PONE-D-20-13546

High CRP, AST, CK, NT-proBNP and leukocytosis after the Norseman Extreme Triathlon (NXTRI)

PLOS ONE

Dear Dr. Nyborg,

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 consider carefully all coments form the reviewers. It seems that ther is a general lck of proper references along the manuscript as well as spelling or grammatical mistakes. Furthermore, more details regarding methodology are required.

Please submit your revised manuscript by Jul 23 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Pedro Tauler, Ph.D.

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 the following in the Competing Interests section:

'The authors have declared that no competing interests exist.' 

We note that one or more of the authors are employed by a commercial company: Aker BioMarine Antarctic AS.

1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. 

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

Additional Editor Comments (if provided):

[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: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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: Yes

Reviewer #3: Yes

**********

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: No

Reviewer #2: No

Reviewer #3: 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: General comment

This is an interesting study on a popular sport, where the authors use correctly scientific methods. I would be happy to recommend it for publication once the authors address a few issues. My major concern is that the English and writing style must be improved throughout the text to meet the standards of PLOS ONE as in its current form some parts are hard to be understood.

Specific comments

1. Title: Adopt a more ‘neutral’ title, e.g. ‘the effect of … on…’ and delete (NXTRI)

2. l.19-24: Reduce this part in 2-3 lines.

3. l.25-30: Increase the information on participants and decrease the information on biomarkers (they are reported in l.31-40)

4. l.31-40: This part must be rewritten as it is hard to follow.

5. l.41: Add a conclusion in 2-3 lines with a practical perspective.

6. l.44: Major literature is missing (DOI: 10.4077/CJP.2016.BAE420).

7. l.49: Changes meters to m.

8. l.45-49: Add references to support the provided information.

9. l.50: The term ‘extreme exercise’ -here and throughout the text- must be revised as it is not precise. Is it prolonged? High-intensity? Extreme cold? Cold water? Large elevations? Eg the ‘prolonged strenuous’ used in l.59 is more precise.

10. l.50-58: Add 3-4 references.

11. l.61: Revise ‘extreme triathlon’.

12. l.61: The gap of the existed literature has not been identified correctly. It needs 3-4 sentences (with references) presenting what is known and what is missing.

13. l.67: Add hypotheses.

14. Introduction: Make this part more specific to the aims to the paper. All biomarkers presented in the methods and results must be introduced here.

15. l.72: When did they receive the email?

16. l.79: …noon of…

17. l.95-96: Delete references 6-13.

18. l.107: Delete ‘significant… 0.001’. Report all p values at 3 decimals, e.g. p=0.364, p<0.001…

19. l.108: Add effect size.

20. l.297: Add a paragraph showing limitations, strength and practical applications.

21. l. 297: Major literature is missing in the discussion, too (DOI 10.3389/fphys.2017.00638; 10.3389/fphys.2018.01959)

Reviewer #2: GENERAL

Thank you for sending me this interesting manuscript in which the authors have described changes in various biomarkers in response to the Norseman Extreme Triathlon. The aim of the study is to elucidate typical changes in these biomarkers, which might assist in risk stratification. The study is simple, with a straight-forward protocol. It provides useful data for Race Directors and medics who might be overseeing the event. I have no major issues with study or analyses, but more in the general write-up and reporting of Methods and Results. Important details are missing from the Methods, and sections need clarifying to help with interpreting the Results. For example, no data has been provided on the precise timescale of post-race blood samples, or in environmental conditions between Norseman and Oslo Olympic. These omissions undermine your interesting results. There are also numerous errors in language and in reporting of Results which may obscure the translation of the data. The study may deserve publication, but the manuscript needs to be considerably revised. Please see Specific comments.

SPECIFIC

Abstract

◦ Line 19-20. “Little is known about the physiological impact of such events on the human body”. I disagree with the statement; there are hundreds of studies looking at physiological responses to ultra- and extreme-endurance competition. Perhaps you could say ‘physiological impact of extreme triathlon’?

◦ Line 21. It seemed to me, from the Introduction and Discussion, that the main aim was to characterize the biomarker response, and that comparison to Olympic was a secondary aim for context. Consider re-ordering the abstract to reflect the primary and secondary aims.

◦ Line 24. Use of the word ‘injured’ here might not be appropriate; implies musculoskeletal injury.

◦ Line 25. Intravenous samples? Many of these biomarkers can be assessed with pin-prick capillary.

◦ Line 31. “Increased levels of clinical significance”. Just say ‘significant increases’.

◦ Line 33. “Clinically significant changes”. Changes could be increase or decrease, be specific.

