Comparative Bioavailability of Synthetic B12 and Dietary Vitamin B12 Present in Cow and Buffalo Milk: A Prospective Study in Lactovegetarian Indians

Round  1

Reviewer 1 Report

This is an interesting paper showing the effectiveness of milk intake in lactovegetarians of low B12 status. However, my questions describe as      below.                                                                                               

In this study, buffalo and cow milk were used, but there is not much description about the difference in composition etc. Please describe the difference and the expected impact in the study.

There is no description about how to procure the milk used for the study. Commercial products ?

Regarding the sterilization of milk, it is thought that if the sterilization time is too long, the amount of B12 will decrease. Please describe the boiling time and method.

Drinking 400 mL of milk may result in excessive calories? Blood markers (triglycerides) after 4 weeks?

What is the intake rate? 100%?

Author Response

Thank you so much for the review and suggestions. We have incorporated the suggestions into the manuscript.                                                                                                

1. In this study, buffalo and cow milk were used, but there is not much description about the difference in composition etc. Please describe the difference and the expected impact in the study.

Response: To our knowledge, hitherto the whereabouts of B12 in Indian cow and buffalo milk has not been reported. However, we have recently undertaken a study comparing the two types of milk available on the Indian market. Besides holding the same amount of endogenous B12, our preliminary data suggest that all B12 in both cow and buffalo milk is bound to the specific B12 binding proteins, transcobalamin (TC) and haptocorin (HC), respectively. As the B12 distribution of B12 between TC and HC is similar, we do not anticipate the bioavailability differs significantly in the two milk types, as supported by the present data. It is our hope in the nearest future to be able to publish a comparative analysis of B12 in milk collected from different species of buffalo and cow.

 2. There is no description about how to procure the milk used for the study. Commercial products?

Response: 60-70% of Pune population buys milk (Cow/buffalo) from M/S Chitale Bandhu dairy. Deenanath Mangeshkar Hospital and M/S Chitale Bandhu dairy firm entered into a M.O.A. stating that Chitale Bandhu will deliver ( 2 X 200 mL x 25) cow milk for 25 participants and 2 x 200 mL x 25) of buffalo milk for other 25 participants) to Deenanath Mangeshkar Hospital and Research Center, Pune. Fifty milk packets each were delivered every morning. Each participant was given two packets to be consumed, one at night and the other next day morning. They poured the milk in a vessel and heated till boiling and cooled immediately and then drank. This procedure continued for 4 weeks.

Milk vitamin B12 was measured on 5 occasions as mentioned in the text during the study period. There was no significant difference between cow and buffalo milk. Table S2 shows the milk B12 concentrations at various time periods.

3. Regarding the sterilization of milk, it is thought that if the sterilization time is too long, the amount of B12 will decrease. Please describe the boiling time and method.

Response: Vitamin B12 resists breakdown even at boiling point of water for several hours. The loss of B12 previously noticed in boiled milk (e.g. Watanabe et al., J. Agric. Food Chem. 1998, 46, 206−210) is likely not due to breakdown of B12, but a result of the centrifugation step usually included before the supernatant is used as the sample for the B12 assay. The centrifugation precipitate denatured protein aggregates, pulling with them a fraction of the endogenous B12. In our settings, the milk was poured into a vessel and heated until boiling only. It was cooled and then consumed. Accordingly, we are content that the oral dose of bioavailable B12 is the same in the capsules and the milk.

4. Drinking 400 mL of milk may result in excessive calories? Blood markers (triglycerides) after 4 weeks?

Response: We measured plasma cholesterol, triglycerides and HDL-Cholesterol at base-line and then at 4 weeks. It is mentioned in the text. Four weeks of milk supplementation, both cow and buffalo, did not show any significant difference.

5.  What is the intake rate? 100%?

Response: Each participant was contacted to confirm capsule/milk intake every day in the morning and evening through Whatsapp..No further measures were taken

   Thank you

Author Response File: Author Response.docx

Reviewer 2 Report

In this longitudinal cohort intervention study the Researchers investigate the effects, on biomarkers of B12 status, of supplementation with equivalent amounts of B12, administered during four weeks in either vitamin capsules (CN–B12), or cow milk (dietary HO-B12), or buffalo milk (dietary HO–B12).

From the methodological point of view the study is adequately appropriate and suitable to scientific rigour and ethical principles. The study design process is valid, both inclusion and exclusion criteria are compliant, as well as data collection; nevertheless the duration of the study is short (4 months), the number of participants is small (68 lactovegetarian Indian individuals) and the study has no control group (taking placebo). These aspects could represent some limits about significativity and scientific relevance of the study.

However, on the other hand, the statistical analysis seems valid; purposes and scientific contents are supported by valid References; editorial structure, figure and tables are good. The results are clear and the “Discussion” section is well structured and interesting.

