The effects of a comparatively higher dose of 1000 mg/kg/d of oral L- or D-arginine on the L-arginine metabolic pathways in male Sprague-Dawley rats

Dain (Raina) Kim; Sarah Martin; Kaushik Desai

doi:10.1371/journal.pone.0289476

Peer Review History

Original SubmissionApril 6, 2023
3 May 2023 Decision Letter - Kanhaiya Singh, Editor PONE-D-23-10457The effects of a comparatively higher dose of 1000 mg/kg/d of oral L- or D-arginine on the L-arginine metabolic pathways in male Sprague-Dawley rats.PLOS ONE Dear Dr. Desai, 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 Jun 17 2023 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. 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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 the following in the Acknowledgments Section of your manuscript: "This research work was funded by the Alexander Graham Bell Canada Graduate Scholarship from Natural Sciences and Engineering Council of Canada (NSERC) to Dain Kim and a Discovery grant from NSERC to Kaushik Desai." We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "KD- Grant # RGPIN-2016-03951, Natural Sciences and Engineering Council of Canada (NSERC), Discovery Grant, https://www.nserc-crsng.gc.ca/index_eng.asp. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. DK - Natural Sciences and Engineering Council of Canada (NSERC), https://www.nserc-crsng.gc.ca/index_eng.asp., Alexander Graham Bell Canada Graduate Scholarship, The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. "Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. Additional Editor Comments: Although the reviewers have found this study of interest, they have recommended to elaborate the results section about the logical flow of information. [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: Yes Reviewer #2: Partly ******** 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 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 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: In this manuscript, the authors are testing the effects of long-duration use of oral L-arginine and D-arginine in nine week old Sprague-Dawley rats. The authors measured the activity of different enzymes such as L-arginine metabolizing enzymes, and also tested the levels of their metabolites in various organs and in the plasma. The results showed that long-duration use of oral L-arginine and D-arginine altered the levels of different metabolites which was not in conjunction with their hypothesis. Thus, the authors specify the dynamic regulation of multiple L-arginine metabolic pathways and propose further studies with different doses and different duration to better assess the use of oral L-arginine. Overall, the manuscript is well written and the results support the conclusions. I have a few minor points that I have highlighted below: 1. In the materials and methods, for most of the sections, the authors write the reasoning and logic behind the methods. I would recommend to mention just the methods in this section. The explanation, logic, reasoning can be added in the results section of the manuscript. 2. Line 93, male is mentioned twice. 3. Instead of using % signs in text, please use the word percent. 4. Line 179, instead of h, please write hour. 5. In the figures, for graphs, please use asterisks for significance, instead of writing the p value. Reviewer #2: The manuscript provides an informative presentation of the study's results and discussions on the effects of oral L-Arginine (L-Arg) and D-Arginine (D-Arg) supplementation. It covers various aspects, including the lack of effect on mean arterial pressure (MAP) and heart rate, the potential benefits of L-Arg supplements in disease conditions, the complexities of L-Arg metabolism and nitric oxide (NO) production, and the impact on arginase, nitric oxide synthase (NOS), and the arginine-glycine amidinotransferase (AGAT) pathway. However, there are areas that could be improved. The interpretation of the results in the discussion section would benefit from further elaboration. For example, if there was no significant effect on MAP and heart rate, exploring potential reasons and discussing influencing factors or variables would enhance the interpretation. Addressing limitations in the study design and their potential impact on the outcomes would also be valuable. Additionally, the discussion could include practical implications of the findings and suggest future research directions. Exploring the application of the results in clinical settings or relevant fields, identifying specific populations or conditions that might benefit from L-Arg supplementation, and proposing avenues for future studies based on the limitations or unanswered questions from the current study would provide valuable insights. Comparative analysis with previous research or existing literature is lacking in the discussion. While previous studies on L-Arg supplementation are briefly mentioned, a comprehensive analysis comparing the current findings to those studies would enhance the understanding of the significance and contextualize the results within the broader scientific landscape. In summary, the manuscript would benefit from a more critical analysis, interpretation, and contextualization of the findings. Providing a comprehensive understanding of the implications and significance of the observed effects of L-Arg and D-Arg supplementation on the various parameters studied, along with comparing the results to previous research, would improve the overall quality of the discussion. ****** 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: No 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.0289476.r001
Revision 1
25 Jun 2023 Author Response PONE-D-23-10457 The effects of a comparatively higher dose of 1000 mg/kg/d of oral L- or D-arginine on the L-arginine metabolic pathways in male Sprague-Dawley rats. PLOS ONE Editor’s comments: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Reply: We have ensured that our manuscript meets PLOS ONE’s style requirements. We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Reply: We have removed funding statement from the manuscript text. Currently, your Funding Statement reads as follows: "KD- Grant # RGPIN-2016-03951, Natural Sciences and Engineering Council of Canada (NSERC), Discovery Grant, https://www.nserc-crsng.gc.ca/index_eng.asp. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. DK - Natural Sciences and Engineering Council of Canada (NSERC), https://www.nserc-crsng.gc.ca/index_eng.asp., Alexander Graham Bell Canada Graduate Scholarship, The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Reply: The above two funding statements are correct. 3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. "Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. Reply: We have uploaded our mimimal underlying data set on DRYAD: https://datadryad.org/stash/share/4jH7mMbb8DzurfoCJzMG1A1pjAyirfB1hJPW9AzPCME DOI https://doi.org/10.5061/dryad.x95x69pqm We will update your Data Availability statement to reflect the information you provide in your cover letter. Additional Editor Comments: Although the reviewers have found this study of interest, they have recommended to elaborate the results section about the logical flow of information. Reply: We have elaborated the results section and the discussion as suggested by the reviewers. Reviewers' comments: Reviewer #1: In this manuscript, the authors are testing the effects of long-duration use of oral L-arginine and D-arginine in nine week old Sprague-Dawley rats. The authors measured the activity of different enzymes such as L-arginine metabolizing enzymes, and also tested the levels of their metabolites in various organs and in the plasma. The results showed that long-duration use of oral L-arginine and D-arginine altered the levels of different metabolites which was not in conjunction with their hypothesis. Thus, the authors specify the dynamic regulation of multiple L-arginine metabolic pathways and propose further studies with different doses and different duration to better assess the use of oral L-arginine. Reply: We are grateful to the reviewer for allocating their time to review our manuscript and provide valuable suggestions to improve it. Overall, the manuscript is well written and the results support the conclusions. I have a few minor points that I have highlighted below: 1. In the materials and methods, for most of the sections, the authors write the reasoning and logic behind the methods. I would recommend to mention just the methods in this section. The explanation, logic, reasoning can be added in the results section of the manuscript. Reply: As suggested by the reviewer, paragraphs with the reasoning and logic behind methods have been moved to appropriate results sections. One paragraph about the dose of arginine used and its relevance has been moved to discussion since there was no appropriate results section for it. We hope the reviewer will find it reasonable. 2. Line 93, male is mentioned twice. Reply: Thank you for pointing out this error. It has been corrected. 3. Instead of using % signs in text, please use the word percent. Reply: This has been corrected throughout the text. 4. Line 179, instead of h, please write hour. Reply: “h” has been replaced with “hour” throughout the text. 5. In the figures, for graphs, please use asterisks for significance, instead of writing the p value. Reply: We have inserted asterisks instead of p value in all the figures. We hope the reviewer will find our corrections satisfactory. Reviewer #2: The manuscript provides an informative presentation of the study's results and discussions on the effects of oral L-Arginine (L-Arg) and D-Arginine (D-Arg) supplementation. It covers various aspects, including the lack of effect on mean arterial pressure (MAP) and heart rate, the potential benefits of L-Arg supplements in disease conditions, the complexities of L-Arg metabolism and nitric oxide (NO) production, and the impact on arginase, nitric oxide synthase (NOS), and the arginine-glycine amidinotransferase (AGAT) pathway. Reply: We grateful to the reviewer for allocating their time to review our manuscript and providing valuable suggestions to improve it. We truly appreciate the encouraging comments. However, there are areas that could be improved. The interpretation of the results in the discussion section would benefit from further elaboration. For example, if there was no significant effect on MAP and heart rate, exploring potential reasons and discussing influencing factors or variables would enhance the interpretation. Addressing limitations in the study design and their potential impact on the outcomes would also be valuable. Reply: As suggested by the reviewer we have now discussed possible reasons and influencing factors for the lack of effect on MAP and heart rate. We have also addressed limitations in study design and their potential impact on the outcomes. We notice that these suggestions have improved our discussion. Additionally, the discussion could include practical implications of the findings and suggest future research directions. Exploring the application of the results in clinical settings or relevant fields, identifying specific populations or conditions that might benefit from L-Arg supplementation, and proposing avenues for future studies based on the limitations or unanswered questions from the current study would provide valuable insights. Reply: We have included paragraphs discussing the practical implications of our findings and suggested future research directions which can add significantly to the knowledge database of L-Arg supplements. We have suggested populations that could benefit from oral L-Arg supplements. Comparative analysis with previous research or existing literature is lacking in the discussion. While previous studies on L-Arg supplementation are briefly mentioned, a comprehensive analysis comparing the current findings to those studies would enhance the understanding of the significance and contextualize the results within the broader scientific landscape. Reply: We have compared our current results with our previous study in greater details now and also provided relevant findings reported in the literature, wherever available. Based on these comparisons we have tried provide the significance of the results as it relates to the use of L-Arg supplements in a broad and varied scientific field. In summary, the manuscript would benefit from a more critical analysis, interpretation, and contextualization of the findings. Providing a comprehensive understanding of the implications and significance of the observed effects of L-Arg and D-Arg supplementation on the various parameters studied, along with comparing the results to previous research, would improve the overall quality of the discussion. Reply: After implementing the suggestions we notice a definite improvement in the manuscript. We hope the reviewer will find our corrections satisfactory. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0289476.r002
20 Jul 2023 Decision Letter - Kanhaiya Singh, Editor The effects of a comparatively higher dose of 1000 mg/kg/d of oral L- or D-arginine on the L-arginine metabolic pathways in male Sprague-Dawley rats. PONE-D-23-10457R1 Dear Dr. Desai, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Kanhaiya Singh, Ph.D 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: Yes ****** 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.0289476.r003
Formally Accepted
24 Jul 2023 Acceptance Letter - Kanhaiya Singh, Editor PONE-D-23-10457R1 The effects of a comparatively higher dose of 1000 mg/kg/d of oral L- or D-arginine on the L-arginine metabolic pathways in male Sprague-Dawley rats Dear Dr. Desai: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Kanhaiya Singh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0289476.r004

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