of ASD, and conducted a comprehensive literature search from 1971 to 2010 investigated the relationship. As a result, it was found that several studies on lactate, pyruvate, carnitine, etc. were reported as mitochondrial-related biomarkers for ASD [. Simultaneous measurement of oxidative stress substances and these mitochondrial-related markers may enable more reliable assessment of ASD stress.

Also, the antioxidant capacity of the body is considered to be affected by the degree of oxidation and the total antioxidant capacity (TAC) that individuals innately possess. However, previous studies have reported only the degree of oxidation of individual indicators. Based on the results of the present study, simultaneous measurements allow the degree of oxidation and antioxidant potential to be used as useful indicators to determine the TAC in children with ASD.

In regard to the relationship between the PARS scores and plasma d-ROMs levels, there was a significant correlation in that the higher the PARS scores, the higher the plasma d-ROMs levels, suggesting an association between subjective and objective assessments. This is likely to increase the reliability of plasma d-ROMs as a biomarker for an intermediate phenotype of ASD. Similarly, the relationship between PARS scores and BAP levels was also investigated, but was not significantly correlated. This suggests that it is difficult to evaluate ASD only by measuring antioxidant activity using BAP.

This study had limitations. First, although various exclusion criteria were used to select the 2 groups of subjects, not all subjects may have been genuinely healthy or had definitive ASD, because the interpersonal relationships and living environment could not be examined in detail. Second, the sample size cannot be said to be sufficient. In particular, in , when the d-ROMs level and the BAP/d-ROMs ratio of TD and ASD of 2–6 years old are analyzed, the d-ROMs level was p = 0.072 and the BAP/d-ROMs ratio was p = 0.063. From the results, the reason why no significant difference was found may be due to the small sample size of the TD 2–6 group. In the future, it is necessary to increase the reliability of the result on the association between ASD and oxidative stress by increasing the sample size and by combined assessment in in vivo and in vitro studies.

The results of this study may be the plasma levels of d-ROMs levels could be an objective easily measured indicator that could be used in clinical practice to assess stress and clinical state in school age children aged 7 to 15 years with ASD.

(DOCX)

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30 Dec 2019

PONE-D-19-26530

Assessment of oxidative stress in autism spectrum disorder using reactive oxygen metabolites and biological antioxidant potential

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Introduction: Line 44 You talk about secondary symptoms in autism, but these are co-morbidities and a certain percentage of patients have those. Please clarify this and add what percentage of patients have insomnia, depression and anxiety.

Line 49: Please reference the last sentence in paragraph 1.

Line 57: Please explain in detail why the markers can not be measured rapidly? Please clarify what you mean from rapidly? How long does it take to run the test and what is the benefit of measuring it fast vs slow?

Line 63:Please explain the biological relevance of d-ROMS and BAP in autism.

Methods: Is "Measurement items" a sub-title? Please exclude this as this section is part of methods. Please follow the author guidelines fro proper sub-section titles.

Line 92: What does ESR stand for?

Figures: Please exclude "This the Fig 1 Title" or "This is the Fig 1 legend" statements from all figures. Please follow the author guidelines for proper figure title, legend and captions. Please explain the results observed in a specific figure in detail in the figure captions.

Results: First paragraph should be moved to Methods section as it is describing the cohort. I would recommend to start the results section with a brief introduction of what was done in the study following with the description of results.

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Reviewer #1: Comments to the Authors:

Autism spectrum disorder (ASD) is a group of complicated neurodevelopmental disorders characterized by difficulties in social communication and interactions with restricted repetitive behaviors or interests. It causes by an interaction between genetic vulnerability and environmental factors. In order to better understand roots of ASD for diagnosis and treatment, efforts to identify reliable biomarkers are growing. This manuscript investigated whether d-ROMs and BAP/d-ROMs ratio could be objective indicators to assess oxidative stress in untreated ASD children. To address this, the authors measured the plasma d-ROMs levels and BAP/d-ROMs ratio in ASD and typical development (TD) children in two different age groups (2-6y and 7-15y) simultaneously. They found that the levels of d-ROMs were significantly higher in the ASD (7-15 years) than in TD (7-15 years). They also found that Parent-interview ASD Rating Scales (PARS) scores were significantly higher in the ASD and were significantly correlated with d-ROMs levels. This work is informative; however, I have a number of observations and comments:

