Review Reports - Effects of a Bioactive Vegetable-Enriched Diet on Autotaxin and Liver Fibrosis in MASLD with Evidence of Sex-Specific Responses: A Pilot Study

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The authors have satisfactorily addressed most of the concerns, however I believe they should explicitly describe the study limitations in the Discussion section, regarding this two comments below. Even though they have been addressed to the peer reviewers, they have not sufficiently been addressed in the manuscript:

Review comment: Figure 1 does show the assessments at baseline and at follow up (control visit). I wonder why physical activity and the psychological general wellbeing index were not included? Surely the authors would want to see if physical activity stayed the same because if it did not, then that is an important confounder in the results. Also given the impact of nutrients in the brain, a follow up psychological assessment would have also been interesting to include.

Authors response: Physical activity and PGWBI were assessed only at baseline to describe the cohort, as the relatively short duration of the intervention (two months) did not allow us to predict significant changes in these parameters. In addition, patients were advised not to change their lifestyle since this was not an endpoint of the study (lines 508–509).

Review comment: How confident are the authors that the findings e.g. decreases in ATX level, are not due to the alternations in body composition? Studies show for example reduced ATX in the setting of weight loss. The ideal set up would have been to assess the hepatic effects in the absence of any changes in body composition. The results show that fat free mass significantly increased in both groups (men and women) post diet. Therefore, there has been a change in body composition during this time. Moreover, body composition data were not included in the statistical models. Please discuss. Also expand the evidence base to include relevant studies (e.g. such as those mentioned in the point above) but not limited to these only.

Author response: Thank you for your comment. We recognise that changes in body composition can influence ATX levels. In our multivariate model, we included fat-free mass (FFM) as a covariate, and the reduction in ATX remained significant, suggesting that the observed effect is not entirely explained by such changes. However, we agree that fat loss may have contributed in part to the result

Author Response

Thank you for your comment. We have clarified the two comments highlighted in the ‘’Limitations of the Study’’ section (lines 479- 488).

Reviewer 2 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

As mentioned earlier, this pilot study presents a novel and timely investigation into the effects of a bioactive vegetable-enriched diet on serum autotaxin (ATX) levels and liver fibrosis in individuals with MASLD, with a particular focus on sex-specific responses. The study is commendable for its originality, practical dietary intervention, and the exploration of ATX as a modifiable biomarker. The manuscript is well-structured and clearly written, with a coherent argument and a sound methodological approach. The findings are significant and contribute meaningfully to the growing field of precision nutrition and metabolic liver disease.

The rationale for selecting specific vegetables could be expanded with more empirical support. Recommendation: Include more references or data supporting the hepatoprotective properties of the selected vegetables.
The absence of a control group limits the strength of causal inferences. Recommendation: Acknowledge this limitation more explicitly and suggest future studies include a control group.
The rationale for excluding certain MASLD-defining variables from the model should be clarified. Recommendation: Provide a more straightforward explanation for the exclusion of these variables in the statistical model.

After reviewing the full manuscript and reference list, I find the presence of inappropriate self-citations concerning. The authors do cite their own previous work (e.g., references [19] (Prior pilot study on vegetable substitution and NAFLD — directly relevant), [37] (autotaxin as a prognostic biomarker — supports the current study’s biomarker focus), and possibly [15]). Still, these citations seem relevant to the current study, especially for establishing the rationale for selecting vegetables and for the role of autotaxin (ATX). The self-citations are not excessive in proportion to the total number of references (40+). Furthermore, they are not used to support controversial claims or to unjustifiably inflate the novelty.

Author Response

The rationale for selecting specific vegetables could be expanded with more empirical support. Recommendation: Include more references or data supporting the hepatoprotective properties of the selected vegetables.
The absence of a control group limits the strength of causal inferences. Recommendation: Acknowledge this limitation more explicitly and suggest future studies include a control group.
The rationale for excluding certain MASLD-defining variables from the model should be clarified. Recommendation: Provide a more straightforward explanation for the exclusion of these variables in the statistical model.

Thank you for your positive comments. I have added a reference and further explanation for the choice of variables in the Statistics Analysis section. Regarding the economy of selecting confounders, given the statistical methodology (one covariate per 13 observations), the confounders were chosen accordingly.

Thank you for your comments. In the manuscript, we have already highlighted among the limitations both the absence of a control group and the lack of detailed phytochemical characterisation of the vegetables used, aspects that we recognise as intrinsic to the preliminary nature of the pilot study. We welcome the suggestion to further clarify the rationale for excluding MASLD variables from the statistical model (lines...). With regard to self-citations, we reiterate that they were used sparingly and only when directly relevant to the scientific context.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

In this manuscript, the authors reported the results of a study aiming at determining the effect of a bioactive vegetable-enriched diet on autotaxin and liver fibrosis in patients with MASLD. For the study, 44 obese (BMI>30 kg/m2) adult patients were recruited and assessed at baseline (time 0) and after a 2 month intervention with a bioactive vegetable-enriched diet. At completion of the treatment, serum autotaxin levels were significantly decreased in the treated patients, and several metabolic parameters showed a marked improvement. The effects were more pronounced in women as compared to men. The conclusion of the authors is that the diet used in the study is effective at lowering the serum levels of autotaxin, especially in women.

Comments: the study appears to be properly conducted, although it would have been helpful to have a control group exposed to the same diet to determine to which extent the diet is beneficial even in the absence of MASLD. It cannot be excluded that the amplitude of the beneficial effects reported here was hampered to some extent by the obesity and or the presence of MASLD in the sampled population. Another limitation acknowledge by the authors is the the pool size is homogeneous and relatively small. It would be interesting to see whether similar results can be obtained in larger, and more heterogenous populations, in presence of an appropriate control group, and over a longer period of treatment.

Overall, the study is properly conducted and no major criticisms were raised at this time.

Abstract is clear and to the point.

Results are properly reported

Discussion is appropriate

Author Response

Overall, the study is properly conducted and no major criticisms were raised at this time.

Abstract is clear and to the point.

Results are properly reported

Discussion is appropriate

Thank you for your positive assessment of our work and for your constructive suggestions. We are pleased that the study design, presentation of results and discussion were considered adequate.

We fully acknowledge the limitations mentioned: the absence of a control group, the relative homogeneity and small size of the sample, and the short duration of the intervention. As indicated in the “Limitations” section, these aspects reflect the preliminary nature of the pilot study, whose main objective was to assess the feasibility of the intervention and the biological variability of the parameters analysed. We also agree that future studies, conducted on larger and more diverse populations, with an appropriate control group and longer follow-ups, will be essential to confirm and generalise the results obtained.

We appreciate the interest shown in our data and believe that the comments provided help to strengthen the rationale for future controlled studies.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting study however there are some serious methodological and analytical concerns that currently reduce the validity of the conclusions drawn. Please see below.

In the introduction and throughout the authors refer to gender related differences. Are they meaning biological sex or gender as reported by participants?
The last paragraph of the introduction (lines 127-135) clearly were not written by the authors and are instead author guidelines which have been pasted in and accidentally not deleted before submitting the manuscript
Figure 1 does show the assessments at baseline and at follow up (control visit). I wonder why physical activity and the psychological general wellbeing index were not included? Surely the authors would want to see if physical activity stayed the same because if it did not, then that is an important confounder in the results. Also given the impact of nutrients in the brain, a follow up psychological assessment would have also been interesting to include.
What did the authors do about advanced LSM values indicative or cirrhosis? Were such patients included?
Please address the limitations of using FibroScan in obese participants
How was obesity defined in this population? The inclusion criteria states patients needed a BMI > 30 kg/m2 however they also conduced BIA to determine body composition. This is a pertinent question because in the results on line 288 the authors state: “Physical activity was <30 min in 8 (20%), >30 288 min in 26 (63%), and sportsman in 7 (17%) of subjects.” Does this mean that the BMI was elevated due to muscle mass in participants in the sportsman category? This should be discussed as it could potentially confound the study results.
Sleeplessness is mentioned for the first time in the results, however there is no reference to how this was measured in the method section at all. This crucial information needs to be added. The same applies to snoring habits.
Table 2 shows significant post diet reductions in BMI. How do the authors explain this and how might this reduction impact the results? Why was this not included in the univariate/multivariate models?
Overall some crucial studies have not been included. The authors need to expand their citations to include all relevant studies, e.g. org/10.1159/000542061, doi: 10.1194/jlr.M014985
How confident are the authors that the findings e.g. decreases in ATX level, are not due to the alternations in body composition? Studies show for example reduced ATX in the setting of weight loss. The ideal set up would have been to assess the hepatic effects in the absence of any changes in body composition. The results show that fat free mass significantly increased in both groups (men and women) post diet. Therefore, there has been a change in body composition during this time. Moreover, body composition data were not included in the statistical models. Please discuss. Also expand the evidence base to include relevant studies (e.g. such as those mentioned in the point above) but not limited to these only.

