Lipid Peroxidation via Regulating the Metabolism of Docosahexaenoic Acid and Arachidonic Acid in Autistic Behavioral Symptoms

Round 1

Reviewer 1 Report

It is reassuring that the authors have considered and incorporated the Role of Ferroptosis into their paper Lipid Peroxidation via Regulating the Metabolism of Docosahexaenoic Acid and Arachidonic Acid in Autistic Behavioral Symptoms. However, while the article is nearly ready for acceptance, a minor modification requires attention. Specifically, conducting an English spell-check to ensure accuracy would be helpful. Once the revision has been addressed, the paper should be deemed suitable for acceptance.

Moderate editing of the English language is required.

Author Response

REVIEWER 1

However, while the article is nearly ready for acceptance, a minor modification requires attention. Specifically, conducting an English spell-check to ensure accuracy would be helpful.

Our response

Thank you so much for your positive comments. This paper was three times received MDPI language editing service. We have carefully conducted English spell-check to ensure accuracy

Reviewer 2 Report

This manuscript provides the association between MDA-LDL levels, lipid peroxidation product and the Aberrant Behavior Checklist (ABC score) in Autism. An imbalance between plasma DHA and ARA levels induced ferroptosis via lips peroxidation.

Authors mentioned that ferroptosis might be an important factor in lipid peroxidation in relation to DHA and ARA

Comments:

1. Many recent studies have found an association between ASD and increased oxidative stress. to detect elevated oxidative stress in ASD, various biomarkers have been developed including lipid peroxides, MDA. 

What is the new finding of this works? 

2. How does you know that the ferroptosis occur in the brain and cause the pathophysiology ? Can the authors detect the iron labile in plasma? 

3. The data presented in table II and III are not clear, need to more organized. Add some pictures like scatter plot and graph plot the correlation between lipid marker and ABC score.

4. There are some incorrect spelling and data. please check such as lines 104 and 113; number 8 should be corrected to 7, line 175, line 241, line 331, line 342.

5. Table 4: why don't collect the nutrient intake from every subjects? please check the data of cholesterol of ASD and control group.

Author Response

Reviewer 4

This manuscript provides the association between MDA-LDL levels, lipid

peroxidation product and the Aberrant Behavior Checklist (ABC score) in Autism.

An imbalance between plasma DHA and ARA levels induced ferroptosis via lips

peroxidation.

Authors mentioned that ferroptosis might be an important factor in lipid

peroxidation in relation to DHA and ARA

Comments:

1. Many recent studies have found an association between ASD and increased

oxidative stress. to detect elevated oxidative stress in ASD, various biomarkers have been developed including lipid peroxides, MDA.

Our response

We examined necessary biomerkers for the purpose of the studuy indicating polyunsaturatedfatty acid -related lipid peroxidation markers in plasma. We did not examine o non closely related biomarkers in this study

What is the new finding of this works?

Our response

We described the new findings in the discussion section as marked with bulu color as fellow: “Importantly, that the present findings indicate that the deviated imbalance between DHA levels and ARA levels in plasma (DHA/ARA ratio eas 0.59 due to decreade DHA levels and increasded ARA levels in plasma) may induce lipid peroxidatioon related ferroptosis and contribute to the pathophysiology of ASD. The present study firstly elucidated that the important role of the imbalance between DHA and ARA in plasma may contribute to the pathophysiology of ASD.”

2. How does you know that the ferroptosis occur in the brain and cause the

pathophysiology ? Can the authors detect the iron labile in plasma?

Our response

We described the pathophysiology of ferroptosisin the duscussion section as marked with blue color as fellow; " that the deviated imbalance between DHA levels and ARA levels in plasma (DHA/ARA ratio was 0.59 due to decreased DHA levels and increased ARA levels in plasma) may induce lipid peroxidation related ferroptosis”

3. The data presented in table II and III are not clear, need to more organized.

some pictures like scatter plot and graph plot the correlation between lipid marker and ABC score.

Our response

We added the graph in the Table 3 as Figure 4. However the data of Table 2 was very wide spacesm, resulting in diffivalty to make graph. Many papers in PubMed did not present graphs of wide space data.

4. There are some incorrect spelling and data. please check such as lines 104 and 113; number 8 should be corrected to 7, line 175, line 241, line 331, line 342.

Our response

We corrected these errors

5. Table 4: why don't collect the nutrient intake from every subjects? please check the data of cholesterol of ASD and control group

Our response

We described that marked with blue color as fellow: .”Because of complicated assessment of DHQ15, only subsample of 7 individuals with ASD and 7 controls participated to this assessment.” In the method section.

Reviewer 3 Report

Dear

1. It is better to capitalize the first letter of keywords.

2. Introduction: Please do not repeat the results of previous articles and add the main results or conclusion and describe the similarity or differences between them.

3. Introduction: Please write the aim clearly and add the biomarkers to measure in this study.

4. Please add a reference for "a fasting time of 3 hours after breakfast is reasonable for applying to blood sampling in the present study."

5. There are several grammatical errors such as Kit in line 185, etc.

6. The number of references is much more and reduce the references based on format of journal.

7. Low number of case is a main issue.

8. Why do you select more cases?

9. Please add graphs for better finding of the results.

10. I think that it is not suitable a high journal.

11.  Please add the reasons for "The small sample size in this study may limit the interpretation of the results.".

Dear

Tehre are several grammatical errors.

Author Response

1. It is better to capitalize the first letter of keywords.

Our response

We have capitalized key words

2. Introduction: Please do not repeat the results of previous articles and add the main results or conclusion and describe the similarity or differences between them.

Our response

We avoided reprated introduction of previous articles in the Introduction.

3. Introduction: Please write the aim clearly and add the biomarkers to measure in this study.

Our answer

We have added the relevant biomarkers to lipid peroxidation-related ferroptosis as shown in marked yellow colors: Moreover, a large sample may not be helpful in other situations because of the higher possibility of errors and reduced validity. Analytical studies may provide more truthful results with a small sample because intensive efforts can be made to control all the confounders; small samples can studied to reach a valid conclusion in certain situations [27]. According to a former clinical study, despite the small sample size  (12 patients), statistically significant improvement was obtained by an intensive neurorehabilitation surface program with electromyography [28[“

4. Please describe a reference for "a fasting time of 3 hours after breakfast is reasonable for applying to blood sampling in the present study."

Our response

We introduced an important clinical study indicating that “After 3-hour fasted interval, whole-blood samples were collected by venipuncture into EDTA tubes and then placed on ice. With respect to the fasting interval, the extent of return to fasting state 2-hours after a glucose challenge among 1879 normoglycemic individuals is associated with lower risk of incident prediabetes/ type 2 diabetes in the Coronary Artery Risk Development [32]. Moreover, nonfasting triglyceride levels may replace fasting levels in assessing cardiovascular disease risk once standard reference values have been developed [33]. Therefore, a fasting time of 3 hours after breakfast is reasonable for applying to blood sampling in the present study. The blood samples were frozen at -80°C until the plasma levels of the variables were analyzed at a clinical laboratory (SRL Inc., Tokyo, Japan). ”

5. There are several grammatical errors such as Kit in line 185, etc.

Our response

We corrected this error

6. The number of references is much more and reduce the references based on the format of the journal.

Our response

We deleted 10 less important references in the revised version

7. Low number of cases is a main issue.

Our response

The small sample size indicated the useful merit in the clinical srydies as described marker with yellow color in section 2.2 as fellows: “Moreover, a large sample may not be helpful in other situations because of the higher possibility of errors and reduced validity. Analytical studies may provide more truthful results with a small sample because intensive efforts can be made to control all the confounders; small samples can studied to reach a valid conclusion in certain situations [27]. Additionally, there is nothing wrong with conducting well-designed small studies [26]. According to a former clinical study, despite the small sample size  (12 parients), statistical significant improvement was obtained by an intensive neuroren\habilitation surface programme with electromyograpy (28)”

8. Why do you select more cases?

Our response

We was not able to obtain more additional participanta in this study.

9. Please add graphs for better finding of the results.

Oue response

We presented three graphs on the main findings

10. I think that it is not suitable a high journal.

Our response

We were not able to answer such comment

11. Please add the reasons for "The small sample size in this study may limit the interpretation of the results.".

Our response

We described such notice in the section 2.2.

Reviewer 4 Report

The altered levels of AA and DHA observed ca be due t altered activities of desaturases that needs to be measured in the study sample. The study sample is small despite the various clarifications offered by the authors.

Measurement of other PUFAs is also needed. Concomitant measurement of n-3 and n-6 PGs, LTs and TXs is recommended and this has to be correlated to the measured PUFAs and desaturases.

Measurement of plasma cytokines is needed.

All these parameters need to be interpreted in a comprehensive manner.

ok

Author Response

The altered levels of AA and DHA observed can be due to altered activities of desaturases that need to be measured in the study sample. The study sample is small despite the various clarifications offered by the authors.

Our response

Thank you for considering our paper

Measurement of other PUFAs is also needed. Concomitant measurement of n-3 and n-6 PGs, LTs and TXs is recommended and this has to be correlated to the measured PUFAs and desaturases.

Our answer

We described our explanation on this issue in the section 2.5.1. on the methods of measurement of plasma variables as follows: “Since the present study aimed to identify the association of lipid peroxidation with ferroptosis, we did not measure eicosanoids, such as prostaglamdin (PG) thromboxanes (TX) and leukotrienes (LT) in the present study. The association between the levels of pro-inflammatory ARA and pro-inflammatory eicosanoids has been reported [34], however, we did not study the association between eicosanoids and peroxidation in the present study.”