1. The major issue of this study is the small sample size. This study included 77 TD children and 98 children with untreated ASD. Based on their age, they were separated into two age groups which made each group less number. Especially the 2-6Y TD group. They didn’t find significant difference in plasma d-ROM level and BAP/d-ROMs ratio between ASD and TD in 2-6Y group. Could that be because of the small sample size?

2. The authors claimed that PARS scores were significantly correlated with the plasma d-ROMs levels. However, for 2-6y old, the correlation is weak (r=0.343). How they calculated p values should be clarified and discussed.

3. The authors thought that increased plasma d-ROMs levels may be caused by mitochondrial dysfunction. Is it possible to test some mitochondria-related biomarkers in these samples?

4. For Fig1: it is confusing to label X axis with A, B, C and D. Labeling each group with the age and TD or ASD will be better. Also, please put mean and error bar in the data too.

5. For Fig2: please add figure legend and r value in the figure.

Reviewer #2: PONE-D-19-26530

The authors have explored oxidative stress in ASD with an approach that appears to bring new elements. It is of interest, but there are some issues regarding the communication of the findings.

A minor point in the abstract. The last line ‘indicator’ should be ‘indicators’.

In the introduction-should point out that ‘suicidal ideation’ is based on rather recent reports, and was not previously known. Also, the last sentence of the first paragraph needs to be fully referenced. Also ‘various biomarkers have been explored’ only cites a paper examining clinical features of children who pass 18-month screening. Not sure how this is related. There are MANY efforts at biomarker exploration that deserve mention, and none are cited here. For example, Amaral’s group looking for metabolomic markers, and the work of Hicks and Middleton’s group looking at RNA markers, both of which have resulted in the formation of companies for ASD biomarkers would seem a starting point.

Methods- it is remarkable that they have found such a sample of ASD patients with no medications, minimizing risk of confounds from drug effects, but should mention in the Discussion the potential limitation of exclusion of patients that were more severe and needed medications. ‘Since age and living environment may effect stress’- should briefly explain the rationale for this initially in paragraph 2 when it is first mentioned. The understanding of this doesn’t become clear until late in the Results. Next section, change ‘Hydeoperoxide, which was’ to ‘Hydroperoxides, which are’, and expand in the background on the ‘produced by oxidation of proteins, amino acids, peptides, glucosides, lipids, nucleotides, and other molecules’- please explain this further so that the reader gets a better understanding of why this is being explored. Later ‘ferric to ferrous’ and ‘reduce ferric’- please make a more complete description of these instead of the shorthand notation here, and give more on how this indicator is used elsewhere to help the reader understand why it is being used here. Briefly describe the PARS. Finally, in the Analysis, was the correlation between PARS and BAP also assessed?

Results- were the ages statistically different between groups? Was there any indicator of intellectual functioning, to show that this is specific to ASD rather than intellectual disability? Also, was gender tracked in the statistics since the groups differed in gender balance? Also, please point out in the text the pertinent negative findings, such as the lack of difference between TD and ASD for ages 2-6 for d-ROMs. Otherwise the reader might not realize this was a negative finding, causing confusion later in reading the paper. Fig. 1 title change ‘levels’ to ‘level’.

