-->PONE-D-26-17120-->-->The impact of pectin supplementation on systemic inflammation pathways, gut microbiome, and metabolic health in patients with Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD): A study protocol for a randomised controlled trial-->-->PLOS One

Dear Dr. Noor Kifah Al-Tameemi,

Thank you for submitting your manuscript to PLOS ONE. 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.

==============================

Dear authors,

I revised carefully the protocol "The impact of pectin supplementation on systemic inflammation pathways, gut microbiome, and metabolic health in patients with Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD): A study protocol for a randomised controlled trial" and listed my comments centered mainly in methodological aspects:

1. Please revise the term methoxyl instead of methoxy.

2. Please revise the dose of pectine recommended by the manufacturer. Some recommend reconstituting 3.5- 5 gr/d by its consistency and osmotic load to treat constipation.

3. Please explain why in healthy adults inflammatory cytokines were augmented and anti- inflammatory IL-10 diminished.

4. Please revise any literature about appropriate outcomes for clinical trials, establishing those relevant for patients and priorized by core outcome set (COS). Consider the clinical over statistical relevance.

5. Please explain and COS- based, why MRI is optional, not mandatory.

6. Please show the sample size calculation

7. Complete in appropriate manner the tables (revise bullets, spaces, and other details).

8. Since my point of view, I consider that you need a pilot study with dose acceptability by participants.

Thank you

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Kind regards,

Sophia Eugenia Martínez-Vázquez, Ph.D.

Academic Editor

PLOS One

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.-->

Reviewer #1: Yes

Reviewer #2: Partly

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-->2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.-->

Reviewer #1: Partly

Reviewer #2: Partly

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-->3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: The elements of this study protocol such as background, rationale , eligibility criteria etc. are provided and will require review of course. My focus is on the statistical aspects of the study. This has a good Statistical Design and Analysis plan and is written as such. The data management and quality control (data validation) looks well in place. The protocol provided in the supplement is well written and helpful. However, there are clarifications and detail which need attention in the sample size and analysis sections as follows:

1. The sample size calculation needs to be checked. It appears that the prior SD assumption seems to be the same for both groups at about 0.036 to arrive at the sample size given by the investigators. This should be an unequal variance calculation according to the background data provided. This will not change the sample size much as such, but should be checked.

2. The missing data assumption being handled by the mixed model approach is reasonable. However, the MAR assumption should have a bit more justification. With regard to recruitment and retention a retention strategy for this type of study should be in place even though the follow up time is short and there is anticipated frequent subject contact. It may minimize the missing data .

3. The analysis via mixed models with random effects looks reasonable. However, the use of a multivariate non parametric alternative should be detailed in the case of non normality.

4. Looking at Figure 1, there are many assessments. Even in this exploratory framework with a small sample size, many analysis ( 6 markers plus the secondary effects) may call for a multiple comparison adjustment of the p-values which the investigators should consider.

Reviewer #2: General comments

The objective of this study is to investigate the effects of daily LM pectin ingestion on inflammatory pathways that drive disease progression, and also to assess changes in anthropometric measures; to evaluate changes in general metabolic indicators, including fasting blood glucose and blood-based biomarkers relevant to MASLD activity and fibrosis. Given the recent growing interest in the relationship between the intake of fermentable dietary fiber and the prevention of MASLD, this study protocol may be of significant importance. However, I have major concerns about this manuscript, particularly regarding the rationale for the dietary fiber intake levels and the lack of data on dietary fiber intake from daily meals.

1. In addition to the dietary fiber intake from regular meals, a daily dose of 15 g of LM pectin may be excessive. Please provide the basis for determining this dosage. The issue of diarrhea must also be taken into account.

2. The placebo formulation must also contain the same amount of dietary fiber, such as cellulose. There is a concern that the effects of adding LM pectin may be confounded by the effects of simply increasing the amount of dietary fiber.

3. It is essential to examine dietary fiber intake, particularly soluble dietary fiber intake, based on daily food records kept throughout the study period.

Specific comments

1. l.83 The author states that HM pectin is generally difficult to ferment in the human body. However, there are reports indicating that HM pectin is a type of fermentable dietary fiber. Please provide evidence that HM pectin has low fermentability.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review?  For information about this choice, including consent withdrawal, please see our Privacy Policy.-->

Reviewer #1: No

Reviewer #2: Yes:  Seiichiro Aoe

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The impact of pectin supplementation on systemic inflammation pathways, gut microbiome, and metabolic health in patients with Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD): A study protocol for a randomised controlled trial

PONE-D-26-17120R1

Dear Dr. Noor Kifah Al-Tameemi,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Sophia Eugenia Martínez-Vázquez, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Dear Authors,

Thank you for your responses, they are adequately. Congratulations.

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.-->

Reviewer #1: Yes

**********

-->2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.-->

Reviewer #1: Yes

**********

-->3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.-->

Reviewer #1: Yes

**********

-->4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

**********

-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

**********

-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: All comments have been addressed and the revisions noted in the manuscript.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review?  For information about this choice, including consent withdrawal, please see our Privacy Policy.-->

Reviewer #1: No

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