Dear Dr. ronghui,

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.

Please submit your revised manuscript by Apr 18 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Nafisa M. Jadavji, PhD, MSc, BSc

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

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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/10.3389/fmed.2023.1269671

- https://doi.org/10.1016/j.scitotenv.2022.156938

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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“Medical Health Research Project of Science and Technology Bureau of Yichang City, Hubei Province (A23-1-021).”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

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6. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

Reviewer #1: Major Issues for Revision

1. Fundamental Conceptual Flaws and Oversimplifications

The study claims that a negative association between MMA and α-Klotho suggests mitochondrial dysfunction's role in aging. However, correlation does not imply causation, especially in a cross-sectional study. The entire premise that MMA directly reflects mitochondrial dysfunction is questionable.

MMA accumulation is more commonly linked to vitamin B12 deficiency and renal impairment rather than mitochondrial dysfunction.

No direct mechanistic link between MMA and α-Klotho is explored—only speculation.

The authors cite literature suggesting MMA as a mitochondrial dysfunction marker, but many of these studies are context-specific (e.g., genetic disorders, severe metabolic conditions) and cannot be extrapolated to the general population.

→ Solution: The authors need to discuss alternative explanations for their findings (e.g., renal impairment, metabolic dysfunction) rather than assuming mitochondrial dysfunction.

2. Weakness in Methodology and Study Design

Cross-sectional design: This prevents causal inference but is not acknowledged adequately. The manuscript implies a directional effect (MMA → α-Klotho decrease), which is misleading.

Many references in the discussion assume causality without direct mechanistic validation.

Longitudinal or interventional studies are needed to confirm such claims.

Serum MMA measurements:

The study relies entirely on NHANES data but does not provide enough quality control details for MMA measurements.

MMA levels fluctuate due to dietary factors, renal function, and transient metabolic changes, which are not sufficiently controlled.

The authors state that MMA was measured by LC-MS/MS, but no intra- or inter-assay variation data are reported.

α-Klotho measurements:

The ELISA method is reported, but batch effects and assay standardization issues are not discussed.

Why were data from 2011–2014 used exclusively when NHANES has additional datasets? This could lead to selection bias.

The reference range used (285.8–1638.6 pg/mL) is outdated and may not reflect inter-laboratory variations.

→ Solution: Acknowledge cross-sectional study limitations, discuss alternative explanations (e.g., renal dysfunction), and provide detailed quality control measures for biochemical assays.

3. Covariate Adjustments Are Superficial and Inadequate

The study controls for confounders (BMI, HbA1c, renal function, etc.), but does not provide a detailed justification for why these specific variables were chosen.

Renal function (eGFR, creatinine levels) is completely missing as a covariate, which is a major oversight.

MMA is strongly influenced by kidney function, and α-Klotho is also a renal protein.

Without renal function adjustments, the reported association could be entirely confounded by kidney disease.

Alcohol intake, medications, and vitamin B12 status are ignored, even though they strongly influence MMA levels.

→ Solution: Add renal function markers (eGFR, creatinine) to models and discuss possible confounders in more depth.

4. Misinterpretation of Statistical Findings

The claim that the MMA-α-Klotho association is "linear" is misleading.

The p-value for linearity (P = 0.181) is not significant, meaning that no strong evidence for a linear relationship exists.

The authors should report additional models (e.g., spline regression) to test for non-linear associations.

The odds ratios (OR) are modest (0.71–0.85), but the manuscript exaggerates their importance.

Even the strongest association (OR = 0.71) is weak in epidemiological terms, and confidence intervals suggest moderate uncertainty.

How much variance in α-Klotho is explained by MMA? This is not reported.

→ Solution: Correct the misrepresentation of linearity, provide alternative statistical models (e.g., cubic splines, mediation analysis), and report variance explained by MMA in α-Klotho levels.

5. Discussion and Interpretation Are Unbalanced

The authors overemphasize the mitochondrial dysfunction hypothesis while ignoring alternative explanations (e.g., renal function, metabolic syndrome, inflammation).

The lack of prior literature on MMA-α-Klotho relationships should be acknowledged as a knowledge gap rather than an implicit confirmation of the hypothesis.

Potential clinical implications are overstated. This is an observational study, and it is misleading to suggest that MMA is a reliable biomarker for aging without further validation.

→ Solution: Rewrite the discussion to:

Acknowledge alternative explanations (e.g., kidney function, inflammation).

Avoid exaggerating the importance of MMA in aging.

Discuss the weaknesses in statistical significance and effect sizes.

6. Figures and Tables Need Improvement

Figure 2 (MMA-α-Klotho association plot) lacks clear interpretation.

The confidence bands are too wide, suggesting uncertainty.

A simple correlation scatter plot would be more informative.

Table 1 (Baseline characteristics by MMA quartiles) should include renal function markers.

Figure 3 (Forest plot for subgroup analyses) is overly complex and needs clearer labeling.

→ Solution: Improve figure clarity, provide scatter plots, and include renal function markers in tables.

Reviewer #2: Dear Authors,

Thank you for submitting your manuscript. I appreciate your efforts in conducting this study and providing valuable insights into the association between methylmalonic acid (MMA) and alpha-Klotho in aging. Kindly find below my comments:

Main Comments:

1. Study Design Limitation: The study only looks at data from one point in time, so it can't prove cause and effect. This limitation is mentioned in the discussion, but it would be better to highlight it earlier in the paper, such as in the abstract and introduction.

2. Results Presentation: In section 3.3, the connection between MMA and α-Klotho could be shown more clearly. Adding more graphs or explaining the data in simpler terms might help.

3. Biological Explanation: The discussion briefly talks about how MMA and α-Klotho might be connected through mitochondrial issues and aging. It would be useful to explain these biological processes in more detail.

Minor Comments:

1. Grammar and Wording:

• There are some grammar mistakes and awkward sentences. For example, in the abstract, the phrase "The correlation between MMA levels and α-Klotho was linear linear" should be fixed.

• In section 4.4, the sentence starting with "While the exact mechanism behind..." could be made shorter and clearer.

2. Figures and Tables:

• Figure 1: Make sure the diagram is easy to read, with all the reasons for excluding participants clearly shown.

• Table 1: Adding a short summary or key points under the table might help readers understand the data better.

Suggestions:

1. Future Studies: Since this study design can't show cause and effect, it would be helpful to suggest future research that could, like studies that follow participants over time.

2. Subgroup Differences: The study finds differences between groups (like based on gender, BMI, and blood pressure). It would be interesting to explore why these differences exist.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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Dear Dr. ronghui,

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.

Please submit your revised manuscript by Jul 12 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Nafisa M. Jadavji, PhD, MSc, BSc

Academic Editor

PLOS ONE

Additional Editor Comment:

Dear Authors,

Your revised submission has been reviewed and there are still several revisions that are needed.

I would encourage your to revise your manuscript and carefully respond to reach reviewer comment.

Sincerely,

Nafisa

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: (No Response)

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #3: No

Reviewer #4: Yes

**********

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

Reviewer #3: No

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #3: Yes

Reviewer #4: No

**********

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

Reviewer #3: No

Reviewer #4: Yes

**********

Reviewer #3: Thank you for submitting this cross‐sectional analysis of NHANES 2011–2014 data examining the inverse association between serum methylmalonic acid (MMA) and α-Klotho. The topic is of potential interest to aging research, but substantial methodological and interpretive weaknesses must be addressed before publication:

A. Confounding and Covariate Adjustment

i. Vitamin B₁₂ and Renal Function: MMA is a sensitive marker of vitamin B₁₂ deficiency and declines in glomerular filtration rate. You must include serum B₁₂ and eGFR (or creatinine) as covariates in all regression models, or at minimum conduct sensitivity analyses stratified by renal function and B₁₂ status. If these data are unavailable, explicitly acknowledge this limitation and discuss its potential impact on your conclusions.

ii. Directed Acyclic Graph (DAG): Provide a DAG or rationale to justify your covariate set, demonstrating how you have controlled for major confounding pathways.

B. Survey Design and Statistical Methods

i. Complex Survey Weighting: Reanalyse using survey‐weighted logistic regression (or linear models for continuous Klotho), incorporating NHANES sampling weights, strata, and PSU. Clearly state the weight variables and software commands used.

ii. Outcome Definition and Modeling Choices: Justify the cutpoint of 704 pg/mL for low α-Klotho, citing normative data or prior studies. Present effect estimates per standard‐deviation increase in MMA, not only quartile comparisons, to enhance interpretability and comparability.

iii. Spline Analyses: Describe knot placement and selection criteria for your restricted cubic spline models.

C. Assay Alignment and Measurement Bias

Temporal Mismatch: Clarify how α-Klotho assays (2007–2016, analyzed 2019–2020) were matched to MMA measurements (2011–2014). Describe any calibration or batch‐effect corrections applied to ensure both biomarkers derive from the same participants under comparable conditions.

D. Interpretation and Causality

i. Cross-Sectional Limitations: Recast all causal language (“mitochondrial dysfunction contributes to aging”) into descriptive associations. Emphasize that temporality cannot be established and discuss reverse causation.

ii. Literature Context: Discuss prior studies linking MMA to aging phenotypes and α-Klotho to age‐related diseases; clearly delineate what novel insight your study adds beyond existing literature.