Discussion- it is stated that ‘plasma d-ROMs levels are highly stable’ but the reason offered is that they are quantitatively measured. Is that why they are stable? Please clarify further the reason why they are stable. Please provide a reference for ‘speculated to be related to rapid cellular apoptosis and regeneration in childhood’, and later, references for ‘children with ASD have high degrees of psychological stress’ and ‘the fact that these children are always exposed to the stress of group living after entering school’. Later, in the discussion on ‘the antioxidant capacity of the body’, should discuss the findings on mitochondrial makers in autism (such as the paper by Rossignol and Frye, Mol Psychiatry 2012;17:290-314), as this is highly relevant. In the paragraph discussing the relation between PARS and d-ROMs, should again note if there was no such relationship for BAP. At the end, this finding is FAR too premature to make the recommendation that this is ‘an objective easily measured indicator that could be used in clinical practice to assess stress’, and it is not reasonable to make a claim such as ‘useful for assessing the clinical severity of ASD’. The actual clinical severity of ASD is inherently more reliable as an indicator of severity-and this marker would not have an impact on this measure.

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Reviewer #2: Yes: David Q. Beversdorf, MD

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12 Feb 2020

3 February, 2020

Dear Reviewer #1

Thank you for inviting us to submit a revised draft of our manuscript entitled, “Assessment of Oxidative Stress in Autism Spectrum Disorder Using Reactive Oxygen Metabolites and Biological Antioxidant Potential” to PLOS ONE. We also appreciate the time and effort you and each of the reviewers have dedicated to providing insightful feedback on ways to strengthen our paper. Thus, it is with great pleasure that we resubmit our article for further consideration. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We also hope that our edits and the responses we provide below satisfactorily address all the issues and concerns you and the reviewers have noted.

Sincerely yours,

Masahito Morimoto

Corresponding author

Our response to the reviewer comment is as follow:

Comment 1:

The major issue of this study is the small sample size. This study included 77 TD children and 98 children with untreated ASD. Based on their age, they were separated into two age groups which made each group less number. Especially the 2-6Y TD group. They didn’t find significant difference in plasma d-ROM level and BAP/d-ROMs ratio between ASD and TD in 2-6Y group. Could that be because of the small sample size?

Response:

We wish to thank the Reviewer for this comment.

Small sample sizes are also described in the study limits in the discussion. We have shown in previous studies that oxidative stress changes with age. So we had to split it into two age groups. Therefore, especially the TD 2-6 Y group has a small sample size.

Analyzing the d-ROMs levels and BAP/d-ROMs ratio for TD and ASD of 2-6 years, the d-ROMs levels were p=0.072 and the BAP/d-ROMs ratio was p=0.063 (Man Whitney U test). From the results, the reason why no significant difference was found may be due to the small sample size of the TD 2-6 Y group.

Add these contents to the discussion of the manuscript.

Comment 2:

The authors claimed that PARS scores were significantly correlated with the plasma d-ROMs levels. However, for 2-6y old, the correlation is weak (r=0.343). How they calculated p values should be clarified and discussed.

Response:

Thank you very much for your point. When statistical analysis was performed again, it was found that there was an error in the p-value. It was changed from p = 0.004 to p = 0.045.

We really appreciate point out important errors.

Comment 3:

The authors thought that increased plasma d-ROMs levels may be caused by mitochondrial dysfunction. Is it possible to test some mitochondria-related biomarkers in these samples?

Response:

We appreciate the Reviewer's comment on this point. The subject's ASD measured lactate and pyruvate as mitochondrial-related biomarkers. I added this to the "Methods" section as follows.

ASD measured lactate and pyruvate as mitochondrial biomarkers and calculated the lactate/pyruvate ratio. As a result, no patients had a ratio>20, and there were no symptoms or complications suspected of mitochondrial disease.

Comment 4:

For Fig1: it is confusing to label X axis with A, B, C and D. Labeling each group with the age and TD or ASD will be better. Also, please put mean and error bar in the data too.

Response:

We wish to thank the Reviewer for this comment.

As you pointed out, the labels on the X axis are difficult to understand, so they have been revised as follows. Group A was changed to "TD 2-6", Group B was changed to "ASD 2-6", Group C was changed to "TD 7-15" and Group D was changed to "ASD 7-15".

We were unable to add the mean and error bars to the scatter plot (Fig 1) created by special processing. So, as an alternative, we added the mean and standard deviation to the figure legend.

