PONE-D-20-22349

Effects of water stably-enriched with oxygen as a novel method of tissue oxygenation on mitochondrial function, and as adjuvant therapy for type 2 diabetes in a randomized placebo-controlled trial

PLOS ONE

Dear Dr. Khoo,

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 address each of the reviewers comments.

Please submit your revised manuscript by 12/15/2020. 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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We look forward to receiving your revised manuscript.

Kind regards,

Gordon Fisher

Academic Editor

PLOS ONE

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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: No

Reviewer #2: No

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

Reviewer #1: No

Reviewer #2: 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: No

Reviewer #2: 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

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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: This paper looks at a study in rats and a randomised controlled trial of ELO vs tap water in individuals with diabetes. At present the design characteristics of the randomised trial are not given - why was 150 participatns chosen; how was randomisation performed etc.

Please give exact p-values throughout and where t-tests etc have been performed give the difference and 95% CI. The dynamite plots currently give less information than a table with the actual data. Numbers are sufficiently small to give actual data points anyway - so the dynamite plots obfuscate the data.

The analyses in Table 1 are wrong. It is entirely incorrect to analyse within group - the between group differences and the p-values should be given and reported. Given the dropouts how many patients were analysed at each follow-up timepoint - was there any imputation of missing data?

Please remove the subgroup analysis unless a proper test for interaction demonstrates heterogeneity.

Reviewer #2: Khoo and colleagues have provided a nicely-written manuscript describing their study examining the effect of oxygen-enriched bottled drinking water (ELO) on metabolic endpoints in HepG2 cells, and also the results of a pilot study of the effects of ELO on HbA1c and other metabolic parameters in a cohort of T2 diabetes patients. Improvements in arterial blood oxygenation have, in some previous reports, provided evidence of improvements in metabolic efficiency and glycemic control, so there is some justification for using ELO in this manner. There are a number of major limitations to the study in its current form which substantially diminish its impact and significance to the field, however. Some of these are described below.

1) The cell model chosen for the in vitro study is not appropriate. HepG2 cells are immortalized and metabolically are not at all similar to primary hepatocytes, and great care must be taken with these cells to make sure they are phenotypically similar to primary heps. There is no mention of these steps at all by the authors, and in any case, the rationale for using hepatocytes is weak, since these cells do not make sense to study in the context of T2 diabetes. Authors should choose consider using myocytes, adipocytes, or even pancreatic beta cells as a model, to determine if ELO has an impact on the metabolism of these cells. And this leads to the next comment,

2) The increases seen in mitochondrial O2 concentration do not seem to be exclusive to ‘proton leak’, but rather all across the board, and this strongly suggests that ELO has simply increased mitochondrial content in these cells, which is not particularly surprising, but somewhat interesting. Authors need to provide evidence for changes in mitochondrial content in these cells.

3) If the authors wish to make the argument that ELO alters redox environment or changes levels of oxidative stress in the body, or in the cells, then they need to provide evidence of this in their study. No markers or readouts of ROS or antioxidants were performed here at all.

4) Why did the authors not examine the effect of ELO more thoroughly in the rat model, specifically in a rat model of T2 diabetes? There are many options available, genetic- or diet-induced obese rat models, that are widely used. This would allow for a much more thorough interrogation of the effect of ELO.

5) The drop in HbA1c seen in the T2 diabetes patients with ELO water is somewhat interesting, but would be hard to argue that a drop from 8.9 to 8.6 is clinically meaningful.

6) It is disappointing that the authors did not measure fasting serum insulin levels in their pilot study, as this would have allowed them to determine if ELO has altered insulin sensitivity in the patients through the HOMA-IR index.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-20-22349R1

Effects of water stably-enriched with oxygen as a novel method of tissue oxygenation on mitochondrial function, and as adjuvant therapy for type 2 diabetes in a randomized placebo-controlled trial

PLOS ONE

Dear Dr. Khoo,

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 address the comments by Reviewer 1

Please submit your revised manuscript by June 15, 2021. 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. 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,

Gordon Fisher

Academic Editor

PLOS ONE

Journal Requirements:

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

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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: (No Response)

Reviewer #2: 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 #1: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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 #2: Yes

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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 #1: Yes

Reviewer #2: 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: Thank you for your responses to my previous comments. I have a few areas where the responses are not clear:

I understand the approach to build the trial based upon a standardised effect size - but such effect sizes are not necessarily the clinically relevant difference which is on the absolute scale. What absolute difference does d=0.5 correspond to, and why is this an appropriate choice?

It is well known (see the papers dating from the mid-1990s onwards) that envelope randomisation is prone to issues with allocation foreknowledge (indeed the classic case given by Altman in his textbook is an envelope randomisation). What steps were taken to ensure that envelopes were opened in sequence only after enrollment?

LOCF can be anti-conservative under certain circumstances - are results robust to method of imputing missing data?

Reviewer #2: The additional mitochondrial content measurements are informative about the potential for ELO to rescue mitochondrial content loss with hyperglycemia.

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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 #1: No

Reviewer #2: Yes: Ethan Anderson

[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.

Effects of water stably-enriched with oxygen as a novel method of tissue oxygenation on mitochondrial function, and as adjuvant therapy for type 2 diabetes in a randomized placebo-controlled trial

PONE-D-20-22349R2

Dear Dr. Khoo,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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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,

Gordon Fisher

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: