Review for 10.1186/s12876-018-0805-z (2018)

Reviewer ’s report  
Title: Response of fibroblast growth factor 19 and bile acid synthes is after a body weight -
adjusted oral fat tolerance test in overweight and obese N AFLD patients: a non -randomized 
controlled pilot  trial 
 
Version: 0 Date: 15 Feb 2017  
 
Reviewer: Yaron Rotman  
 
Reviewer's report:  
FGF19 is a major regulator of bile acid synthesis and a crucial arm in the enterohepatic signaling 
circuit. In this work the author s aim to characterize the changes in FGF19 blood levels after a fat 
challenge, and compare those between healthy controls and subjects with non -alcoholic fatty 
liver disease (NAFLD).  
 
The data presented by the authors is valid, worthy of publication and co ntributes to the scientific 
knowledge of the metabolic changes occurring in the post -prandial period. No bias was noticed. 
Although we would like to see the data published, several concerns need addressing prior to 
publication:  
 
Major Comments:  
1. Hypothes is: The authors should clearly state the study hypothesis and aims, preferably in the 
introduction section. It would be relevant to understanding the study design and especially 
the choice to group subjects by obesity status and not by other criteria.  
2. Clinical trial participants:  
a. Please specify how subjects with NAFLD were recruited (advertisements, inpatients, 
outpatient clinics, community -based health care providers) and similarly what was the source 
for healthy controls.  
b. Please clarify how h ealthy controls were defined beyond having a normal BMI and whether 
fatty liver was excluded by US and liver enzymes.  
c. Were cirrhotics excluded?  
d. Were NAFLD subjects on medications? Specifically medications that could have influence 
on metabolic pathwa ys (fibrates, statins, diabetes medications).  
3. The only reliable method to differentiate steatosis from NASH is a liver biopsy. Since only 5 
biopsies are available (and the indication for biopsy is not specified), there can be no reliable 
evaluation of h istology or its impact on FGF19 in the paper. The authors should omit all 
references to histology in the manuscript and acknowledge the absence of sufficient 
histological data (beyond a few patients) in the discussion.  
4. Statistical analyses:  
a. A primary  end-point was not defined. In a small study such as this, spurious false -negative 
and false -positive significance tests are likely given the many different analyses. Knowing 
what the initially defined primary end point helps assessing the validity and giv es appropriate 
weight to it and to secondary end points.  
b. Given the study design of 3 groups, the appropriate statistical approach is to have a Kruskal -
Wallis test to determine differences between groups and only apply Mann -Whitney two -way 
analyses as a secondary post -hoc analysis. Currently as the data is reported in the tables and 
texts, it does not appear to have been the approach, and in some cases only a two -way 
analysis is reported, and only for significant pairs.  
c. Secondary analyses where signif icance was only found for one subgroup (i.e. figure 3 for 
men only and figures 8 -9 for controls only) should be minimized. These only make sense if 
there is a rationale for having significance only in a sub -group, otherwise they represent the 
result of mul tiple testing, are likely to be affected by outliers and introduce bias. The authors 
should very clearly address this limitation whenever presenting the data. Results should be 
shown for the whole cohort (i.e. in figure 3 show correlation of fasting FGF19 and body fat 
over the whole group); subgroup analyses can be presented in supplementary figures and 
should include both significant and insignificant groups (i.e. for figure 3 the supplementary 
should be one panel for men and another for women).  
5. Is the effect due to NAFLD? The authors recruited from two populations - obese/overweight 
subjects with NAFLD or normal weight presumably without NAFLD. There is no reason or 
proof to suggest that the differences they are seeing in FGF19 secretion is due to NAFLD  and 
not due to obesity or metabolic syndrome. One important way to hint at NAFLD as an 
important factor would be to analyze FGF19 secretion patterns by grade of steatosis. In the 
absence of that, the authors should tone down the implication that NAFLD is the important 
factor in the paper, abstract and title. They definitely cannot claim (p. 17, line 55) that 
decreased FGF19 is involved in the pathogensis of NAFLD based on the results in this 
manuscript.  
6. The authors chose to provide a weight -based dose o f fat in the challenge test, with somewhat 
limited justification of increased caloric intake in NAFLD subjects (which does not 
necessarily imply weight -based increase). This markedly limits their ability to do weight -
based analyses, since some of their fin dings could actually reflect the dose provided. This 
cannot be experimentally corrected now that the study is over, but the authors should 
comment on that and discuss this as a limitation of their study beyond the cursory discussion 
on p. 16 line 44.  
7. The discussion is not very well organized. Although it discusses several other studies of post -
prandial FGF19, it fails to mention the recent one by Sonne et al (JCEM 2016) and fails to 
clearly explain what the current manuscript contributes beyond those oth er studies.  
 
