PONE-D-20-21640

Plasma trimethylamine N-oxide and its metabolic precursors and risk of mortality, cardiovascular and renal disease in individuals with type 2-diabetes and albuminuria

PLOS ONE

Dear Dr. Winther,

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PLOS ONE

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PR reports having given lectures for Astra Zeneca, Bayer, Novo Nordisk and Boehringer Ingelheim, and has served as a consultant for AbbVie, Astra Zeneca, Bayer, Eli Lilly, Boehringer Ingelheim, Astellas, Gilead, Mundipharma, Vifor, and Novo Nordisk, all fees given to Steno Diabetes Center Copenhagen. SAW, TWH, BJvS, TSA and PR own stocks in Novo Nordisk A/S and TSA in Zealand Pharma A/S. ZW and SLH are named as co-inventors on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics, and have the right to receive royalty payment for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland Heart Lab, Quest Diagnostics and Proctor & Gamble. SLH also reports having been paid as a consultant from Proctor & Gamble, and having received research funds from Proctor & Gamble and Roche. The other authors report no conflicts of interest.  "

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

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: Partly

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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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

Reviewer #3: No

Reviewer #4: Yes

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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

Reviewer #3: Yes

Reviewer #4: Yes

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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 study investigators look at possible associations between plasma levels of trimethylamine-N-oxide and its metabolic precursors, and CVD and renal disease in patients with type 2 diabetic and albuminuria. This metabolic pathway and the role its metabolites may play in disease is still relatively underexplored. The outcomes of the studies that have been published appear to be relatively inconclusive and controversial. This study uses long term data from a reasonably large patient cohort to come to the conclusion that choline, carnitine and the weighted sum of the TMAO pathway metabolites may be risk markers for deteriorating renal function. They also show that only a higher weighted sum score of TMAO pathway metabolites shows an association with increased risk of CV mortality while individual metabolites alone do not. The manuscript is well written and data analyses appear to be well executed. The authors’ conclusions are reasonable and supported by the data.

Minor comments

1. The sentence on line 198 should be rephrased. Perhaps “In addition, at baseline individuals with higher TMAO………”

2. Line 208 onwards. Last sentence in particular is difficult to follow. “During follow-up a total of 106 patients died. Of 116 patients that suffered CVD events, 44 of these events were fatal.” Perhaps?

3. Line 289. “ Consistent with our study……".

Reviewer #2: I have to commend the authors on a well documented and high impact research. The problem statement was complimented with a focussed introduction and provided the reader with sufficient background leading to the motivation and rationale for the current study. A large cohort of patients that participated in 2 clinical trials were recruited for this study. Four plasma metabolites that are linked to the TMAO pathway was investigated with respect to their risk with all-cause mortality, CVD and deterioration in n=311 renal function in individuals with type 2-diabetes (T2D) and albuminuria.

Appropriate and sensitive measurements were performed to quantify the variables.

I would like to know what the inclusion criteria was for this study? This is not clearly described.

All other aspects of the manuscript is in my opinion flawless. I was curious as to why cystatin C was not estimated in this study? Not much is reported on this but I was just curious.

I really enjoyed the variety of figures and tables that clearly indicated the contribution of the biological markers toward risk of mortality,cardiovascular and renal disease.

Reviewer #3: Here is a list of specific comments. Note: line and page numbering in reviews and comments is based on ruler applied in Editorial Manager-generated PDF.

1. Page 7, line 141: In the Endpoints section, please make sure all four endpoints, all-cause mortality, CV mortality, CVD and eGFR decline >=30%, for survival analyses were properly introduced.

2. Page 7, lines 148–149: “Censored” might not be an appropriate word here. I suggest writing ‘individuals experiencing multiple events stopped their follow up at time of the first event in the analyses’.

3. Page 8, lines 165–166: Please clarify “analyses” referred to ‘correlation and regression analyses’, not including summary statistics in Table 1.

4. Page 8, line 166: For the non-normal continuous variables (those reported in median and IQR), please use a non-parametric version of analysis of variance (e.g., Kruskal-Wallis analysis of variance).

5. Page 8, lines 170–172: For all-cause mortality, Cox proportional hazards models were appropriate. For CV mortality, CVD and eGFR decline >=30%, please describe whether or not there were competing events for each endpoint. If competing events existed, the models in Table 2 were technically called cause-specific hazards models (by censoring the competing events). You do not have to change the analyses in Table 2, but, for each endpoint, clarify the presence/absence of competing events and the use of cause-specific hazards models when the competing events present.

6. Page 8, lines 173–174: Please clarify if these variables were the candidates of the forward selection approach or the results of the selection. Table 2 suggested the latter. For the latter, how many and what variables did you include at the beginning of the forward selection? Different endpoints should result in different sets of selections. How did you determine the final adjusted covariates for all endpoints? Please specify responses to the above questions in this paragraph. Please comment on why treatments and cohorts were not included; I would suggest including them even if they did not pass the inclusion criteria of the forward selection approach.

7. Page 8, line 179: Because of the competing events and the use of cause-specific hazards models (see Comment #5 above), I suggest replacing “Cox regression model” with ‘proportional hazards models’ such that both Cox and cause-specific hazards models are included. Apply this accordingly for the rest of the manuscript.

8. Page 9, line 203: Please confirm the correlation of 0.33 were r or r-squared. The Statistics section described the use of Pearson correlation. I assumed it was r, not r-squared.

9. Page 9, line 203: I suggest including p-values in S1 Fig.

10. Page 10, line 220: Please include this information in Table 2.

11. Page 11, line 226: I suggest combining eGFR decline >=30% with S1 Table into Table 3. If it exceeds the limit of tables/figures, I would move Fig. 1 to S2 Fig.

12. Table 1: I suggest including cohorts and the weighted metabolite sum score in the table.

13. Table 2: For each endpoint, if the sample size was not 311, please specify in the table.

Reviewer #4: Review on the manuscript entitled: “Plasma trimethylamine N-oxide and its metabolic precursors and risk of mortality, cardiovascular and renal disease in individuals with type 2-diabetes and albuminuria” by Winther SA et al

The manuscript’s aim is to investigate the associations between four plasma metabolites in the TMAO pathway and risk of all-cause mortality, cardiovascular disease, and deterioration in renal function in individuals with type 2 diabetes and albuminuria. Levels of plasma metabolites were measured at baseline in individuals with type 2 diabetes, while other information was obtained from registries. Associations were analysed using Cox models. The results showed that in type 2 diabetes and albuminuria, higher choline, carnitine, and a weighted sum of four metabolites from the TMAO pathway were risk markers for deterioration in renal function in the follow up.

This is a beautifully written manuscript with all aspects very well clarified. I find it easy to follow from the title, the abstract, and the rest of the article. The authors did address all their findings, and also highlighting the limitations of the study accordingly.

Something minor I noticed is that in their subjects 77% were females. Was wondering if perhaps there were any variations in the TMAO pathway with gender of the patients?

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Reviewer #1: Yes: Eugene du Toit

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Plasma trimethylamine N-oxide and its metabolic precursors and risk of mortality, cardiovascular and renal disease in individuals with type 2-diabetes and albuminuria

PONE-D-20-21640R1

Dear Dr. Winther,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

M. Faadiel Essop

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 #3: All comments have been addressed

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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 #3: Yes

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

Reviewer #3: Yes

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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 #3: (No Response)

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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 #3: Yes

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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 #3: (No Response)

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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 #3: No