Measurement of plasma cytokines is needed.

Our response

We added the sentences with marked yellow color as fellow: With response to the association between cytokine and lipid peroxidation, a few references indicated that nanoparticles administration induced a maladaptive irreversible structural remodeling starting with increased pro-inflammatory cytokines levels and lipid peroxidation, resulting in upregulation of the main pro-fibrotic cardiac gene [35]. However, this association appeared to be rare case, and therefore, we did not assay plasma cytokine and did not consider the association between cytokine and lipid peroxidation

All these parameters need to be interpreted in a comprehensive manner

Our response

We responded your comments in a comprehensive manner

Round 2

Reviewer 2 Report

Review Reports

A brief summary of Manuscript ID: cimb-2648184

Type of manuscript: Article
Title: Lipid Peroxidation via Regulating the Metabolism of Docosahexaenoic
Acid and Arachidonic Acid in Autistic Behavioral Symptoms: The role of
ferroptosis
Authors: Kunio Yui *, George Imataka, Tadashi Shiohama           

General concept comments

Reviewer 4

This manuscript provides the association between MDA-LDL levels, lipid peroxidation product and the Aberrant Behavior Checklist (ABC score) in Autism. An imbalance between plasma DHA and ARA levels induced ferroptosis via lipid peroxidation. Authors mentioned that ferroptosis might be an important factor in lipid peroxidation in relation to DHA and ARA.

Comments:

Comment 1 Many recent studies have found an association between ASD and increased oxidative stress. to detect elevated oxidative stress in ASD, various biomarkers have been developed including lipid peroxides, MDA.

Our response 

We examined necessary biomerkers for the purpose of the studuy indicating polyunsaturatedfatty acid -related lipid peroxidation markers in plasma. We did not examine o non closely related biomarkers in this study 

Q1 What is the new finding of this work?

Our response

We described the new findings in the discussion section as marked with bulu color as fellow: “Importantly, that the present findings indicate that the deviated imbalance between DHA levels and ARA levels in plasma (DHA/ARA ratio eas 0.59 due to decreade DHA levels and increasded ARA levels in plasma) may induce lipid peroxidatioon related ferroptosis and contribute to the pathophysiology of ASD. The present study firstly elucidated that the important role of the imbalance between DHA and ARA in plasma may contribute to the pathophysiology of ASD.”

Q2 How does you know that the ferroptosis occur in the brain and cause the pathophysiology? Can the authors detect the iron labile in plasma?

Our response

We described the pathophysiology of ferroptosisin the duscussion section as marked with blue color as fellow; " that the deviated imbalance between DHA levels and ARA levels in plasma (DHA/ARA ratio was 0.59 due to decreased DHA levels and increased ARA levels in plasma) may induce lipid peroxidation related ferroptosis”

Comment to the response:

There are three hallmarks of ferroptosis which are iron overload, the consequent lipid peroxidation and inflammation.

Since the title of this manuscript stated about “the role of ferroptosis”, So the iron store or plasma iron such as serum ferritin, plasma transferrin saturation or non transferrin bound iron (NTBI), should be measured. How can the authors prove the correlation between the DHA/ARA ratio and iron level. Although, transferrin was measured but it was not significant different from healthy control.

I suggested the authors adjust the title by deleting the word “the role of ferroptosis”.

Line 192: the authors discuss that higher plasma MDA-LDL levels in the ASD group indicated ferroptosis.

Ferroptosis is a type of regulated cell death associated with the accumulation of lipid peroxides. However, it's important to note that the relationship between MDA-LDL levels and ferroptosis is not straightforward, and the statement alone does not provide enough information to make a definitive conclusion.

Line 416-618 the authors state that “Furthermore, ARA transport may be an important contributor to ferroptosis sensitivity [41]. These previously reported findings support the present findings in that increased DHA levels and decreased ARA levels in plasma may induce ferroptosis in the ASD group.”

And Line 514-517 “Furthermore, upon exceeding the buffering capacity of triglyceride storage into lipid droplets, both omega-3 and omega- 6 PUFAs induce peroxidation, leading to cytotoxic effects in the presence of diacylglycerol acyltransferase inhibitors (DGATis) [41].

Reference No. 41. Dierge, E.; Debock, E.; Guilbaud, C.; Corbet, C.; Mignolet, E.; Mignard, L.; Bastien, E.; Dessy, 774 C.; Larondelle, Y.; Feron, O. Peroxidation of n-3 and n-6 polyunsaturated fatty acids in the acidic 775 tumor environment leads to ferroptosis-mediated anticancer effects. Cell Metab. 2021, 33, 1701- 776 1715. doi: 10.1016/j.cmet.2021.05.016

I found that this references was in vitro assay in acidic cancer cells and in vivo involved cancer and found that dietary LC-PUFA as an adjuvant modality to take advantage of acidosis-driven induction of ferroptosis in tumors. The authors may use more relevant study to support the present study, such as ferroptosis in ASD not in tumor cells.