In summary, the Authors conclude that two servings of vitamin capsules with CN–B12, cow milk or buffalo milk (containing mostly HO–B12) are equally efficient in improving biomarkers of B12 deficiency, but the supplemented dose of 1.5 μg per day (used in this study) is insufficient to reach a fast replenishment of B12 stores.

But then, how much of B12 daily supplement should take Indian lactovegetarians? This could be a question of a common Reader and I think this is the main goal of the work. I would expect stronger focus on this question by the Authors.

Author Response

 Thank you very much for reviewing the article. The response to the comment is  as follows:

 But then, how much of B12 daily supplement should take Indian lactovegetarians? This could be a question of a common Reader and I think this is the main goal of the work. I would expect stronger focus on this question by the Authors.

 Response: We demonstrated in our earlier study that daily oral treatment for 8 weeks with 3 ug CN-B12 or HO-B12 caused an increase in plasma total vitamin B12 and holotranscobalamin plus minor changes in the metabolic markers, methylmalonic acid and homocysteine. We also demonstrated that CN-B12 has a tendency to fall behind OH-B12 in the rates of tissue uptake at 3 ug/day. We agree that further study is warranted with higher concentration of vitamin B12 for supplementation However, in this work our main goal was to compare the biological effect of CN-B12 with that of endogenous OH-B12 in milk, available in daily servings not exceeding relevant amounts.

Reviewer 3 Report

The manuscript by Mahalle et al. presents a novel study on the comparability of supplemental versus dietary vitamin B12 on improving B12 status in a deficient population. The research question is timely, the study is well designed, and the outcomes have public health relevance.

Overall the manuscript is well written and presents important findings.

Comments and Suggestions:

Abstract – the abstract starts of nicely, yet the methods and results section could be described with more detail, and may benefit from minor editing. E.g., “Baselines differed in holotranscobalamin…” should be reworded for English language and flow; the results in parentheses are for the entire study sample, but differences are mentioned – the authors may want to consider mentioning the magnitude of significance rather than presenting the global mean. The conclusion could be stronger. 

Introduction - the background summarizes nicely the general theme of the work. The knowledge gap identified is whether different B12 forms "are of equal value for maintenance of B12 homeostasis". However, it can be questioned whether the blood biomarkers and a 4-week intervention can answer the introduced research question about B12 homeostasis. How would the authors define homeostasis in case of B12 and especially how can homeostasis be measured in light of the limitations of the individual B12 biomarkers? A 4-week intervention was formerly shown to not be sufficient in altering B12 biomarkers after low-dose B12 supplementation. Further, CN-B12 tends to accumulate in blood – this should be taken into consideration when interpreting the results of the supplemental B12 group. The authors should get back to the topic of homeostasis and related questions in the Discussion section.

For the final paragraph of the introduction, the authors ideally present their specific research objectives, rather than providing a sneak-preview of the study design, participants, results, and conclusion. 

Methods: It would have been important confirming the actual milk B12 content. The amount of B12 in supplements was aligned with that measured in milk samples by the group; however, the source cited dates back to 2013. Does B12 content in buffalo, or cow milk differ between herds, seasons, years? A justification should be provided why the B12 content measured in 2013 can be assumed to being the same as the one in the milks provided for this study. A reference/link to supplemental tables with B12 concentration in milk is provided at the end of the manuscript; however, these tables are not mentioned or referred to in the manuscript text. Also, the method section does not describe how B12 content in milk was measured.

Power calculation: Please provide more information - what were the dependent and independent variables for the multiple linear regression model selected for power calculation? Why was a regression chosen and not a comparison test for power calculation? The power calculation could be connected to the other parts of the data analysis section (which eases then the justification of the selection of test).

Study design: the study is described as a longitudinal cohort intervention study; however, participants were randomized. While it was an open-design, a randomized intervention trial may be the more accurate definition. It is unclear, why the 4-week intervention study is described as longitudinal cohort study. 

Dietary B12: Because participants were allowed to follow their usual lactovegetarian diet, it would be important to monitoring the dietary B12 intake in all groups, especially the milk intake. Did dietary B12 intake, and milk intake, change in the groups over time? If the dietary intake was not assessed, this should be remarked as study limitation in the Discussion section, and results should be interpreted with higher caution. 

Statistics: The data analyses are described in exceptional detail.

Results: The results are equally well presented, in great detail, and sufficiently explained.

Discussion: The discussion may benefit from some restructuring for better flow. 

The difference in the change of functional biomarkers, i.e., tHcy decreased but MMA did not (lines 391-393), requires further elaboration. The difference cannot be referred to the fact that B12 needs to reach the mitochondria (versus the cytosol) to impacting MMA concentrations versus tHcy concentrations. MMA was shown to be a sensitive indicator and responds promptly to B12 supplementation, so the argument is weak for explaining the difference by intracellular trafficking over a 4-week time window. Bacterial overgrowth was described to be prevalent in low-income countries and may influence MMA concentrations, given propionic acid production by gut microbiota. This could be one argument, but others, e.g., difference between confounding factors of tHcy versus MMA, should be discussed as well.

