-->PONE-D-26-01139-->-->Plasma vitamin C levels are associated with brain structural networks on MRI: A large cohort study-->-->PLOS One

Dear Dr. Shintaku,

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Academic Editor

PLOS One

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2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

- https://doi.org/10.1016/j.acra.2025.04.048

- https://www.ncbi.nlm.nih.gov/pubmed/40531396

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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“I have read the journal's policy and the authors of this manuscript have the following competing interests: A company (KAGOME CO., LTD.) established a department at Hirosaki University. Two authors (Daichi Kokubu and Yusuke Ushida) are employees of the company. Two authors hold stock in the company. This study reflects the views of the scientists, not the company.”

We note that one or more of the authors are employed by a commercial company: KAGOME CO., LTD

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Additional Editor Comments:

The study is well designed, leverages a large community-based cohort, and addresses a timely topic. However, several points require clarification to strengthen interpretative balance and reproducibility. Causal language should be tempered given the cross-sectional design, and conclusions regarding maintenance of brain structure or DMN connectivity should be reframed as associative. The magnitude and clinical relevance of the reported effect sizes should be more explicitly discussed.

Additional clarification is needed regarding the statistical approach, including the rationale for using both correlation and regression analyses and confirmation that model assumptions were assessed. The identification and interpretation of DMN-related components—particularly the opposing direction observed for one posterior component—require clearer explanation. The limitations section should more explicitly acknowledge potential residual confounding and the use of a single plasma vitamin C measurement. Clarification of the clinical relevance, including the relationship with MMSE, would further strengthen the manuscript.

These issues are considered minor and primarily involve clarification and refinement of interpretation. We look forward to receiving the revised version.

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

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Reviewer #1: 1. Causality and interpretation

The study is cross-sectional, yet some statements in the Discussion and Conclusion imply a causal role of vitamin C in maintaining brain structure and DMN connectivity.

The authors should consistently temper their language and clearly emphasize that causal inferences cannot be made.

Statements suggesting “interventions” or “maintaining brain integrity” should be reframed as hypothesis-generating rather than evidence of effect.

2.Effect size and clinical relevance

Although several associations reach statistical significance, the standardized β coefficients and correlation coefficients are relatively small.

The authors should explicitly discuss the magnitude and potential clinical significance of these effects, rather than focusing primarily on p-values.

A brief comparison with effect sizes reported in prior MRI–nutrition studies would strengthen the interpretation.

2a)Statistical clarity

The rationale for using both Spearman correlation and multiple regression should be briefly clarified.

It would be helpful to specify whether assumptions for regression analyses were formally checked.

3.DMN component interpretation

One DMN component (posterior DMN-II) shows a negative association with plasma vitamin C levels, while others show positive associations.

This opposing directionality requires clearer biological interpretation. At present, this finding is described but not adequately explained.

The authors should discuss whether these components represent distinct functional or structural sub-networks and how vitamin C might differentially influence them.

3a)DMN identification

DMN components were identified by expert neuroradiologists. While acceptable, reporting inter-rater agreement or referencing a standardized atlas-based confirmation would improve reproducibility.

4.Potential confounding variables

Important lifestyle and nutritional factors (e.g., total dietary intake, physical activity, BMI, smoking, alcohol consumption, and socioeconomic status beyond education) were not included.

While some of these may not be available, the authors should explicitly acknowledge them as potential residual confounders and discuss how they may influence the observed associations.

5.Plasma vitamin C measurement

Only a single plasma vitamin C measurement was used.

Given the known variability of vitamin C levels, the authors should more clearly discuss how measurement variability and short-term dietary effects might influence results.

Reviewer #2: Overall assessment

This manuscript leverages a large, community-based cohort and combines global volumetric measures (GMV/ICV, WMV/ICV) with ICA-based source-based morphometry (SBM) to examine associations between plasma vitamin C and brain structure in older adults. The sample size and imaging pipeline are clear strengths, and the topic is timely given growing interest in links between nutritional factors and brain health. That said, several methodological and interpretive points would benefit from clarification and, where feasible, additional analyses to improve reproducibility and strengthen the biological and clinical interpretation. In particular, some conclusions regarding “maintenance of DMN connectivity” appear stronger than what is directly supported by the current results, especially considering the negative association observed for one DMN-related component.

Major Comments

1) Clarify the procedure for identifying DMN-related components

Although the authors cite prior studies, the manuscript would benefit from a more detailed description of how DMN-related components were identified among the 147 ICA-derived GM structural networks. The statement that “two neuroradiologists identified DMN-related networks based on previous studies” is helpful, but additional detail is needed to ensure reproducibility. At minimum, please specify the anatomical criteria used to label a component as DMN-related and outline the decision process.

2) Consider adding voxel-wise localization analyses (VBM)

The study reports significant associations between plasma vitamin C levels and global brain volume ratios (GMV/ICV and WMV/ICV). While these global indices are informative, voxel-wise analyses could provide additional anatomical specificity and strengthen the biological interpretation. Since SPM12/CAT12 processing has already been performed, it may be feasible to include voxel-wise VBM analyses (GM and, if appropriate, WM) to localize regions associated with plasma vitamin C.

3) Please reconcile the interpretation of DMN findings (including Component 135)

In the Abstract and Discussion, the narrative suggests that plasma vitamin C may be important for “maintaining DMN connectivity” and could be linked to cognition. However, Component 135 (posterior DMN-II) shows a negative correlation with plasma vitamin C, and this point would benefit from clearer interpretation.

4) Strengthen clinical relevance by reporting associations with MMSE

Given that MMSE is available and included as a covariate, the clinical relevance of the imaging findings could be enhanced by reporting:

• the association between plasma vitamin C levels and MMSE (e.g., correlation and/or adjusted regression); and

• the associations between the extracted DMN-related components (Components 85/100/135) and MMSE.

Showing whether the vitamin C–DMN-related structural network associations relate (or do not relate) to cognitive performance would help clarify the clinical implications.

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Reviewer #1: No

Reviewer #2: Yes: Kazuhide Seo

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<p>Plasma vitamin C levels are associated with brain structural networks on MRI: A large cohort study

PONE-D-26-01139R1

Dear Dr. Shintaku,

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.

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Kind regards,

Branko Aleksic, MD, PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Excellent paper

Reviewers' comments: