Untargeted lipidomic analysis of metabolic dysfunction-associated steatohepatitis in women with morbid obesity

Laia Bertran; Jordi Capellades; Sonia Abelló; Cristóbal Richart

doi:10.1371/journal.pone.0318557

Peer Review History

Original SubmissionSeptember 4, 2024
4 Sep 2024 Author Response https://doi.org/10.1371/journal.pone.0318557.r001
20 Oct 2024 Decision Letter - Anna Di Sessa, Editor PONE-D-24-36282Untargeted Lipidomic Analysis of Metabolic Dysfunction-Associated Steatohepatitis Associated with Morbid ObesityPLOS ONE Dear Dr. Richart, 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. As listed below, Reviewers' comments need to be carefully addressed before considering the manuscript for potential publication. Please submit your revised manuscript by Dec 04 2024 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. Please include the following items when submitting your revised manuscript: 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, Anna Di Sessa, PhD, MD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf . 2. Thank you for stating in your Funding Statement: “This work was supported by Fundació URV (grant number IT20041S tu C.R.)” Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). 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: No Reviewer #2: No ****** 4. 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 ******** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript titled "Untargeted Lipidomic Analysis of Metabolic Dysfunction-Associated Steatohepatitis Associated with Morbid Obesity" presents a significant contribution to the study of Metabolic Dysfunction-Associated Steatohepatitis (MASH) in the context of morbid obesity. The authors conducted an untargeted lipidomic analysis using LC/MS on serum samples from 216 women, identifying lipid profiles across different histological stages of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD). The findings provide valuable insights into potential non-invasive biomarkers for MASH. While the manuscript is well-structured and addresses an important gap in the literature, there are several areas that require improvement to enhance clarity, methodological transparency, and overall impact. Below are major and minor revisions to consider. Major Corrections - The introduction would benefit from a more comprehensive discussion of the key lipid metabolites implicated in MASH and MASLD, with reference to current literature. By providing this background, readers will better understand the study's significance and how it builds on or diverges from previous work. - The manuscript references previous work regarding LC/MS methods, but a more detailed explanation of cleaning data, quality controls, preprocessing steps (transformation, normalization, scaling and handling missing data) and software used, is needed. Ensuring full methodological transparency is critical for the reproducibility of the findings. - Table 1 would be more informative if global p-values and specific p-values were included for specific comparisons, along with statistical significance notation (e.g., * for p < 0.05, for p < 0.01, * for p < 0.005). This would make the statistical relevance of the data clearer and easier to interpret. - Since Tables 2, 3, and 4 focus, it would be beneficial to merge them with the S1 Table, where p-values and fold changes are already provided. This consolidation will streamline data presentation, making it easier for readers to access all relevant information in one location. - Consider performing Partial Least Squares Discriminant Analysis (PLS-DA) to improve the discriminatory power between the different experimental groups of the study. Furthermore, the best biomarkers could be evaluated in sensitivity analyses that account for potential confounders, such as alcohol consumption and comorbidities, would strengthen the study’s conclusions. - Adding a Venn diagram or other visual representation of overlapping lipid metabolites across different experimental groups (e.g., NL, SS, MASH) would clarify the common and distinct lipid profiles. This would help visualize key findings and their relationships more effectively. - A critical issue is the lack of a clear Data Availability Statement in the current manuscript draft. According to PLOS ONE guidelines, data availability is a requirement for publication. The authors should ensure that they provide a detailed data availability section that explains how the underlying data can be accessed, either through a repository link or within the manuscript itself. Minor Corrections - The title should clearly reflect that the study was conducted exclusively on women. A more accurate title could be: "Untargeted Lipidomic Analysis of Metabolic Dysfunction-Associated Steatohepatitis in Women with Morbid Obesity". - It is recommended to include the geographical details of the cohort, such as the hospital, city, and country, to provide context and ensure the generalizability of the findings. - Including a correlation plot between clinical variables (e.g., BMI, glucose levels) and metabolites would offer deeper insights into the relationships within the data. A Principal Component Analysis (PCA) plot to visualize group separation based on lipidomic profiles would also be a useful addition. The manuscript offers significant insights into the lipidomic profiles associated with MASH and MASLD. However, addressing the suggested revisions will improve the manuscript's clarity and adherence to publishing standards. These revisions will also enhance the study’s overall methodological rigor and impact. I recommend that the manuscript undergoes revision before it is considered for publication. Reviewer #2: In this study titled "Untargeted Lipidomic Analysis of Metabolic Dysfunction-Associated Steatohepatitis Associated with Morbid Obesity" by Bertran et al, authors have identified the key lipid changes associated with MASH. Although there are other lipidomics papers in similar lines, but this study has used a different cohort and presents some new findings. I think manuscript is straightforward and nicely written. My only suggestion is that authors must provide the complete lipidomics data set as the supplementary information. ******** 6. 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: Yes: Álvaro Torres-Martos Reviewer #2: 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 . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0318557.r002