Comment 2 The data presented in table II and III are not clear, need to more organized. Some pictures like scatter plot and graph plot the correlation between lipid marker and ABC score.

Our response

We added the graph in the Table 3 as Figure 4. However the data of Table 2 was very wide spacesm, resulting in diffivalty to make graph. Many papers in PubMed did not present graphs of wide space data.

Comments: Figures 1, 2 and 3 are the same data as table 1, no need to duplicate. However, every graph should be made to have the same scale. The correlation graph is preferred.

Comment 3 There are some incorrect spelling and data. please check such as lines 104 and 113; number 8 should be corrected to 7, line 175, line 241, line 331, line 342.

 Our response

We corrected these errors

Q3 Table 4: why don't collect the nutrient intake from every subject? please check the data of cholesterol of ASD and control group.

Our response

We described that marked with blue color as fellow: .”Because of complicated assessment of DHQ15, only subsample of 7 individuals with ASD and 7 controls participated to this assessment.” In the method section.

More Comments from reviewer 4:

1.      In the response: there are too many errors in spelling, authors should be more awareness.

2.      In the manuscript, there are still some mistakes about the number of healthy control which is 7 healthy control in lines 96, 104. Please check the error thoroughly.

3.      The reference No 25 have 2 references, please correct.

4.      Line 196: add the figure number.

5.      Line 235: correct the spelling of “figure”.

6.      Lines 563-578 and Lines 605-621, Both paragraphs stated repeatedly about the limitations.

7.      Line 629: This biomedical mechanism may be due to the rate-limiting enzyme of DHA synthesis, cell elongase 2 (ELOVL2). In my opinion, ELOVL2 did not perform in this study, so this statement should be in the discussion only.

Author Response

1. In the response: there are too many errors in spelling, authors should be more awareness.

Our response 

This revised version was three times examined by the MDPI Language Editing Servise. Three words in tebtext were miss-pelling and we improved these spellings

2. In the manuscript, there are still some mistakes about the number of healthy control which is 7 healthy control in lines 96, 104. Please check the error thoroughly.

Our response

We corrected to 7 healthu controls

• The reference No 25 have 2 references, please correct.

Our response

Reference one of 25 was deleted and different reference was presented.

4. Line 196: add the figure number.

Our response

We inserted “Figure”

NO 5. Line 235: correct the spelling of “figure”

Line 563-578

Our response

We corrected the spelling

Lines 563-578 and Lines 605-621, Both paragraphs stated repeatedly about the limitations

Our response

We delated this sentences and used in the Discussion section

Line 629: This biomedical mechanism may be due to the rate-limiting enzyme of DHA synthesis, cell elongase 2 (ELOVL2). In my opinion, ELOVL2 did not perform in this study, so this statement should be in the discussion only

Our response

We provided the mechanisms on DHA ass fellows: “DHA is synthesized from ALA via ELOVL Fatty Acid Elongase 2 (ELOVL2), a rate-limiting enzyme of DHA synthesis in cells [59]. Therefore, the increase in plasma DHA levels in the present study may reflect a comparable increase in the activity of ELOVL2. Moreover. since metabolism of omega-3 PUFA) are synthesized  delta-12 and delta-15 desaturase enzymes [60], these enzyme may be lowered, resulting in low DAH levels” in the Discussion section

Reviewer 4 Report

In this modified version, authors answered some of the concerns.

It may be brought to the notice of the authors that DGLA is the precursor of PGE1, an anti-inflammatory molecule and that is involve din neurotransmission.  Similarly HA is also the precursor of resolvins, protectins and maresins that are potent anti-inflammatory compounds. It is possible that altered levels of AA and DHA may result in reduced formation of these anti-inflammatory compounds that may have a role in autism. From the study it is not clear why AA and DHA levels are altered. Is it due to desaturases enzymes altered activity? 

THe study is only a partial study. Much more in depth studies are needed to know as to why AA and DHA and lipid peroxidation changes have occurred. Measuring SOD alone is not sufficient. One need to look at GPX, and other anti-oxidants. Similarly, measurement of other PUFAs and the activities of desaturases is also needed. Thus the study is incomplete.

ok

Author Response

This study examined the role of PUFAs in the lipid peroxidation. Moreover, A lot of studied reported that ferroptosis has been reported the occurrence ferroptosis accompanied with decreased SOD levels. Therefore, we did not assay the CPX levels.