◦ See my comment below in Results; the post-race absolute values need context. The absolute vealues presented here don't mean anything. Why not express the Results as percentage increase from baseline? This way you provide context against baseline values.

◦ Abstract needs a concluding statement. How might the data be used?

Keywords. These contain several misspelled words, e.g., ‘Extreeme’ and ‘Thriathlon’. Also, keywords are generally those not included in the title. Reconsider.

Title. The title is not descriptive, and also contains technical abbreviations. Please amend the title congruent with PLOS ONE submission guidelines (https://journals.plos.org/plosone/s/submission-guidelines). Titles should be “Specific, descriptive, concise, and comprehensible to readers outside the field. Avoid specialist abbreviations if possible”. This should have been highlighted by the journal before being sent for review.

Introduction

◦ Line 45. Given that the citations are non-academic resources, please caveat that Norseman has been rated by commercial/media/popular outlets as one of the world’s toughest triathlons.

◦ Line 46. Please use SI units for meters (m), here and throughout.

◦ Line 51. Please change ‘world’ to possessive world’s.

◦ Line 61. Others have already evaluated biomarkers in response to Ironman (Danielsson et al - PMID: 28609447; Carlsson et al - PMID: 27483401; Neubauer et al - PMID: 18548269). Why haven’t these studies been mentioned here?

Line 61. I think much of the rationale for the study is based on the idea that Norseman is in a different category of triathlon. For your paper to be unique, please expand upon why Norseman is more extreme than ‘normal’ Ironman distance tri.

Methods

◦ Line 71. It would serve you to mention that Norseman and Oslo Olympic were both contested in August (i.e., similar anticipated weather); I had to look this up as the data weren’t provided. What time of day were the starts? Do you have any data on the weather conditions during both races? This would support your argument that the races were comparable in all but distance/terrain, and would lend validity to your comparisons.

◦ Line 79. Please state an approximate timescale for the first post-race blood sample; i.e., ‘within 5 min of race completion’ or ‘within 1 h of race completion’.

◦ Line 79. Why ‘noon’ the following day? Surely you didn’t collect >100 samples at noon? Be specific. How many hours on average after the finish were samples collected? Include means and STDEV. When assessing biomarkers, and in a study designed to elucidate the time-course of recovery, these details are paramount.

◦ Blood Sampling. For clarity, I would recommend integrating the first and last paragraphs (the protocol) and then following with the second paragraph (the measures). Also, I’m assuming samples were taken from an antecubital vein, but please state in the text.

◦ Line 104. No problem with the stats, but I’m confused why you’ve used Bonferroni post-hocs only on AST, CRP, and CK. Did you not assess each variable at three timepoints at both events? Sorry if I’m missing something.

◦ Line 104. I think the stats section needs a little clarity on the ‘why’. Be specific with your reasons for performing a given test, spell it out. For example, ‘to assess biomarkers among the three time-points (pre-, post-, recovery), a Wilcoxon Signed Rank Test…’. Moreover, line 105 is the first mention of correlations. State why you’re doing these: ‘to assess for associations among the variables, a Spearman’s Rank…’. Assessing associations should be in the aims.

◦ Line 104. Please capitalize ‘Spearman’s’.

◦ Line 107. To avoid repetition, consider replacing the first sentence by saying that ‘critical alpha level was set as 0.05’.

Results

◦ Line 112. Please use ‘before, after, +24h’, or ‘pre, post, post-day’, or some equivalent, but avoid mixing your time-points as you’ve done here ‘pre, after, post-day’. There needs to be consistency here for clarity. In the tables you’ve used ‘before, finish, after’. Even this is vague. What about ‘Start, Finish, +24’?

◦ Line 114. These things happen. Is this necessary information, though? i.e., do you anticipate there being differences among the years? If not, consider removing this statement.

◦ Line 116. Please clarify what is meant by ‘in some instances, single vials were discarded’. For what reason?

◦ Line 117. ‘There are differences in the total number of analyses…’. This line seems to contradict line 112 where you mentioned that ‘113 subjects were included for analysis with full sets of samples’. I think what you mean is that 113 subjects provided blood samples start, finish, and +24, but that not every measure was assessed in each participant. Again, be specific. Why were all measures not available from all subjects?

◦ Table 1. I’m sure this will be adjusted in the journal formatting, but title should always be above tables and below figures.

◦ Table1. Why provide height to 2 decimal places, but performance times and ages to none?

◦ Table 1. Consider changing ‘weight’ to ‘mass’. Also, provide mass to 1 d.p.

◦ Table 1. Age SI unit is ‘y’

◦ Interesting in itself that males / females had comparable swim times.

◦ Line 124. WBC increased to 14.2 *109/L after the race. This is different to what’s reported in Table 2. What am I missing here?

◦ Results. The post-race absolute values are meaningless if not given in the context of the baseline values. E.g., CK up at 2450 U/L doesn’t tell me about the magnitude of the increase. Seeing as you provide the raw data in Table 2, why not express the Results as percentage increase from baseline? This way you are providing something different to what I can find myself in the table, but it also provides context against baseline values.

◦ Line 139. The correlations; are these the R values? If so, please clarify in the text.

◦ Line 140. ‘Between’ variables implies only two. ‘Among’ variables means more than two.

◦ Line 154. Non-significant correlations aren’t technically correlations.

◦ Table 2. Caption, please use ‘Wilcoxon’ instead on Wilcox.

◦ Fig 2. I appreciate your desire to show individual values, but with such a large sample there is far too much data on this graph. It is difficult to read. Either use a light grey line for individual data and a thick dark line for means, or just display the means and SD/IQ.

◦ Fig 2. Given the female sample is 1/3 of the male, do you expect there to be meaningful differences among the variables? Also, is a comparison between sexes an aim of the study? If not, why present the data as two cohorts on the graphs?

◦ Fig 1/correlations. I’ve not seen correlations shown this way before. I think it’s an interesting and unique way of showing the data. Does it really add to what’s been written in the text? At your discretion.

Discussion

◦ I would like to see some more comparisons of your data against values reported in other Ironman distance triathlons. Also, there is very little mention of the factors that make Norseman so unique; e.g., the cold-water temperatures, the tough ascents/descents (presumably greater muscle damage with downhill components in running). How might these factors influence your measured variables. This is important to distinguish your study from others looking at ‘normal’ Ironman.

◦ Line 200. ’Measurements of WBC show that extreme exercise is related leukocytosis’; please check, is the word ‘to’ missing here?

◦ Line 205. Needs a citation.

◦ Line 209. You have switched from US to UK spelling of leukocyte/leucocyte. Be consistent.

◦ Line 221. Could the difference be due to the relatively greater stress in ultra-marathon due to impact forces?

◦ Line 223. Do you mean that IL6 production elevates CRP, or is CRP produced by CRP in a positive feedback mechanism?

◦ Line 250. The 24 h race you cite comprised loops of a flat course, so I could believe that exercise intensity and/or strain was greater in Norseman, but I’m not sure about this for many/most 24-hour races performed on trails, mountains, in heat, etc. Could other conditions like the cold water have influenced the values?

◦ Line 263. Swap the word ‘thesis’ for ‘notion’ or ‘idea’.

◦ Line 289-290. Possibly the result of slower races times and, therefore, lower intensity? Although I see you found no correlation here between race time and BMI, which is unusual.

Reviewer #3: Line 22: the aim of the study was

Line 26: explain that n=98 was for 3 years

Lines 45-49: I suggest adding references such as Chin J Physiol. 2016 Oct 31;59(5):276-283. doi: 10.4077/CJP.2016.BAE420 or Springerplus. 2015 Sep 2;4:469. doi: 10.1186/s40064-015-1255-5

Line 50: add a reference

Lines 52-53: add a reference

Lines 60-61: add a reference

Line 61: the aim of the study was

Line 67: what is the hypothesis of your study?

Line 69: what were the criteria of inclusion/exclusion to the study?

Line 200: is related to

Lines 204-205: add a reference

Lines 206-207: add a reference

Lines 207-208: add a reference

Lines 220-221: add a reference

Lines 221-222: add a reference

Lines 222-223: add a reference

Line 232: add a reference

Lines 232-233: add a reference

Lines 250-252: add a reference

Lines 252-253: add a reference

Line 261: was not related to

Line 261-262: add a reference

Lines 262-265: add a reference

Lines 265-266: add a reference

Line 269: add a reference

Lines 269-270: add a reference

Lines 270-271: add a reference

Lines 271-271: add a reference

Lines 284-285: incomplete sentence

Line 289: showed a negative

Line 289: indicating that a higher

Lines 293-295: add a reference

Lines 295-296: add a reference

**********

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

Reviewer #3: 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.

Biochemical markers after the Norseman Extreme Triathlon

PONE-D-20-13546R1

Dear Dr. Nyborg,

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,

Pedro Tauler, 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

Reviewer #3: 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

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: 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

Reviewer #3: 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

Reviewer #3: 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 commentno commentno commentno commentno commentno commentno commentno commentno commentno comment

Reviewer #3: The authors have addresssed all my comments and improved the manuscript accordingly which is now ready to be accepted for publication

**********

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

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

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

Reviewer #1: No

Reviewer #3: No