Revision 1
22 Nov 2024 Author Response Response to Reviewers Review Comments to the Author: Reviewer #1: The manuscript titled "Untargeted Lipidomic Analysis of Metabolic Dysfunction-Associated Steatohepatitis Associated with Morbid Obesity" presents a significant contribution to the study of Metabolic Dysfunction-Associated Steatohepatitis (MASH) in the context of morbid obesity. The authors conducted an untargeted lipidomic analysis using LC/MS on serum samples from 216 women, identifying lipid profiles across different histological stages of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD). The findings provide valuable insights into potential non-invasive biomarkers for MASH. While the manuscript is well-structured and addresses an important gap in the literature, there are several areas that require improvement to enhance clarity, methodological transparency, and overall impact. Below are major and minor revisions to consider. Major Corrections: - The introduction would benefit from a more comprehensive discussion of the key lipid metabolites implicated in MASH and MASLD, with reference to current literature. By providing this background, readers will better understand the study's significance and how it builds on or diverges from previous work. Response: Thank you for this suggestion. We have revised the introduction to include a more comprehensive review of the lipid metabolites associated with MASH and MASLD. The updated text now incorporates additional references [14-16] and a contextual discussion on ceramides, triacylglycerols, phospholipids, and their roles in MASLD progression and MASH pathogenesis. - The manuscript references previous work regarding LC/MS methods, but a more detailed explanation of cleaning data, quality controls, preprocessing steps (transformation, normalization, scaling and handling missing data) and software used, is needed. Ensuring full methodological transparency is critical for the reproducibility of the findings. Response: We have expanded the Materials and Methods section to include a thorough explanation of our data preprocessing steps, including data cleaning, quality control measures, normalization, and scaling techniques. The specific software tools and packages used in the preprocessing have also been identified to ensure full methodological transparency. - Table 1 would be more informative if global p-values and specific p-values were included for specific comparisons, along with statistical significance notation (e.g., * for p < 0.05, for p < 0.01, * for p < 0.005). This would make the statistical relevance of the data clearer and easier to interpret. Response: We did not consider it appropriate to add global p-values among the three groups, as they may lead to confusion and do not provide relevant significance, potentially confusing the reader. However, we have updated Table 1 to include specific comparisons across groups, with appropriate statistical notation (* for p < 0.05, for p < 0.01, and * for p < 0.005). This addition enhances the interpretability and statistical relevance of the findings. - Since Tables 2, 3, and 4 focus, it would be beneficial to merge them with the S1 Table, where p-values and fold changes are already provided. This consolidation will streamline data presentation, making it easier for readers to access all relevant information in one location. Response: We agree that consolidating these tables would facilitate a more comprehensive view of the lipidomic data. Thus, we have merged the tables as suggested to include family names, p-values, fold changes, and statistical significance indicators for all groups in new S2, S3, S4 and S5 Tables. Consolidated tables will provide a more accessible resource for readers. - Consider performing Partial Least Squares Discriminant Analysis (PLS-DA) to improve the discriminatory power between the different experimental groups of the study. Furthermore, the best biomarkers could be evaluated in sensitivity analyses that account for potential confounders, such as alcohol consumption and comorbidities, would strengthen the study’s conclusions. Response: We have generated a PLS-DA with all the variables and another one with the significant ones, but it does not show separation between the groups. We believe the model is weak, so we decided to perform a PCA with the significant variables, which we have added as the new Fig 1. Since there is also no clear separation between the groups, we cannot conduct a sensitivity analysis and have postponed this for future studies when this analysis can be performed with more concrete results. This was a preliminary study. - Adding a Venn diagram or other visual representation of overlapping lipid metabolites across different experimental groups (e.g., NL, SS, MASH) would clarify the common and distinct lipid