PONE-D-22-03128Human lipoproteins comprise at least 12 different classes that are lognormally distributed.PLOS ONE

Dear Dr.Tomokazu Konishi

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 ensure your supplemental material can be easily identified.

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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: 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: 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: In this work, Tomokazu and colleagues provide an alternative classification of lipoprotein subclasses in human plasma after HPLC-based separation. The conclusions that are drawn by the authors, despite interesting, clash against multiple well established aspects of lipoprotein metabolism and thus should be supported by a larger and more detailed corpus of data, which is not present in the current work. In particular the manuscript lacks any mechanistical data supporting the key claims about e.g. HDL cholesterol content as well as TG-rich lipoprotein catabolism. My comments are as follows:

Methods:

Line 210: “No postmeal time was specified for blood collection.” Is repeated twice

Line 211: “The age of the volunteers and the sex of the subjects were 50/50, as shown in Figure S1”. This statement is in my opinion unclear. On the one hand, there is not information regarding sex in Figure S1. What is the female to male ratio in the study? How many females/how many males? Additionally, what is meant here with “50/50”? Was the mean age in the study 50 years?

Line 214: here “HDL” should be used instead than “HLD”. Also please indicate what were the “conventional methods” used to determine them. E.g. what commercial kits were used

Line 216: What are the parameters used in the HPLC analysis? What column? What flow rate? What medium? Where different conditions used for analytical VS preparative HPLC? What methods were used to monitor TG and cholesterol?

Line 217: what do the authors mean here by hyperlipidaemic? The plasma lipid parameters of these patients as well as the type of hyperlipidaemia should be specified in the manuscript

Line 225: “class., with The amount”. Take dot away. Do not capitalize T in “The”

Line 236-237: please either specify the MALDI-TOF-MS methods or provide a reference to a previous work where they were used.

Line 243: “which has better sensitivity than the biuret reaction commonly used for serum samples”. This assertion is not correct, as the BCA method is based on the biuret reaction as well. Do the authors intend that the BCA method is more sensitive compared to the Bradford method? If yes, they should change this sentence accordingly

Figures:

Fig 1. As far as I understand, in the figure two curves are shown that display the raw data for TG as well as for cholesterol. Both are shown in gray, which in my opinion is quite confusing, as curve-fitted data for TGs and choelesterol are then shown in red and blue. Curves for raw data should also be colored (e.g. in a different shade of red and blue), to indicate whether they refer to TG or cholesterol.

Additionally, the SDS-PAGE gel in the left panel should be shown for clarity in a separate panel within the same figure. The SDS-PAGE panel should also indicate what are the numbers on the axes referring to (Molecular weight and elution time?)

Last, in the SDS-PAGE the ApoB100 peak seems to reach its maximum between 10 to 19min, while no ApoB is visible after the 21 minutes fraction. Nevertheless, the authors depict a high “LDL1” peak with mean elution time of about 23 minutes. Could the authors elaborate on this? It is especially difficult to reconcile the band intensities in this silver-stained gel with the ApoB quantification data should in Fig S3, which show instead a clear ApoB100 peak around 23 minutes. A similar discrepancy between Figure 1 and S3 seems to affect the elution curve of ApoB48 as well.

Figure 2B: (see also comment to Fig S6). Based on this graph, the HDL fractions combined seem to contain much less cholesterol compared to the LDL fractions than expected for “normal” plasma LDL-C and HDL-C values. The authors should elaborate on this point.

Fig S2: the abbreviation “TR” should be explained again in the corresponding line of the Results section (line 61) as well as in this figure’s legend. A definition is given instead only in line 80 of the results. Additionally, measurement units should be indicated on the y axes in both panels. The expression “It is unique in that it is smaller and very low in cholesterol. However, fitting is difficult without this.” Is quite unclear without having read line 80.

Fig S3: please see comment given for Fig 1. The authors should indicate what the two panels shown in this figure indicate. Are they curves obtained from two different samples? This should be mentioned in the legend. Additionally, what to the small gray numbers in the middle of each panel indicate?

Fig S6 The authors show a “LDL2” fraction, which nevertheless seems to appear clearly after the bulk of ApoB100 has been eluted. The HDL-cholesterol peak seems also to be much smaller than expected for an individual with an average “healthy” lipoprotein profile. Could the authors elaborate on this? How can the authors be sure that the “LDL2” peak does not in fact depict the main HDL-cholesterol peak? Additionally, what does the number “401” in the left panel indicate? In this figure as well, the SDS-PAGE gel should be shown as a separate panel, indicating what is shown in each axis.

Fig S8: the authors should provide correlation coefficients and p-values for linear regression for each panel.

Results:

Lines 62-63: “The attributes of the classes were determined based on the elution pattern of the major protein components (Fig. 1, S3-S6). The elution patterns of these proteins coincided with the distribution of the corresponding classes”. If the classes were assigned based on the elution pattern of the major protein components, it would then be logical that they would elute at the same time. The authors should thus delete the second statement. Also, please see my comments regarding figures 1, S3 and S6 on apparent incongruences regarding the elution times of different proteins and lipoprotein subclasses

Line 63: “As the positions and scales coincided between TG and cholesterol”. The meaning of this sentence is unclear to me. Do the authors intend to say that cholesterol and TG followed a similar elution pattern within each subclass, thus suggesting a stable cholesterol-to-TG ration within that class?

Line 68: “and the LDL2 fraction is devoid of B48”. Please see my comments on Figures 1, S3 and S6. Besides being devoid of ApoB48, this fractions seems not to contain ApoB100 either based on the data in Fig S3, which would clash against their classification by the authors as LDL

Line 71: “they may supply cholesterol to the thrombus and protect it from lysis by plasmin”. This statement belongs in my opinion to the discussion section. Additionally, the authors should indicate on what this statement is based (other data? literature?)

Lines 80-83: the authors should provide a statistical evaluation of correlation of the parameters analyzed in Fig S8, including correlation coefficients and p-values.

Line 114: “HDLs were minor cholesterol carriers, contradicting the estimations of the previous study”. I find this statement problematic, and the reference to Gordon et al., (doi: 10.1021/pr100520x) who show how the ApoA-I-containing HDL fractions also carry large amounts of cholesterol in normolipidemic patients as contradicting the findings of the current study. The fact that Gordon and colleagues did not measure ApoA-I levels in each HDL fraction doesn’t cancel the fact that they nevertheless identified it as the most abundant HDL-associated protein in their MS experiments.

Line 118: “the structure of HDL is surrounded by ApoA-1 and therefore has a limitation in size”. The authors refer to Wu et al., doi: 10.1074/jbc.M110.209130. The authors should elaborate more on why the structural findings on this paper set a limit on HDL size based on the conformation of ApoAI.

Lines 123-126: these data seem in contrast, among others, with those of Gordon et al (see comment above). The authors should explain how their findings compare to other HPLC-based studies in this regard.

Discussion:

Lines 149-152: these conclusions contradict data about lipoprotein metabolism gathered through multiple decades and should thus be supported by a much larger amount of evidence as well as a more detailed interpretation of the data. Among others, the selective modification/knockdown of key players in each lipoprotein metabolism pathway in mice as well as genetic studies in human mutation carriers have allowed to establish the current model of lipoprotein metabolism, which, despite not perfect or complete, cannot be discarded as quickly as done in this section of the current manuscript. Thus, the authors should attenuate their claims and provide them in the context of what is known e.g. in relation of the catabolism of TG-rich lipoproteins in humans.

Lines 148-186: please see my previous comments about how cholesterol peaks were assigned to each subclass.

Reviewer #2: The manuscript describes the results of a rapid HPLC method to separate human lipoproteins. The authors identify 12 lipoprotein classes log-normally distributed. The study technically sounds and the main conclusions are supported by data. I have no major concerns but some points should be addressed.

The manuscript is hard to follow. Many data are included in the supplementary figures, this reviewer tried to download these suppl data and it was impossible to identify the different figures cited in the text.

The levels of VLDL, Lp(a), and TR (triglyceride-rich lipoproteins) showed no relationship, suggesting that they were produced independently (Fig. S8AB). I cannot download this figure, but it is difficult to believe that the levels of VLDL and TR were produced independently. How? VLDL is one of the main determinants of TR.

Define mHDL (mature HDL?)

Since there is significant overlap in several lipoprotein classes, TG and cholesterol distribution for these lipoproteins is a critical point. This point should be clearly addressed.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-22-03128R1Human lipoproteins comprise at least 12 different classes that are lognormally distributed.PLOS ONE

Dear Dr. Konishi,

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 submit your revised manuscript by Sep 25 2022 11:59PM. 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: https://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,

Jérôme Robert, PhD

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:

Dear Authors,

After carefully reading your manuscript, the academic editor and two reviewers recommend publication of your work in PLOS One.

According to the new reviewer we ask you to address the following two points:

1. In the last paragraph of the introduction, the authors mention that ultracentrifugation causes loss of membrane proteins. It is an important detail for the present story. The authors should check the reference they have added here and provide more references, if available, supporting this claim.

2. In the discussion section, the authors appear to presume a wider role of the different LPP. Although plausible, this is not part of the present work. It would be good that the authors lower the claims and describe more on the results from the present work.

Best regards

[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: All comments have been addressed

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 #2: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

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 #2: Yes

Reviewer #3: 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 #2: Yes

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 #2: The authors addressed the concerns of this Rw. I have no further comments.

Maybe the authos could add subsections to facilitate the reading of the paper.

Reviewer #3: In the present study, Konishi et al, aim to identify a wider range of LP particles exists in human plasma. The premise of the study is that the currently established and widely used methods of LPP purification or separation alter the protein composition or cause degradation of particle constituents that would eventually affect adversely the class separation or identification. They previously identified 10 different classes of serum LPP in rats and now show the existence of 12 such classes in humans. They use lipid quantification, Western blotting and protein sequencing for identification of each class post HPLC separation. The results, in general, are interesting and the MS that this reviewer received is an improved version over the first submission from the authors. These observations are intriguing given the importance of LPP in metabolic disease.

I have, however, some minor points for the authors.

1. In the last paragraph of the introduction, the authors mention that ultracentrifugation causes loss of membrane proteins. It is an important detail for the present story. The authors should check the reference they have added here and provide more references, if available, supporting this claim.

2. In the discussion section, the authors appear to presume a wider role of the different LPP. Although plausible, this is not part of the present work. It would be good that the authors lower the claims and describe more on the results from the present work.

Barring these minor points, I recommend publication of this work in PLOS ONE.

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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

Reviewer #3: No

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[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.

Human lipoproteins comprise at least 12 different classes that are lognormally distributed.

PONE-D-22-03128R2

Dear Dr. Konishi,

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,

Jérôme Robert, PhD

Academic Editor

PLOS ONE