Reviewer 2 Report

Comments and Suggestions for Authors

This is an interesting and detailed study in which participants were extensively evaluated.

Major issues:

(1) The main limitation is that, as the authors correctly explain, the study does not have a control group. That makes impossible to attribute to the intervention any change in measured variable like ATX, insulin sensitivity, or body weight.

(2) The intervention describe is to replace starch with specific vegetables. Vegetable servings (2 or more per day) adds 1 point to the Mediterranean diet score. Thus the maximum effect of the intervention would be to increase the score by 1. The Mediterranean diet adherence index increased 3 points in average, from 8 to 11. Therefore it seems that the diet of the subjects changed in additional ways.

In addition, the subjects lost more than 5% of their body weight in 2 months. That would require a sustained caloric deficit.

These two findings suggest that the subjects underwent other changes in addition to the prescribed intervention. Therefore it is difficult to attribute outcome to the intervention.

Minor issues

(1) The authors write ""One of the key strengths of this study is the high feasibility and adherence of the intervention": Was adherence assessed?

(2) Why do authors call their intervention a " plant enriched diet"? Are not starch and the indicated vegetable both of plant origin?

(3) The last paragraph of the introduction are instructions.

Reviewer 3 Report

Comments and Suggestions for Authors

General Assessment

This manuscript presents a prospective interventional study evaluating the effects of a plant-enriched diet on serum autotaxin (ATX) levels and liver fibrosis in individuals with obesity and MASLD. The study is well-conceived and addresses a relevant and timely topic in nutritional hepatology, with a novel focus on gender-specific responses.

Originality and Novelty

- Originality: The study explores the modulation of ATX—a relatively novel biomarker in MASLD—through dietary intervention, which is an under-investigated area.

- Novelty: The gender-stratified analysis and the use of specific bioactive-rich vegetables (Brassicaceae and Asteraceae families) add a unique dimension to the research.

Assessment: High originality and novelty. The study contributes meaningfully to the field of precision nutrition and metabolic liver disease.

Scientific Soundness and Methodology

- Design: A two-month prospective study with pre- and post-intervention assessments. While the design is appropriate, the absence of a control group limits causal inference.

- Sample Size: Moderate (n=44), with 9 dropouts. A retrospective power analysis is mentioned but not detailed.

- Statistical Analysis: Robust use of Generalized Estimating Equations (GEE) to account for repeated measures and confounding variables. Stratified models by gender are well-justified.

Assessment: Scientifically sound with appropriate methodology. Minor improvements needed in reporting power analysis and discussing the lack of control group.

Scope and Relevance

The manuscript aligns well with the scope of Nutrients, addressing dietary interventions for metabolic disorders. The focus on ATX as a modifiable biomarker and the gender-specific outcomes are highly relevant to current research trends.

Assessment: Fully within scope and of high relevance to the journal’s readership.

Quality of Writing and Language

- Clarity: The manuscript is generally well-written and logically structured.

- Grammar and Style: Minor grammatical errors and occasional awkward phrasing (e.g., “salutary model”) should be revised.

- English Proficiency: Acceptable for publication, though professional language editing is recommended for polish.

Assessment: Good overall writing quality. Minor language editing suggested.

Figures and Tables

- Tables: Comprehensive and well-organized. Statistical annotations are clear.

- Figures: Limited but effective. Figure 2 (scatter and linear plot) is informative.

- Suggestions: Consider adding more visual summaries (e.g., bar graphs or box plots) to enhance data presentation.

Assessment: High-quality tables; figures are adequate but could be expanded.

Discussion, Strengths, and Limitations

Discussion

- Thoughtful interpretation of findings.

- Gender-specific differences are well contextualized with biological plausibility.

- Integration of clinical relevance and future research directions is commendable.

Strengths

- Feasible and practical dietary intervention.

- Use of validated tools (FibroScan, ELISA).

- Gender-stratified analysis enhances depth.

Limitations

- No control group.

- Short duration (2 months).

- Small sample size.

- Lack of phytochemical quantification.

- Gender analysis was post hoc and not powered.

Assessment: Balanced discussion with clear strengths and acknowledged limitations.

Citations and Literature Review

- Coverage: Extensive and up-to-date references.

- Style: Consistent and appropriate.

- Depth: Demonstrates strong familiarity with the field.

Assessment: Excellent citation practice and literature integration.