Overall, the manuscript is an important contribution to existing knowledge of B12 metabolism, nutrient accessibility, and to our need to identify effective measures on combating the high prevalence of B12 deficiency.

Author Response

Thank you very much for reviewing the article and the suggestions. The response is as follows. The response to the suggestions are added in the text.

Comments and Suggestions:

(1)   Abstract – the abstract starts of nicely, yet the methods and results section could be described with more detail, and may benefit from minor editing. E.g., “Baselines differed in holotranscobalamin…” should be reworded for English language and flow; the results in parentheses are for the entire study sample, but differences are mentioned – the authors may want to consider mentioning the magnitude of significance rather than presenting the global mean. The conclusion could be stronger. 

           Response: We have edited the abstract highlighting the important finding trying to keep close to the number of words allowed for the abstract.

(2)   Introduction - the background summarizes nicely the general theme of the work. The knowledge gap identified is whether different B12 forms "are of equal value for maintenance of B12 homeostasis". However, it can be questioned whether the blood biomarkers and a 4-week intervention can answer the introduced research question about B12 homeostasis. How would the authors define homeostasis in case of B12 and especially how can homeostasis be measured in light of the limitations of the individual B12 biomarkers? A 4-week intervention was formerly shown to not be sufficient in altering B12 biomarkers after low-dose B12 supplementation. Further, CN-B12 tends to accumulate in blood – this should be taken into consideration when interpreting the results of the supplemental B12 group. The authors should get back to the topic of homeostasis and related questions in the Discussion section.

            Response: We agree that a four-week supplementation with low dose B12 is insufficient to normalize B12 status in B12 deplete individuals 

            irrespective of the form of B12 employed. We have corrected the introduction accordingly and emphasized this in the discussion.

(3)   For the final paragraph of the introduction, the authors ideally present their specific research objectives, rather than providing a sneak-preview of the study design, participants, results, and conclusion. 
Response: We have removed the sneak-preview as suggested.

 (4)   Methods: It would have been important confirming the actual milk B12 content. The amount of B12 in supplements was aligned with that measured in milk samples by the group; however, the source cited dates back to 2013. Does B12 content in buffalo, or cow milk differ between herds, seasons, years? A justification should be provided why the B12 content measured in 2013 can be assumed to being the same as the one in the milks provided for this study. A reference/link to supplemental tables with B12 concentration in milk is provided at the end of the manuscript; however, these tables are not mentioned or referred to in the manuscript text. Also, the method section does not describe how B12 content in milk was measured.
Response: Method employed for milk B12 measurement has been included in the method section (lines 74-75), and is already mentioned in the result section (lines 237-240).

 (5)   Power calculation: Please provide more information - what were the dependent and independent variables for the multiple linear regression model selected for power calculation? Why was a regression chosen and not a comparison test for power calculation? The power calculation could be connected to the other parts of the data analysis section (which eases then the justification of the selection of test).
Response: We have edited the section providing more details on the data used for the sample size calculation.

 (6)   Study design: the study is described as a longitudinal cohort intervention study; however, participants were randomized. While it was an open-design, a randomized intervention trial may be the more accurate definition. It is unclear, why the 4-week intervention study is described as longitudinal cohort study. 

            Response: We agree that a randomized intervention trial may present an adequate description and have changed the text.

(7)   Dietary B12: Because participants were allowed to follow their usual lactovegetarian diet, it would be important to monitoring the dietary B12 intake in all groups, especially the milk intake. Did dietary B12 intake, and milk intake, change in the groups over time? If the dietary intake was not assessed, this should be remarked as study limitation in the Discussion section, and results should be interpreted with higher caution. 
Response: We do not have data on dietary intake. A sentence addressing this has been included in the discussion under the weaknesses of the study.

(8)   Statistics: The data analyses are described in exceptional detail.

(9)   Results: The results are equally well presented, in great detail, and sufficiently explained.: Thank you. No change has been made

      (10) Discussion: The discussion may benefit from some restructuring for better flow. 

           The difference in the change of functional biomarkers, i.e., tHcy decreased but MMA did  not (lines 391-393), requires further elaboration. The difference cannot be referred to the fact that B12 needs to reach the mitochondria (versus the cytosol) to impacting MMA concentrations versus tHcy concentrations. MMA was shown to be a sensitive indicator and responds promptly to B12 supplementation, so the argument is weak for explaining the difference by intracellular trafficking over a 4-week time window. Bacterial overgrowth was described to be prevalent in low-income countries and may influence MMA concentrations, given propionic acid production by gut microbiota. This could be one argument, but others, e.g., difference between confounding factors of tHcy versus MMA, should be discussed as well.
Response: We have included a sentence on the food intake, see above. We agree that the possible explanation for the difference in response amongst the two metabolic markers is speculative and has chosen to remove the sentence.

 Thank you.