Comment 5:

For Fig2: please add figure legend and r value in the figure.

Response:

As you pointed out, the figure legend and r value are added to Fig2.

Again, thank you for giving us the opportunity to strengthen our manuscript with your valuable comments and queries. We have worked hard to incorporate your feedback and hope that these revisions persuade you to accept our submission.

Masahito Morimoto (Corresponding Author)

Japanese Red Cross Tokushima Hinomine Rehabilitation Center for People with Disabilities

Address: 4-1Shinbiraki, chuden-cho, Komathushima-shi, Tokushima, 773-0015 Japan

Phone : +81-885-32-0903 Fax : +81-885-33-3037

E-mail address: morimoto@hinomine-mrc.jp

3 February, 2020

Dear Prof. David Q. Beversdorf, MD (Reviewer #2)

Thank you for inviting us to submit a revised draft of our manuscript entitled, “Assessment of Oxidative Stress in Autism Spectrum Disorder Using Reactive Oxygen Metabolites and Biological Antioxidant Potential” to PLOS ONE. We also appreciate the time and effort you and each of the reviewers have dedicated to providing insightful feedback on ways to strengthen our paper. Thus, it is with great pleasure that we resubmit our article for further consideration. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We also hope that our edits and the responses we provide below satisfactorily address all the issues and concerns you and the reviewers have noted.

Sincerely yours,

Masahito Morimoto

Corresponding author

Our response to the reviewer comment is as follow:

Comment 1:

The last line ‘indicator’ should be ‘indicators’.

Response:

Revised as pointed out.

Comment 2:

Introduction- In the introduction-should point out that ‘suicidal ideation’ is based on rather recent reports, and was not previously known.

Response:

We strongly appreciate the Reviewer's comment on this point. As you pointed out, "suicidal ideation" has been clarified in a recent report and will be removed from the text.

Comment 3:

Introduction- The last sentence of the first paragraph needs to be fully referenced.

Also ‘various biomarkers have been explored’ only cites a paper examining clinical features of children who pass 18-month screening. Not sure how this is related. There are MANY efforts at biomarker exploration that deserve mention, and none are cited here.

Response:

We thank the Reviewer for this pertinent comment.

Yes, I added references. As you pointed out, reference number 5 was not a valid reference. Therefore, we removed this reference.

Comment 4:

Methods- it is remarkable that they have found such a sample of ASD patients with no medications, minimizing risk of confounds from drug effects, but should mention in the Discussion the potential limitation of exclusion of patients that were more severe and needed medications. ‘Since age and living environment may effect stress’ - should briefly explain the rationale for this initially in paragraph 2 when it is first mentioned. The understanding of this doesn’t become clear until late in the Results.

Response:

We thank the Reviewer for this insightful comment.

Regarding 'Since age and living environment may effect stress', the following content was added to paragraph 2 and explained.

In our previous study, younger children had higher oxidative stress levels. And it has been reported that the stress level of children differs due to changes in the environment between preschool and school life.

Comment 5:

Methods- Next section, change ‘Hydeoperoxide, which was’ to ‘Hydroperoxides, which are’, and expand in the background on the ‘produced by oxidation of proteins, amino acids, peptides, glucosides, lipids, nucleotides, and other molecules’- please explain this further so that the reader gets a better understanding of why this is being explored.

Response:

We appreciate the Reviewer's comment on this point.

‘Hydeoperoxide, was changed to ‘Hydeoperoxides,.

The text has been revised as follows to help readers understand why hydroperoxides were measured in this study.

For "oxidative stress evaluation", the level of excess free radicals produced in the body must be accurately measured. However, free radicals are difficult to measure in vivo because of their short lifetime and high reactivity. Thus, hydroperoxides, stable chemicals produced by oxidation of proteins, amino acids, peptides, glucosides, lipids, nucleotides and other molecules, were measured. Hydroperoxides produce free radicals (alkoxy radical, peroxy radical) in the presence of metal ions; therefore, “amount of hydroperoxides in the blood = amount of free radicals in the body.”

Comment 6:

Methods- Later ‘ferric to ferrous’ and ‘reduce ferric’- please make a more complete description of these instead of the shorthand notation here, and give more on how this indicator is used elsewhere to help the reader understand why it is being used here.

Response:

We strongly appreciate the Reviewer's comment on this point.

The BAP test has been revised with detailed explanations. Also added with references to how BAP is used in other studies. The details of the revision are described below.

The BAP test measures the ability to reduce Fe3+ to Fe2+ and is evaluated as the ability to stop the peroxide chain reaction caused by free radicals. The principle of measurement is to first add human serum to ferric chloride (FeCl3) and thiocyanate derivative (uncolored) to create a colored complex of ferric chloride with the thiocyanate derivative. Next, adding “molecule of blood plasma barrier with reducing / electron giving/antioxidant activity against ferric ions (BP (e-))”, ferrous chloride (FeCl2), thiocyanate derivative (uncolored) and oxidized form of BP (e-) generates. The BAP test evaluates the amount of Fe3+ reduced to Fe2+ in human serum by optical measurement at the bleaching level. Based on these facts, we defined “the amount of Fe ions reduced by the sample = antioxidant power”. The unit is μmol / L [Ref].

The BAP test has a report on metabolic syndrome [Ref]. In addition, various studies conducted in combination with d-ROMs have been reported to evaluate both oxidation and reduction. e.g. diabetes, nonalcoholic steatohepatitis, epilepsy, carotid atherosclerosis [Ref].

Comment 7:

Methods- Briefly describe the PARS.

Response:

The following explanation has been added.

PARS-TR is an evaluation scale that interviews parents about the developmental and behavioral symptoms of ASD, and evaluates the presence or absence and degree. The evaluation items consist of 57 items in 6 areas, including interpersonal, communication, commitment, banding, difficulty, and irritability. The levels of each items are evaluated on a three-point scale (0, 1, 2), and a higher total score strongly suggests ASD.

Comment 8:

Methods- In the Analysis, was the correlation between PARS and BAP also assessed?

Response:

We appreciate the Reviewer's comment on this point. We investigated and analyzed the correlation between PARS and BAP. PARS and BAP were not significantly correlated in both the 2-6 and 7-15 years groups. This result is shown in Fig. 3. This content was added to "Methods", "Results" and "Discussion".

Comment 9:

Results- Were the ages statistically different between groups?

Response:

We appreciate the Reviewer's comment on this point. Age did not differ between the TD group and the ASD group when tested by the chi-square distribution. Analysis results were 2-6 years: p=0.276, 7-15 years: p=0.425. This has been added to the subjects section of the methods.

Comment 10:

Results- Was there any indicator of intellectual functioning, to show that this is specific to ASD rather than intellectual disability?

Response:

We thank the Reviewer for this insightful comment. ASD had been tested for intelligence. Defining IQ <70 as intellectual disability and statistically analyzing whether intellectual functions affected d-ROMs, BAP and BAP / d-ROMs, there was no significant difference. Therefore, this study does not appear to be affected by intellectual disability. This has been added to the Methods and Results section.

Comment 11:

Results- Was gender tracked in the statistics since the groups differed in gender balance?

Response:

Table 2 shows the gender, measurements, and analysis results for each group. Statistical analysis revealed no gender differences in each of the groups.

Comment 12:

Results- Please point out in the text the pertinent negative findings, such as the lack of difference between TD and ASD for ages 2-6 for d-ROMs.

Response:

We thank the Reviewer for this pertinent comment. Added the results of this study, especially the negative findings in Fig 1 and Fig 2.

Comment 13:

Results- Fig. 1 title change ‘levels’ to ‘level’.

Response:

The title of Fig.1 has been changed from "levels" to "level".

