PONE-D-20-39903

Profiling Immuno-Metabolic Mediators of Vitamin B12 Deficiency Among Metformin-Treated type II Diabetes Patients in Ghana

PLOS ONE

Dear Dr. Sakyi

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 you can see from the Reviewers' comments below, the principal areas of concern are in the study design and the applicability of the Fedosov methodology

The Reviewers' would want you to include a comparator group consisting of either patients without a diagnosis of type 2 diabetes, or patients with type 2 diabetes but were not treated with metformin.

There is also the need to exclude the diagnosis of type 1 diabetes in the subjects because of high prevalence of other autoimmune conditions in your cohort.

Please submit your revised manuscript by March 13, 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

We look forward to receiving your revised manuscript.

Kind regards,

Benjamin Udoka Nwosu, 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

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2. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.

In your Methods section, please provide a justification for the sample size used in your study, including any relevant power calculations (if applicable).

3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section.

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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: Yes

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

Reviewer #1: Yes

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: Yes

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: The study addresses an important problem of B12 deficiency associated with metformin use. However, there are many, well conducted prior studies (close to 200) across the globe which have conclusively demonstrated this association. This study is from Ghana, and the authors acknowledge another study from Ghana that demonstrated similar conclusions (This reviewer could not retrieve the citation for this study on Pubmed as referenced by Yakubu at al which showed a lower prevalence of B12 deficiency).

There are several limitations to the study:

1. It is a relatively small study (n=200) and there is no control group.

2. There is a very high prevalence of Vitamin B12 deficiency (40.5%) reported, but in the absence of any control (for example, age/gender matched individuals not on Metformin) it is difficult to attribute this high prevalence only to metformin use. There are numerous other prior studies that are larger, with appropriate controls, which show lower prevalence.

3. In the absence of a control group, a before treatment B12 compared to B12 after treatment with Metformin would provide useful data. This was not done as this is an observational study.

4. The study uses a relatively little used and poorly validated computational model (Fedosov combined marker) to diagnose Vitamin B12 deficiency. Low B12 levels alone are a sensitive indicator of deficiency ( up to 95% sensitive depending on cutoff) (NEJM, 2013. 368:149-60) . In those with borderline low Vitamin B12 (200-300 pg/ml), an elevation of MMA and Homocysteine add to the sensitivity, but both need to be elevated as homocysteine alone can be high in folate deficiency. MMA elevations may not be specific as other conditions including renal disease can cause elevations. Therefore these should be used in situations where Vitamin B12 is borderline low. Holotranscobalmin is neither specific or sensitive but can be useful in pregnancy. There is little data on validity of Fedosov model (Reference 18 in current article) which has been cited only 15 times since its publication in 2013. Reading the original paper reveals this is a theoretical construct which is validated against cognitive impairment in the elderly and hematological impairments.

5. The data correlating B12 deficiency with neurological impairments (two clinical measures of peripheral neuropathy) and immunological parameters does not provide any new information

6. This study shows rates of Vitamin B12 deficiency (40.5%) which is far higher than almost all prior studies, but the non-standard measure (Fedosov wellness quotient) may be the reason for this discrepant finding.

7. Since the average B12 in the group, on page 14, line 287 is reported as 142 pmol/L (which is below the threshold for severe Vit B12 deficiency 148 pmol/L or 200 pg/ml), it would be informative to know what was the percentage of B12 deficiency using just the threshold of 148 pmol/L which is very specific cutoff

8. The study needs to emphasize what new knowledge it adds to the topic of B12 deficiency in Metformin treated patients.

Minor Problems:

Labelling/legends in Figure 1 need to be clearer

Reviewer #2: In the manuscript “Profiling Immuno-Metabolic Mediators of Vitamin B12 Deficiency Among Metformin-Treated type II Diabetes Patients in Ghana” Sakyi et al investigated the immuno-biochemical mediators of vitamin B12 deficiency using Fedosov age normalized wellness quotient. Fedosov proposed the utilization of combined indicator of vitamin B12 status using two or more of the following markers: total serum B12, methylmalonic acid, holo-transcobalamin, and total homocysteine. Comparison of immuno-metabolic parameters based on vitamin B12 status was performed using one-way analysis of covariance (ANCOVA) with adjustment for age, sex, BMI, duration of diabetes, and dosage and duration metformin therapy. The authors report that the prevalence of metformin induced vitamin B12 deficiency was 40.5% whilst the prevalence of diabetic neuropathy was > 67%. Also, Vitamin B12 deficient patients had higher levels of intrinsic factor, gastric parietal cell antibody, TNF-α, total cholesterol and LDL-cholesterol. They conclude that prevalence of Vitamin B12 deficiency and diabetic neuropathy are high among metformin-treated patients with type 2 diabetes and suggest that routine measurement of Vitamin B12 should be included in the management of patients with type 2 diabetes treated with metformin.

Comments:

Strength: a reasonably elaborate study on treatment with metformin and Vitamin B12 deficiency

Major

1. It would be appropriate to clarify how the diagnosis of type 2 diabetes was made. The presence of gastric parietal cell antibody makes it imperative to exclude type 1 diabetes

2. A comparator group such as patients without diabetes or patients with type 2 diabetes but who were not treated with metformin would be required to justify the conclusion that the prevalence of Vitamin B12 deficiency and peripheral neuropathy are high in patients treated with metformin in Ghana. Could there be the possibility that B12 deficiency may be high in the general population.

3. Is there any correlation between the dose/duration of treatment with metformin and Vitamin B12 deficiency/peripheral neuropathy?

4. 2017 ADA guidelines recommended periodic monitoring of Vitamin B12 in patients treated with metformin over a long period of time, suggesting routine measurement in all patients treated with metformin may not be cost effective.

Minor

1. Type II diabetes should read Type 2 diabetes

2. “on metformin” is better rendered as “treated with metformin” throughout the manuscript

3. Figure 4 appears confusing, a table might be easier to understand

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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: Samir Malkani

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.

PONE-D-20-39903R1

Profiling Immuno-Metabolic Mediators of Vitamin B12 Deficiency Among Metformin-Treated type II Diabetes Patients in Ghana

PLOS ONE

Dear Dr. Sakyi,

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 provide a detailed discussion of the limitation of your study with respect to the lack of a comparator group. This is critical. Your current two-sentence response is not sufficient: 'Another limitation of this study is the relatively low sample size and the cross-sectional nature of the design. Further case-control and prospective studies are warranted.'

You have to discuss the concerns about his lack of comparator group, and what your group should have done in terms of including a control group of healthy subjects, or a group of patients with diabetes but were not receiving metformin.

Please submit your revised manuscript by March 15th, 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

We look forward to receiving your revised manuscript.

Kind regards,

Benjamin Udoka Nwosu, MD

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.

Additional Editor Comments (if provided):

Please provide a detailed discussion of the limitation of your study with respect to the lack of a comparator group. This is critical. Your current one sentence response, is not sufficient: 'Another limitation of this study is the relatively low sample size and the cross-sectional nature of the design. Further case-control and prospective studies are warranted.'

You have to discuss the concerns about his lack of comparator group, and what your group should have done in terms of including a control group of healthy subjects, or a group of patients with diabetes but were not receiving metformin.

[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 #2: (No Response)

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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 #2: Yes

**********

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

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 #2: 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 #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 #2: "It is imperative Ghana include periodic measurement of Vitamin B12 deficiency using the more sensitive combined indicators (4cB 12 ), in the management of T2DM patients treated with metformin" in concluding sentence of the abstract should read "if the findings of this study are verified in a prospective case-control study, it may be beneficial to include periodic measurement of Vitamin B12 using the more sensitive combined indicators (4cB 12 ) in the management of patients with T2DM treated with metformin in Ghana.

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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 #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.

Profiling Immuno-Metabolic Mediators of Vitamin B12 Deficiency Among Metformin-Treated type II Diabetes Patients in Ghana

PONE-D-20-39903R2

Dear Dr. Sakyi,

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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Kind regards,

Benjamin Udoka Nwosu, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

My comments have been addressed.

Reviewers' comments: