Exploring In Vivo Dynamics of Bovine Milk Derived Gangliosides

Round 1

Reviewer 1 Report

This is a solid study about the influence of nutrition and storage on the medical evaluation of GM3 concentration in blood samples. The study shows no scientific excellence or novelty but it provides medical personal with some important information.

 

Despite the solid study, the manuscript shows some discrepancies which need to be corrected before publication:

GM3 levels are analysed but the high-ganglioside meal contains mainly GD3. This makes no sense to me. please explain the reason of this setup.

 

the data sometimes reveals an rather impossible increase of Gangliosides during storage. Please provide an explanation for this.

 

How was the concentration of GM3 evaluated by ESI-MS? The complete mass spectrometry method and the complete evaluation of the MS-data is missing.

 

The MS-data is missing. Only the evaluated results are presented. This way, the study is not comprehensible.

 

the lowest BMI value as well as the number of participants varies between summary and manuscript.  Please check your data and use the correct value.

 

information about the study population is repeated several times. Please cite your  text instead of repeating yourself.

Author Response

Reviewer 1:

This is a solid study about the influence of nutrition and storage on the medical evaluation of GM3 concentration in blood samples. The study shows no scientific excellence or novelty but it provides medical personal with some important information.

Despite the solid study, the manuscript shows some discrepancies which need to be corrected before publication:

GM3 levels are analysed but the high-ganglioside meal contains mainly GD3. This makes no sense to me. please explain the reason of this setup.

This is an important observation. For clarity we have revised the text in the discussion to make this clearer (page 17, lines 508-521). Other studies have showed increases in circulating GM3 in response to chronic high GD3 feeding. Hence, it’s not a simple case of what is consumed in the diet is reflected in the circulation. Gangliosides seem to undergo conversion to other ganglioside species in the gut before release into the circulation.

 

the data sometimes reveals an rather impossible increase of Gangliosides during storage. Please provide an explanation for this.

An important aspect of this study is to understand (and report on) stability of different storage scenarios. We report here out of 11 GM3 species, two indicated increased levels over time (GM3 32:1, GM3 40:2), and GM3 42:2 showed inconsistent patterns of change over time. While 7 GM3 species consistently showed reductions over time. The anomalies were likely due to analytical variation. These are minor GM3 species in the circulation and low concentrations generally leads to greater variability and greater chance of false positives. Indeed, inter-run CV’s for these species ranged from 19-23%. This explanation was added to the manuscript (page 16, lines 454-457)

How was the concentration of GM3 evaluated by ESI-MS? The complete mass spectrometry method and the complete evaluation of the MS-data is missing.

We have expanded on the mass spectrometry method and added a chromatogram to illustrate the MS-data (page 5-6, lines 234-246).

The MS-data is missing. Only the evaluated results are presented. This way, the study is not comprehensible.

See above comment.

the lowest BMI value as well as the number of participants varies between summary and manuscript.  Please check your data and use the correct value.

The information was corrected in the abstract and are now aligned with the manuscript (page 1, lines 18-19).

 

information about the study population is repeated several times. Please cite your  text instead of repeating yourself

Unnecessary reference to the study population were deleted throughout the manuscript (page 2, line 94, page 3, lines 97-101 was moved to P3, lines 125-129, P16, lines 416-417, P19, line 572).

Reviewer 2 Report

The authors studied GM3 ganglioside levels in blood sample type (serum/plasma), storage conditions, day-to-day variation and acute effects of consuming bovine-derived gangliosides on circulating monosialylated gangliosides. Authors concluded, that samples stored at -20- or -70◦C for up to 6 months, are acceptable for GM3-ganglioside analysis. Blood samples can be collected any time with no fasting requirement. This is very systematic methodology development study to quantify gangliosides by giving more details in introduction the manuscript importance will increase.

1. Authors should discuss the importance of different gangliosides and how these gangliosides are important.
2. What is the optimum range of the GM3 in healthy women and children.

Author Response

Reviewer 2:

The authors studied GM3 ganglioside levels in blood sample type (serum/plasma), storage conditions, day-to-day variation and acute effects of consuming bovine-derived gangliosides on circulating monosialylated gangliosides. Authors concluded, that samples stored at -20- or -70◦C for up to 6 months, are acceptable for GM3-ganglioside analysis. Blood samples can be collected any time with no fasting requirement.

This is very systematic methodology development study to quantify gangliosides by giving more details in introduction the manuscript importance will increase.

Thank you for this comment. We have expanded the introduction to include more background information regarding gangliosides (page 2, lines 46-58) as well as how the results from the current investigation could be useful in terms of designing robust human clinical studies (page 2, lines 67-73).

Authors should discuss the importance of different gangliosides and how these gangliosides are important.

Information regarding different gangliosides were added to the introduction (Page 2, lines 51-58).

What is the optimum range of the GM3 in healthy women and children.

The optimal range for serum/plasma GM3 in humans is unknown due to a limited number of human studies reporting links between serum/plasma GM3 and health outcomes, a lack of standardisation of ganglioside methodology across laboratories and methodological gaps like those investigated in the current study. The current study addresses some of these barriers in determining optimal ranges. Page 17 discusses the limited number of studies that have measured serum/plasma gangliosides and we have also added information to further clarify the reviewer’s query (page 17, lines 476-479).

Round 2

Reviewer 1 Report

The authours have corrected everything that was critizised in the first submission. I have no further concerns regarding a publication of  this paper.