Dietary Omega-3 Fatty Acids from Fish and Risk of Metabolic Dysfunction-Associated Steatotic Liver Disease in a Mediterranean Population: Findings from the NUTRIHEP Cohort

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Evaluation of manuscript nutrients-3943468

The manuscript addresses a relevant topic by investigating the association between omega-3 fatty acid intake (EPA and DHA) and the risk of MASLD in an Italian cohort. I will now present my point-by-point considerations.

The abstract is overly descriptive. I believe that in addition to the odds ratios, the main means and their respective statistical values from the comparison between the two groups could be added.

The current introduction adequately establishes the clinical importance of MASLD but could better contextualize the investigation. One point that deserves further development is the pathophysiological rationale for the hepatoprotective effects of omega-3s. It would be valuable to elaborate on how exactly EPA and DHA act on the central mechanisms of the disease. For instance, their role as PPAR-α agonists, promoters of beta-oxidation, and inhibitors of SREBP-1c could be detailed, thereby reducing lipogenesis and triglyceride accumulation in the hepatocyte. Additionally, a mention of their ability to modulate inflammatory signaling through the production of mediators that balance omega-6 concentration would be welcome.

Another aspect to be incorporated is the specific justification for focusing on dietary seafood intake, in contrast to the extensive literature on supplements. The current introduction does not explicitly state why it is relevant to study the nutrient in its natural food source. A clear argument on how the consumption of whole fish can offer synergistic effects due to the presence of co-factors (such as other micronutrients and high-quality proteins), and how this translates into a real-world context for public health recommendations, would differentiate this study and highlight its practical contribution. Furthermore, there are studies questioning the efficacy of omega-3 consumption in supplement form.

Finally, the knowledge gap should be more explicit, and the tested hypothesis/hypotheses should be added.

The cross-sectional design, using data from the NUTRIHEP cohort follow-up, is appropriate for generating hypotheses about this specific association. The methodological description is generally robust. The diagnosis of MASLD via standardized and semi-quantitative ultrasound confers clinical validity to the outcome. The use of the validated EPIC food frequency questionnaire, verified by nutritionists, is another positive point.

However, the cross-sectional nature of the study is its main limitation, as it prevents causal inference. It is impossible to determine whether lower omega-3 intake is a cause or a consequence of MASLD, or if it is related to other residual confounding factors. Please include this in the "Strengths and Limitations" subsection.

The exclusion of variables that are part of the MASLD definition (such as BMI and glycemia) from the adjusted models was a statistically sound decision to avoid collinearity but may have removed important confounding factors, potentially underestimating the final model adjustment.

Overall, I found the methods very robust; however, the inclusion and exclusion criteria need to be clearer.

Regarding the results, the analysis by food source is particularly enlightening. By observing a stronger protective effect for Group 2 fish (such as salmon and sardines) and a weaker or absent effect for mollusks and processed fish (Groups 1 and 3), the authors present a relevant finding that challenges the common sense notion of simply "eating seafood."

However, the Tables do not allow for a complete understanding of the results. For example, for the variable "Family income assessment (%)" we have a 5x2 comparison (insufficient, just sufficient, sufficient, more than sufficient, good vs. yes or no); a p-value of 0.025 was found, but it is not clear which groups differed from each other. Please make the results in the Tables clearer.

The discussion is well-structured, contextualizing the findings within the existing literature on NAFLD/MASLD and omega-3. The exploration of plausible mechanisms (hepatic lipid metabolism, anti-inflammatory, antioxidant) is appropriate and informative. I believe the authors should further explore why Group 1 (mollusks and crustaceans) showed a weaker association. It would be interesting to present indicators of absolute EPA/DHA content, differences in total fatty acid profiles, or other dietary components that accompany the consumption of these foods.

In the limitations section, I believe it is important to mention that although adjustment for adherence to the Mediterranean diet (rMED) was performed, residual dietary factors not captured by the score could act as confounders.

Author Response

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Reviewer 2 Report

Comments and Suggestions for Authors

The authors investigated the relationship between eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) intake and the risk of metabolic dysfunction–associated steatotic liver disease (MASLD). The findings indicate that higher EPA and DHA intake is associated with a lower risk of MASLD. The study is strengthened by a large cohort size, comprehensive participant data, and clearly described analytical parameters. The statistical analyses, including regression modeling, are appropriate and well presented.

If feasible, the inclusion of n-6 fatty acid intake derived from the dietary questionnaire would provide additional context regarding the balance between n-3 and n-6 fatty acids. Moreover, the authors could consider conducting a combined analysis of the three groups to further explore overall trends or dose–response associations.

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

Dear author,

Thank you to give the opportunity to review your manuscript. I have some suggestions to improve it.

Title and abstract

I recommend replacing “risk” with “odds” and “associated”. Include “cross-sectional analysis of NUTRIHEP follow-up, 2015-2018”

Introduction

Update MASLD prevalence with precise Italian estimates and MASLD vs NAFLD terminology. Limit the literature to observational studies on fish/EPA/DHA and steatosis in Mediterranean settings.

Methods

Provide detailed eligibility criteria, recruitment methods, participation rates, and any non-responder analysis for the NUTRIHEP cohort follow-up. Add a flow diagram of participant inclusion/exclusion.

Results

Resolve inconsistencies in reported statistical significance vs. text statements. Harmonize use of quantiles (tertiles or quartiles), unify decimal and unit formats, and ensure tables and figures are self-contained with clear units and legends.

Discussion

Explicitly acknowledge the limitations of cross-sectional design including potential reverse causality. Discuss clinical relevance considering the modest effect sizes and the applicability to populations with different dietary habits.

Limitations

Expand discussion to include measurement error in dietary assessment, absence of physical activity measures, single timepoint dietary data, and generalizability limits.

Best regards,

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors,

Please, correct the manuscript based on the suggestions below:

Introduction - the authors provided a good contextualization on MASLD and provided a good justification for the research. However, have concerns on lines 56-58: Please, elaborate the sentences on the recent findings on the possible correlation between omega 3 and MASLD

On Lines 61-64: is it the first work on evaluating the correlation in this scale? 

Figure 2 - Make appropriate differentiation on the items described. For example, use letters "A", "B", and "C". Also, improve the resolution/increase the font size and standardize for Calibri or Arial (the visualization gets easier)

Experimental design/statistical analysis: please, explain why the authors did not implemented a control group (no intake); this is also another limitation of the study. 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I see in this second version that the authors have improved the manuscript, but some justifications are not applicable. The abstract is 213 words long; they could indeed meet my recommendation, which would make the abstract more informative. I believe the text is suitable; however, I recommend that the authors make the changes to the abstract first.

Author Response

Thanks for the suggestion. We've implemented the abstract as requested.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for your prompt review. The changes have been successfully implemented. 

Best regards, 

Author Response

Thanks for the suggestions

Best regards