Minor Comments:  
1. FGF19 measurement - please state (p. 9) if levels were measured in single/duplicate/triplicate 
form.  
2. Statistical: Defining strength of correlation, and especially the use of the word "sufficient" is 
unnecessary. Please r emove. Given that the authors are using the non -parametric Spearman 
test (appropriately), please change "r" to "rho". Non -parametric correlation does not denote a 
linear relationship (p. 10, line 9). Please report p -values for non -significant tests  
3. Clinical trial: please confirm if the trial was registered in clinicaltrials.gov or a similar 
database.  
4. Suggestion for additional secondary analysis - the authors have looked appropriately at total 
AUC and incremental AUC of FGF19. It may be worth to look a t relative IAUC (i.e. 
incremental AUC normalized to baseline values) to see if it is more informative.  
5. Style - the introduction is lengthy, contains too many references (38!) and is not well 
organized. There is no real need to discuss elements that are irrelevant to the study question 
(i.e NAFLD genetics with 6 references), intestinal failure, etc. Would suggest shortening it 
significantly and only mention elements that are pertinent to the study design and hypothesis.  
6. Would argue that the hallmark o f NASH (p. 4) is hepatocyte injury (ballooning) and not 
inflammation, but maybe even worth omitting that incorrect sentence altogether given its 
irrelevance to the paper.  
7. Results (p. 11 -12): Data that is present in table 1 and is not part of the primary  endpoint does 
not need to be repeated extensively in the test. The sections on body fat mass and metabolic 
parameters can be eliminated, or summarized in 2 sentences. Similarly, the correlations 
between bile acids and cholesterol (fig 6) and fat mass (p. 14) are not part of the study 
hypothesis and should not be included, unless they can be tied to the discussion of FGF19.  
8. Serum bile acid - the data in figures 5 -6 suggest 6 subjects are outliers and have fasting bile 
acid levels that are 2 -4 times highe r (5uM or greater) than the rest of the cohort which have a 
much lower levels. These in turn drive the apparent (but likely false -positive) correlation 
between FGF19 and bile acid levels noted in figure 5. The authors should verify that this is 
not due to a technical issue, and identify what is different about those subjects.  
9. It appears that the authors measured cholesterol levels only once (in the fasting state) and 
when calculating C4/cholesterol ratios, use the same fasting cholesterol with all of the  post-
prandial C4 values. Please clarify if this is the case and comment on the validity of this, as 
opposed to using concomitant cholesterol values.  
10. References:  
a. Some of the references are not the most updated ones. Examples include references from 
2004 and 2008 (refs 18 -19) to justify limited knowledge of NASH pathogenesis or a review 
from 2007 on the function of FGF19 (ref 24) when newer reviews are available (see for 
example a review by Jahn et al from 2015).  
b. Several references are in German an d not readable by the readers of this journal. Although 
great science is done and published in non -English languages, it is more appropriate to prefer 
English references, when available, for an English publication that has an international 
readership.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
Yes 
Are the conclusio ns drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
No 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy what aspects require further 
assessment in your comments to the editors.  
I am able to assess the statistics  
Quality of written English  
Please indicate the quality of language in the manuscript : 
Acceptable  
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that  may in any way gain or lose financial ly from the publication of this 
manuscript, either now or in the future?  
2. Do you hold any stocks or shares in an organisation t hat may in any way gain or lose 
financially from the publication of this manuscript, eith er now or in the future?  
3. Do you hold or are you currently applying for any patents  relating to the content of the 
manuscript?  
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the content of the manuscript?  
5. Do you have any other financial competing interests?  
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
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