profiles. This would help visualize key findings and their relationships more effectively. Response: A Venn diagram illustrating overlapping and distinct lipid profiles among NL, SS, and MASH groups has been added to the Results section as Fig 2, as you recommended. - A critical issue is the lack of a clear Data Availability Statement in the current manuscript draft. According to PLOS ONE guidelines, data availability is a requirement for publication. The authors should ensure that they provide a detailed data availability section that explains how the underlying data can be accessed, either through a repository link or within the manuscript itself. Response: We have added a Data Availability Statement as per PLOS ONE’s requirements, detailing how the data supporting our findings can be accessed. Minor Corrections: - The title should clearly reflect that the study was conducted exclusively on women. A more accurate title could be: "Untargeted Lipidomic Analysis of Metabolic Dysfunction-Associated Steatohepatitis in Women with Morbid Obesity". Response: To clarify the study’s focus on a female cohort, we revised the title to: “Untargeted Lipidomic Analysis of Metabolic Dysfunction-Associated Steatohepatitis in Women with Morbid Obesity.” - It is recommended to include the geographical details of the cohort, such as the hospital, city, and country, to provide context and ensure the generalizability of the findings. Response: We added the specific location details of the cohort in the Materials and Methods section to provide context regarding generalizability. - Including a correlation plot between clinical variables (e.g., BMI, glucose levels) and metabolites would offer deeper insights into the relationships within the data. A Principal Component Analysis (PCA) plot to visualize group separation based on lipidomic profiles would also be a useful addition. Response: Given your advice, we have conducted multiple linear regressions with significant lipid metabolites against clinical variables, but the R^2 values do not exceed 0.25, indicating that the regressions are poor and not worth including (I attach it in the file "Response to Reviewers"). This is better, as it suggests that the trends of these lipids are not influenced by these variables. However, we have included a PCA plot showing the separation of variables between the groups in the new Fig 1. The manuscript offers significant insights into the lipidomic profiles associated with MASH and MASLD. However, addressing the suggested revisions will improve the manuscript's clarity and adherence to publishing standards. These revisions will also enhance the study’s overall methodological rigor and impact. I recommend that the manuscript undergoes revision before it is considered for publication. Reviewer #2: In this study titled "Untargeted Lipidomic Analysis of Metabolic Dysfunction-Associated Steatohepatitis Associated with Morbid Obesity" by Bertran et al, authors have identified the key lipid changes associated with MASH. Although there are other lipidomics papers in similar lines, but this study has used a different cohort and presents some new findings. I think manuscript is straightforward and nicely written. My only suggestion is that authors must provide the complete lipidomics data set as the supplementary information. Response: We have included the complete lipidomics dataset as the new Supporting Information (S1 Table). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0318557.r003
23 Dec 2024 Decision Letter - Anna Di Sessa, Editor <div>PONE-D-24-36282R1Untargeted Lipidomic Analysis of Metabolic Dysfunction-Associated Steatohepatitis in Women with Morbid ObesityPLOS ONE Dear Dr. Richart, 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 Feb 06 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. Please include the following items when submitting your revised manuscript: 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, Anna Di Sessa, PhD, MD Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). 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: No ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Thank you for addressing the comments and suggestions provided in the initial review. Your efforts to improve the manuscript are evident, and the inclusion of new visualizations and tables has enhanced the overall presentation of the study. The research offers important insights into the lipidomic profiles associated with MASH and MASLD and provides valuable groundwork for non-invasive biomarker identification. That said, there are still several aspects that can be refined to improve the manuscript's scientific rigor, reproducibility, and clarity. Below, I provide suggestions categorized as major and minor revisions. Major Revisions - The statement "Finally, we have reported a panel of lipid metabolites that are discriminatory and specific to patients with MASH compared to SS patients, made up of increased levels of 9-HODE, some PC and PE, the LPI (16:0), and decreased levels of DG (36:0)." suggests strong discriminatory power of the identified metabolites. To support such claims, supervised analyses such as PLS-DA should be performed to calculate classification metrics, including accuracy, sensitivity, specificity, etc along with cross-validation. Similar studies, such as those outlined in https://www.mdpi.com/2073-4425/14/2/248, highlight the importance of validating panels with these metrics. In the conclusion, the statement "Thus, this panel of lipid metabolites could be used as a non-invasive diagnostic tool" should be revised to "might be used" unless robust classification results