Comment 14:

Discussion- it is stated that ‘plasma d-ROMs levels are highly stable’ but the reason offered is that they are quantitatively measured. Is that why they are stable? Please clarify further the reason why they are stable.

Response:

We appreciate the Reviewer's comment on this point. The description of the reason why the d-ROMs level is stable has been changed and the reason why the level is stable has been added as follows.

d-ROMs level (hydroperoxides) are substances that are converted to alkoxy and peroxy radicals in the presence of iron ions. Because it is not a reactive oxygen species or free radical, it is a stable substance with low reactivity with oxygen.

Comment 15:

Discussion- Please provide a reference for ‘speculated to be related to rapid cellular apoptosis and regeneration in childhood’, and later, references for ‘children with ASD have high degrees of psychological stress’ and ‘the fact that these children are always exposed to the stress of group living after entering school’.

Response:

We thank the Reviewer for this pertinent comment. As you pointed out, we provided references for ' These findings are speculated to be associated with rapid synaptic pruning and regeneration in childhood'. And the text was changed to "These findings are speculated to be associated with rapid synaptic pruning and regeneration in childhood."

‘Children with ASD have high degrees of psychological stress' and ‘the fact that these children are always exposed to the stress of group living after entering school' was changed as follows and added reference.

‘They consider after entering school life to be more stressful due to impaired social communication and interpersonal interactions that are characteristic of ASD'.

Comment 16:

Discussion- In the discussion on ‘the antioxidant capacity of the body’, should discuss the findings on mitochondrial makers in autism (such as the paper by Rossignol and Frye, Mol Psychiatry 2012;17:290-314), as this is highly relevant.

Response:

We strongly appreciate the Reviewer's comment and suggestion. We have read the review of Rossignol DA and Frye RE. The findings of oxidative stress and mitochondrial-related markers in ASD were discussed with the results of this study and added to the Discussion.

Comment 17:

Discussion- In the paragraph discussing the relation between PARS and d-ROMs, should again note if there was no such relationship for BAP.

Response:

In connection with the response to comment 8, added the following to the paragraph pointed to you.

Similarly, the relationship between PARS scores and BAP levels was also investigated, but was not significantly correlated. This suggests that it is difficult to evaluate ASD only by measuring antioxidant activity using BAP.

Comment 18:

Discussion- This finding is FAR too premature to make the recommendation that this is ‘an objective easily measured indicator that could be used in clinical practice to assess stress’, and it is not reasonable to make a claim such as ‘useful for assessing the clinical severity of ASD’. The actual clinical severity of ASD is inherently more reliable as an indicator of severity-and this marker would not have an impact on this measure.

Response:

We thank the Reviewer for this pertinent comment. As you pointed out, the conclusions are overstatement that cannot be proved by the results of this study. Therefore, the last sentence, “In addition, the plasma d-ROMs levels were correlated with the PARS scores, suggesting that measurement of the plasma d-ROMs levels could also be useful for assessing the clinical severity of ASD.” was deleted.

Again, thank you for giving us the opportunity to strengthen our manuscript with your valuable comments and queries. We have worked hard to incorporate your feedback and hope that these revisions persuade you to accept our submission.

Masahito Morimoto (Corresponding Author)

Japanese Red Cross Tokushima Hinomine Rehabilitation Center for People with Disabilities

Address: 4-1Shinbiraki, chuden-cho, Komathushima-shi, Tokushima, 773-0015 Japan

Phone : +81-885-32-0903 Fax : +81-885-33-3037

E-mail address: morimoto@hinomine-mrc.jp

Submitted filename: Response to editor comments.docx

Click here for additional data file.

13 Apr 2020

PONE-D-19-26530R1

Assessment of oxidative stress in autism spectrum disorder using reactive oxygen metabolites and biological antioxidant potential

PLOS ONE

Dear Dr. Morimoto,

Thank you for submitting your manuscript to PLOS ONE. I apologize for the delay in our decision. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please review the comments from the Reviewers. Please proof-read the text and change some of the poor wording commented by Reviewer 2.

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Reviewer #2: PONE-D-19-26530 R1

The authors have explored oxidative stress in ASD with an approach that appears to bring new elements. It is of interest, and the authors have been responsive to comments. Only a few issues remain with the new text.

In the introduction-line 61 -wording is clumsy, change ‘that tests that’ to ‘that tests which’- less redundant. Also, change ‘illnesses’ to ‘conditions’- as ASD is not really an ‘illness’. Also be consistent on capitalization for ‘Fenton’.

Methods- Wording is bad on lined 103-104 ‘Analysis results were 2-6 years p = 0.276, 7-15 years p = 0.425. ASD measured lactate and pyruvate’ I don’t know what the first sentence means at all, and how did ‘ASD’ measure lactate and pyruvate? Do the authors mean ‘Lactate and pyruvate was measured in ASD participants’? Please clarify. Line 134- change ‘generates’ to ‘is generated’. Line 138- change ‘has a report on metabolic syndrome’ to ‘has been reported in metabolic syndrome’ and insert ‘in conditions including’ before ‘diabetes’ on line 140. Lines 159-160, change ‘items’ to ‘item’ and don’t need ‘strongly’. Line 162, change ‘ASD conducted an intelligence test’ to ‘an intelligence test was conducted in the ASD participants’.

Results- Table 3 (line 286) change ‘intellectual functions of ASD’ to ‘intellectual function among ASD participants’.

Discussion- at the end- still over stated- this finding is FAR too premature to make the recommendation that this is ‘an objective easily measured indicator that could be used in clinical practice to assess stress and clinical state’. It may certainly be worthy of further exploration, but this study is not sufficient to make such a clinical practice recommendation at this time.

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Reviewer #2: Yes: David Q. Beversdorf, MD

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17 Apr 2020

April 17, 2020

Dear Reviewer #2

We thank the reviewer for fruitful suggestions, especially for suggesting the better terms and sentences. We have revised the manuscript PONE-D-19-26530R1 “Assessment of oxidative stress in autism spectrum disorder using reactive oxygen metabolites and biological antioxidant potential” on the basis of the reviewer's comments.

We look forward to a publication of our manuscript in PLOS ONE.

Sincerely yours,

Masahito Morimoto

Corresponding author

Our response to the reviewer comment is as follow:

Comment 1:

In the introduction-line 61 -wording is clumsy, change‘that tests that’ to ‘that tests which’- less redundant.

Response:

We corrected as you pointed out.

Comment 2:

Also, change ‘illnesses’ to ‘conditions’- as ASD is not really an ‘illness’.

Response:

We corrected as you pointed out.

Comment 3:

Also be consistent on capitalization for ‘Fenton’.

Response:

We become consistent on capitalization for ‘Fenton’.

Comment 4:

Wording is bad on lined 103-104 ‘Analysis results were 2-6 years p = 0.276, 7-15 years p = 0.425.

Response:

We deleted the sentence ‘Analysis results were 2-6 years p = 0.276, 7-15 years p = 0.425.’, because it was irrelevant to the explanation in Table 1.

Comment 5:

ASD measured lactate and pyruvate’ I don’t know what the first sentence means at all, and how did ‘ASD’ measure lactate and pyruvate? Do the authors mean ‘Lactate and pyruvate was measured in ASD participants’? Please clarify.

Response:

We appreciate the Reviewer's comment on this point.

I changed the text as follows.

`Lactate and pyruvate was measured in ASD participants, and calculated the lactate/pyruvate ratio. As a result, no patients had a ratio>20, and there were no symptoms or complications suspected of mitochondrial disease.'

And since it is not related to Table 1, we wrote it in another paragraph.

Comment 6:

Line 134- change ‘generates’ to ‘is generated’.

Response:

We corrected as you pointed out.

Comment 7:

Line 138- change ‘has a report on metabolic syndrome’ to ‘has been reported in metabolic syndrome’ and insert ‘in conditions including’ before ‘diabetes’ on line 140.