E. Presentation and Writing

i. Language: Address duplicated words, inconsistent tense, and grammatical errors through professional copyediting.

ii. Figures/Tables: Ensure each legend defines all abbreviations, models, sample sizes, and indicates survey‐weight usage. Provide high-resolution images and clear axis labels.

iii. References: Use a citation manager (Zotero/Mendeley) to enforce consistent formatting, include DOIs and issue numbers, and eliminate duplicate entries.

F. Conclusions and Future Directions

Soften conclusions to:

“These cross-sectional associations between serum MMA and α-Klotho warrant confirmation in longitudinal and mechanistic studies before inferring a causal role for mitochondrial dysfunction in aging.”

Reviewer #4: Your research titled “Association Between Methylmalonic Acid and Alpha-Klotho in American Adults: Cross-Sectional Study” was reviewed and the following comments were made for your consideration.

Abbreviations/Acronyms

• When acronyms/abbreviations are stated in full, initial letters for such acronyms should be capitalized. Sentence 2, covariates section, “body mass index (BMI)” should be rectified as “Body Mass Index (BMI)”. This was similarly observed in sentence 3, covariate section, “Glomerular filtration rate (GFR)”. Please rectify these throughout the research protocol.

Title

• Please ensure consistency in the case of all proper nouns in the title. “methylmalonic”, “acid” and “adults” inconsistent with “Alpha-Klotho” in the title. Please ensure consistency of the case throughout the title.

Abstract

• Sentence 3 of materials and methods section, The author stated that “MMA is a continuous variable,” but in sentence 3 of the results section, the author categorized the MMA levels. Could the author please clarify.

• Sentence 1 of the conclusion, the author stated “significant association”. Could the author provide the corresponding p-value to corroborate the significance.

• Sentence 2 of the conclusion section, the author stated “mitochondrial dysfunction”, this makes the conclusion complex for comprehension, the author didn’t name or include any information about mitochondrial dysfunction through ought the abstract but only to include it at the conclusion. The author should please provide an information of how the “mitochondrial dysfunction” is connected to the study right from the beginning of the abstract but not include it at the conclusion without providing prior information and its linkages to the research.

Materials and Methods

• Sentence 1, materials and methods section, the author indicated “This cross- sectional study utilized NHANES data collected by Centers for Diseases Control and Prevention between 2011 and 2014”. But sentence 1, measurement of serum alpha-klotho levels section, the author stated,” Serum alpha-klotho levels were analyzed in frozen samples from individuals aged 40-79 years, which were collected during NHANES from 2007 to 2016, over the period from 2019 to 2020”. The timelines stated are inconsistent or complex, could the author please clarify.

• Sentence 5, covariates section, could the author indicate or cite the source of the standards used in the BMI categorization. Could the author indicate the acceptable labels given to the categorizations. For instance, according to the World Health Organization (WHO), BMI< 18.5kg/m2 is underweight. Please provide more details.

• Similarly, smoking status, vitamin B12, among others. The author should please indicate the sources for the categorization including the standards. The author excellently showcased that with household income. The author should repeat that across all categorical variables.

Results

• The author should kindly report Standard deviations in parenthesis.

General Comments

• Even though, I was expecting the author to employ global acceptable tools for the assessment of certain variables in this study, For instance, Global Adult Tobacco Survey (GATS) for assessing participants smoking status, Global Physical Activity Questionnaire (GPAQ) to assess participants physical activity levels among others, nonetheless the author had done impressive work.

• Despite the author indicated the source of the data, much efforts should have been made to provide better detailed source of the data. The url link provided by the author indicating the source of data does not specifically exposed the data for download. Could the author provide recent specific link to the dataset.

**********

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Reviewer #3: No

Reviewer #4: Yes:  Hardi Adam

**********

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: REVIEW.docx

Dear Dr. ronghui,

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.

Reviewer #5 has a detailed list of minor revisions that need to be made. I would request that these edits be made. We look forward to reading the revised version of the mansucript.

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

Please submit your revised manuscript by Nov 21 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Nafisa M. Jadavji, PhD, MSc, BSc

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #4: All comments have been addressed

Reviewer #5: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #4: Yes

Reviewer #5: Yes

**********

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

Reviewer #4: Yes

Reviewer #5: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #4: Yes

Reviewer #5: Yes

**********

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

Reviewer #4: (No Response)

Reviewer #5: No

**********

Reviewer #4: I am satisfied with the revisions made by the authors. The data underlying the findings have been made fully available, and the manuscript is clearly presented in standard English. The work is technically sound, with the data appropriately supporting the conclusions. Furthermore, the statistical analyses have been conducted rigorously and appropriately. I therefore find the manuscript suitable for acceptance.