are presented. - Detailed information on how missing values were handled should be included. Additionally, sharing scripts or pipelines for data preprocessing and analysis is critical for ensuring reproducibility. - Citations for all software and tools employed, such as Proteowizard’s MSConvert, RHermes, and XCMS, should be added. Suggested references include: https://pubs.acs.org/doi/10.1021/ac051437y , https://www.nature.com/articles/nbt.2377 and https://www.nature.com/articles/s41592-021-01307-z. - PCA, while useful for exploratory purposes, focuses solely on variance and may not reveal discriminatory patterns between experimental groups. Supervised methods like PLS-DA, which leverage covariance to enhance group separation, are more suitable for this purpose. PLS-DA analysis should be conducted, and its results presented with proper classifications metrics to confirm discriminatory power. A Venn diagram based on supervised analysis results, highlighting metabolites specific to each comparison, would clarify findings further. - Perform functional enrichment analyses to provide deeper biological insights into the pathways and physiological implications of the identified metabolites. Tools like MetaboAnalyst (https://www.metaboanalyst.ca/) are recommended, and the tool should be cited if used. - Include database identifiers (e.g., HMDB, KEGG, PubChem, Wikipathways) for the detected metabolites. Specify the confidence levels of identification, following established standards for reporting, such as those outlined in https://www.mdpi.com/2218-1989/10/1/8. Minor Revisions - The improved statistical notation in the main tables is commendable and enhances readability. Extending this notation to supplementary tables would ensure consistency. In supplementary tables where p = 0 is listed, it should be replaced with a threshold such as p < 0.0001. - Replace all occurrences of “triglycerides” with “triacylglycerols” and adopt the abbreviation TAG throughout the manuscript to align with international biochemistry guidelines. - Ensure consistent use of abbreviations (e.g., PC for phosphatidylcholine) across the manuscript and supplementary materials. - Revise the sentence "Since we have found a common pattern of lipid metabolites in the comparisons between groups but have also observed that some metabolites are distinctive to the studied groups, we wanted to conduct a PCA to illustrate the distribution of these lipid metabolites among the groups (Fig. 1)." for clarity. Highlight that PCA reflects general variance and may not indicate group-specific patterns. Similarly, revise the discussion sentence "We could also mention that no exclusive lipid metabolite profile was shown in the PCA, so in this regard, the discriminative potential of the model is limited". - Enhance the Venn diagram by including the names or abbreviations of metabolites directly in the figure for better visualization. - Add a paragraph to the discussion summarizing the strengths and limitations of the study to provide a balanced perspective. - Include details about the commercial biochemical analyzer used for biochemical measurements, including the manufacturer and model. - Incorporate this information into Table 1: "In this sense, the percentage of patients in each group treated with lipid-lowering drugs was 19.4% of NL, 41.5% of SS, and 23.4% of MASH." with the statistical significance for clarity. - Provide all figures in a vector format (e.g., PDF or SVG) to ensure high-quality images suitable for publication. This manuscript is a valuable contribution to lipidomics research, and the revisions made thus far have strengthened its presentation. Addressing these additional suggestions will further improve the manuscript’s scientific rigor and impact. ****** 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 ******** [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 . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0318557.r004
Revision 2
16 Jan 2025 Author Response Response to reviewers: Reviewer #1 Major Revisions - The statement "Finally, we have reported a panel of lipid metabolites that are discriminatory and specific to patients with MASH compared to SS patients, made up of increased levels of 9-HODE, some PC and PE, the LPI (16:0), and decreased levels of DG (36:0)." suggests strong discriminatory power of the identified metabolites. To support such claims, supervised analyses such as PLS-DA should be performed to calculate classification metrics, including accuracy, sensitivity, specificity, etc along with cross-validation. Similar studies, such as those outlined in https://www.mdpi.com/2073-4425/14/2/248, highlight the importance of validating panels with these metrics. In the conclusion, the statement "Thus, this panel of lipid metabolites could be used as a non-invasive diagnostic tool" should be revised to "might be used" unless robust classification results are presented. Response: We appreciate your comment, so we have changed the sentence to be less conclusive. - Detailed information on how missing values were handled should be included. Additionally, sharing scripts or pipelines for data preprocessing and analysis is critical for ensuring reproducibility. Response: We included the method used in statistical analysis section. Our bioinformatics said that with all the information from the supporting information documents the reproducibility is ensured. - Citations for all software and tools employed, such as Proteowizard’s MSConvert, RHermes, and XCMS, should be added. Suggested references include: https://pubs.acs.org/doi/10.1021/ac051437y , https://www.nature.com/articles/nbt.2377 