Response:

We corrected as you pointed out.

Comment 8:

Lines 159-160, change ‘items’ to ‘item’ and don’t need ‘strongly’.

Response:

We corrected as you pointed out.

Comment 9:

Line 162, change ‘ASD conducted an intelligence test’ to ‘an intelligence test was conducted in the ASD participants’.

Response:

We corrected as you pointed out.

Comment 10:

Results- Table 3 (line 286) change ‘intellectual functions of ASD’ to ‘intellectual function among ASD participants’.

Response:

We corrected as you pointed out.

Comment 11:

Discussion- at the end- still over stated- this finding is FAR too premature to make the recommendation that this is ‘an objective easily measured indicator that could be used in clinical practice to assess stress and clinical state’. It may certainly be worthy of further exploration, but this study is not sufficient to make such a clinical practice recommendation at this time.

Response:

We appreciate the Reviewer's comment on this point.

As you pointed out, the results of this study do not currently have any solid evidence to use in clinical practice. Therefore, we changed "suggest" to "may be" to soften the expression.

Again, thank you for giving us the opportunity to strengthen our manuscript with your valuable comments and queries. We have worked hard to incorporate your feedback and hope that these revisions persuade you to accept our submission.

Masahito Morimoto (Corresponding Author)

Japanese Red Cross Tokushima Hinomine Rehabilitation Center for People with Disabilities

Address: 4-1Shinbiraki, chuden-cho, Komathushima-shi, Tokushima, 773-0015 Japan

Phone : +81-885-32-0903 Fax : +81-885-33-3037

E-mail address: morimoto@hinomine-mrc.jp

Submitted filename: Response to reviewers.docx

Click here for additional data file.

8 May 2020

Assessment of oxidative stress in autism spectrum disorder using reactive oxygen metabolites and biological antioxidant potential

PONE-D-19-26530R2

Dear Dr. Morimoto,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

12 May 2020

PONE-D-19-26530R2

Assessment of oxidative stress in autism spectrum disorder using reactive oxygen metabolites and biological antioxidant potential

Dear Dr. Morimoto:

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on behalf of

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PLOS ONE

Table 1

Background of TD children and ASD children subjects.

		TD children (n = 77)	ASD children (n = 98)
Age (y)	means ± SD	8.49±3.75	7.08±2.87
	median	8	7
	maximum	15	15
	minimum	2	2
Sex	male	39 (50.6%)	73 (74.5%)
	female	38 (49.4%)	25 (25.5%)
Gender comparison by age group
2–6 y (n)	male	15	37
	female	13	13
7–15 y (n)	male	24	36
	female	25	12

SD = Standard deviation; y = years.

Table 2

Comparison of gender differences among each measured value and age groups.

	Age groups (y)	Sexa	TD children (n = 77)		ASD children (n = 98)
			Mean ± SD	p value	Mean ± SD	p value
d-ROMs level	2–6	M	401.4±57.1	0.75	435.2±73.6	0.79
		F	409.5±63.6		427.0±53.3
	7–15	M	296.7±27.8	0.78	418.4±70.2	0.18
		F	293.3±26.8		393.4±41.4
BAP/d-ROMs ratio	2–6	M	5.45±1.30	0.66	6.40±1.15	0.53
		F	5.71±0.96		6.13±0.83
	7–15	M	8.11±1.06	0.77	6.23±1.18	0.43
		F	7.97±0.93		6.59±1.34

SD = standard deviation; y = years

aSex M = male, F = female.

Table 3

Relationship between intelligent function and d-ROMs, BAP and BAP/d-ROMs.

	IQ≧70 (n = 85) mean±SD	IQ<70 (n = 13) mean±SD	p value
d-ROMs level	422.2±66.8	426.5±66.5	0.821
BAP level	2627.6±436.2	2698.0±408.4	0.987
BAP/d-ROMs ratio	6.316±1.162	6.405±0.960	0.749

IQ = intelligence quotient, SD = standard deviation