Reviewer #5: Abstract

1. Add “the US National Health and Nutrition Examination Survey (NHANES)” to the abstract

2. ‘Marriage’ should be replaced with ‘Marital status’ throughout the manuscript

3. American adults or U.S. adults? Please be consistent throughout the manuscript

4. Please ensure there is a space after a full stop, a comma, and before an open bracket throughout the manuscript.

Introduction

1. “Methylmalonic acid (MMA) is a byproduct of the metabolic pathway of propionic acid[1].” The reference is incorrect; please replace it.

2. “A growing compilation of research indicates that MMA plays a significant role in the development of mitochondrial dysfunction and oxidative stress in both in vitro and in vivo studies[2].” Please add a reference to the in vitro research.

3. “Numerous investigations have established that increased circulating levels of MMA are reflective of the aging process.” Please add references.

4. Reference 8 does not mention α-Klotho. Please remove or fix the text.

5. “Limited studies have systematically investigated the association between α-Klotho levels and methylmalonic acid levels.” Are there any? If so, please reference.

Materials and Methods

1. “In our study, participants aged 40 and above were interviewed” Should this be “included” or did the authors also interview the participants?

2. Reference 11 refers to MMA analysis, not blood collection. Please move to the correct location.

3. *****The Authors say they re-analysed some serum/plasma samples for α-Klotho levels. Why didn’t they analyse more of the samples to fill in the missing data to strengthen their results?

4. “final values of all samples exceeded the limit”. Do you mean “Sensitivity limit”?

Results

1. The first paragraph does not make sense. Is this better? “A total of 11,329 participants aged 40 years or older completed the interview. We excluded participants for several reasons: pregnant women (n=122); missing methylmalonic acid data (n=1,125); and missing α-Klotho data (n=4,866). Ultimately, this cross-sectional study included 5,216 participants from the NHANES dataset collected between 2011 and 2014. Figure 1 illustrates the detailed inclusion and exclusion process.”

2. Figure 1. Flow diagram of the study’s inclusion and exclusion criteria. – Please update the figure title to include more detail.

3. Figure 1 has “No-pregnant” – please fix the typographic mistake to “Non-pregnant”

4. Figure 1 “72.1% were man” – please fix the typographic mistake to “72.1% were men”

5. Table 1 - Variables are listed as 1, 2, 3 and 4 in the columns. Please change to Q1, Q2, Q3 and Q4 to align with the Legend.

6. Typographic mistakes in Table 1. – “Smoke” should be “Smoker”. “now” should be “current” and α-Klotho.

7. Table 2 – Replace “Smoke” with “Smoking”

8. Make the Figure 2 title more detailed. For example: “Figure 2. The relationship between methylmalonic acid levels and the Odds Ratio of serum α-Klotho.”

9. Figure 2 - Fix y-axis title: Odds Ratio of α-Klotho

10. Figure 2 - Fix x-axis title: MMA (nmol/L)

11. Figure 2 – Replace “Smoke” with “Smoking”

Discussion

1. “α-Klotho is a well-known gene associated with anti-aging, mitochondria influence or regulate a number of key aspects of aging [16].” – This statement lacks clarity. Is this what you mean… “α-Klotho is a well-known protein linked to anti-aging, while mitochondria play a crucial role in regulating various key aspects of the aging process [16].”

2. Reference 18 doesn’t mention MMA. Please replace with the correct reference.

3. “The human KL gene encodes the alpha-Klotho protein, recognized as an anti-aging gene[24].” Do you mean “The human KL gene encodes the α-Klotho protein, which is recognized as an anti-aging protein [24].”

4. Remove the sentence “In mice,KL gene overexpression can prolong lifespan, while KL gene mutation can shorten lifespan” as it is repeated.

5. “our results are substantiated by biological evidence” Please expand on this comment, as it would be interesting and relevant to the discussion.

Conclusion

1. “As a biomarker of mitochondrial dysfunction, mitochondrial dysfunction may be an influencing factor in the aging process.”– This statement lacks clarity. Is this what you mean… “Considering that MMA serves as a biomarker for mitochondrial dysfunction, the results indicate that mitochondrial dysfunction might be a contributing factor in the aging process”

References

1. References 2 and 20 are the same.

2. References 4 and 5 are not relevant to the text.

**********

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 #4: Yes:  Hardi Adam

Reviewer #5: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org

<p>Association Between Methylmalonic Acid and Alpha-Klotho in American Adults:a Cross-Sectional Study

PONE-D-25-04628R3

Dear Dr. ronghui,

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,

Nafisa M. Jadavji, PhD, MSc, BSc

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #5: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #5: Yes

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

Reviewer #5: I Don't Know

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #5: Yes

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

Reviewer #5: Yes

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Reviewer #5: (No Response)

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Reviewer #5: No

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