and https://www.nature.com/articles/s41592-021-01307-z. Response: We have included these references. - PCA, while useful for exploratory purposes, focuses solely on variance and may not reveal discriminatory patterns between experimental groups. Supervised methods like PLS-DA, which leverage covariance to enhance group separation, are more suitable for this purpose. PLS-DA analysis should be conducted, and its results presented with proper classifications metrics to confirm discriminatory power. A Venn diagram based on supervised analysis results, highlighting metabolites specific to each comparison, would clarify findings further. Response: We have already generated a PLS-DA with all the variables and another one with the significant ones, but it does not show separation between the groups. So, we believe the model is weak and we decided to perform a PCA (Figure 1) with the significant variables. In addition, we already included the Venn diagram as Figure 2. - Perform functional enrichment analyses to provide deeper biological insights into the pathways and physiological implications of the identified metabolites. Tools like MetaboAnalyst (https://www.metaboanalyst.ca/) are recommended, and the tool should be cited if used. Response: We are currently developing a manuscript for the functional analysis of the findings from the metabolomics, lipidomics, and proteomics study of this project. Therefore, we are working with that data for a new study and cannot include it in this one. - Include database identifiers (e.g., HMDB, KEGG, PubChem, Wikipathways) for the detected metabolites. Specify the confidence levels of identification, following established standards for reporting, such as those outlined in https://www.mdpi.com/2218-1989/10/1/8. Response: Given the lipid linkage, the lipidic metabolite name does not match its identifier, which is less specific. This is why we have included the exact formula of the lipid species in the supporting information S1 Table. Minor Revisions - The improved statistical notation in the main tables is commendable and enhances readability. Extending this notation to supplementary tables would ensure consistency. In supplementary tables where p = 0 is listed, it should be replaced with a threshold such as p < 0.0001. Response: Accordingly, we have replaced it. - Replace all occurrences of “triglycerides” with “triacylglycerols” and adopt the abbreviation TAG throughout the manuscript to align with international biochemistry guidelines. Response: Accordingly, we have changed it. - Ensure consistent use of abbreviations (e.g., PC for phosphatidylcholine) across the manuscript and supplementary materials. Response: Done. - Revise the sentence "Since we have found a common pattern of lipid metabolites in the comparisons between groups but have also observed that some metabolites are distinctive to the studied groups, we wanted to conduct a PCA to illustrate the distribution of these lipid metabolites among the groups (Fig. 1)." for clarity. Highlight that PCA reflects general variance and may not indicate group-specific patterns. Similarly, revise the discussion sentence "We could also mention that no exclusive lipid metabolite profile was shown in the PCA, so in this regard, the discriminative potential of the model is limited". Response: Done. - Enhance the Venn diagram by including the names or abbreviations of metabolites directly in the figure for better visualization. Response: We attempted to include the variables in the same diagram, but it became overloaded, so we added a supplementary table in Supporting Information (S6 Table) with the overlapping variables between groups represented in the Venn diagram. - Add a paragraph to the discussion summarizing the strengths and limitations of the study to provide a balanced perspective. Response: Done. - Include details about the commercial biochemical analyzer used for biochemical measurements, including the manufacturer and model. Response: Done. - Incorporate this information into Table 1: "In this sense, the percentage of patients in each group treated with lipid-lowering drugs was 19.4% of NL, 41.5% of SS, and 23.4% of MASH." with the statistical significance for clarity. Response: Done. - Provide all figures in a vector format (e.g., PDF or SVG) to ensure high-quality images suitable for publication. Response: Done. This manuscript is a valuable contribution to lipidomics research, and the revisions made thus far have strengthened its presentation. Addressing these additional suggestions will further improve the manuscript’s scientific rigor and impact. Attachments Attachment Submitted filename: Response to Reviewers_R2.docx https://doi.org/10.1371/journal.pone.0318557.r005
20 Jan 2025 Decision Letter - Anna Di Sessa, Editor <p>Untargeted Lipidomic Analysis of Metabolic Dysfunction-Associated Steatohepatitis in Women with Morbid Obesity PONE-D-24-36282R2 Dear Dr. Richart, 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. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Anna Di Sessa, PhD, MD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). 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. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) ****** 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 ******** https://doi.org/10.1371/journal.pone.0318557.r006
Formally Accepted
Acceptance Letter - Anna Di Sessa, Editor PONE-D-24-36282R2 PLOS ONE Dear Dr. Richart, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Anna Di Sessa Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0318557.r007

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .