A Current Approach to Non-Alcoholic Steatohepatitis in Type 2 Diabetes Mellitus Patients

Round 1

Reviewer 1 Report

This retrospective and open study included 59 patients with NAFLD and T2DM who were divided into two groups: 44 (74.57%) were diagnosed with hepatic steatosis (HS) and 15 (25.42%) with NASH. I'd like to make the following observations.

1. Abbreviations such as HS must be clearly indicated.

2. The sample size for each group must be indicated in the legends of figures or tables.

3. What statistical difference is used in each table to compare the variations between what? Three groups were included in each table.

4. What does HS stand for in Figure 4 or 5? Please double-check it.

5. Insulin resistance (IR) has been discussed in the absence of data. Why?

6. The differences between patients with HS who have NAFLD and NASH must be described in detail.

7. There was no study of the time-dependent progression of HS. Why?

8. The serum ferritin and neutrophil percentage-to-albumin ratio (NPAR) as important biomarkers require reference(s).

It seems better to check through the professional editing.

Author Response

Dear Reviewer,

Thank you for your comment and questions!

I appreciate your efforts to improve the quality and content of our article.

 

Next, I will try to answer the questions you asked me.

 

1.Abbreviations such as HS must be clearly indicated.

We have implemented the necessary modifications regarding the abbreviation HS (hepatic steatosis). We apologize for instances where the abbreviation might not have been explicitly indicated. Thank you for your feedback.

 

2.The sample size for each group must be indicated in the legends of figures or tables.

We have specified the sample size in both the figure and table legends. Thanks for the advice.

 

3. What statistical difference is used in each table to compare the variations between what? Three

groups were included in each table.

We acknowledge our initial lack of clarity and apologize for any confusion. In each table, the initial group of patients encompasses the entire cohort (59 patients with NAFLD and T2DM). Subsequently, statistical analysis was conducted between column 2 (comprising 44 patients with HS and T2DM) and column 3 (consisting of 15 patients with NASH and T2DM) to ascertain differences. Thank you for your observation.

 

4. What does HS stand for in Figure 4 or 5? Please double-check it.

We regret any confusion caused by not clearly specifying the abbreviation. To rectify this, we have revised the figure legend to explicitly define the abbreviation HS (hepatic steatosis).

 

5. Insulin resistance (IR) has been discussed in the absence of data. Why?

Thank you for your question. As IR serves as a key element connecting the two conditions, NAFLD and T2DM, we have opted to discuss it in our paper. Regrettably, due to the selected study timeframe, conducting insulinemia assessments for inpatients at the Diabetes and Nutrition Diseases Clinic was unfeasible, thus hindering our ability to evaluate insulin resistance. Given its significant role in NAFLD's pathogenesis, this parameter will undoubtedly be a subject of evaluation and exploration in our future studies.

 

 

6. The differences between patients with HS who have NAFLD and NASH must be described in detail.

Thank you for your advice. We have provided clearer elaboration on the distinctions in the discussion section. Additionally, presented here is a succinct summary of these differences.

 

Considering the distinct progressions of NAFLD's defining entities, HS and NASH, along with the current limited diagnostic methods that differentiate these forms, our study aimed to identify easily accessible blood biomarkers to distinguish between them.

From the gathered data, several distinctions emerged between the two patient groups. Regarding comorbidities, those with hepatic steatosis and diabetes displayed higher rates of obesity, dyslipidemia, and metabolic syndrome. Conversely, patients with steatohepatitis and type 2 diabetes exhibited a greater prevalence of hypertension.

In ultrasound findings, all study participants showed hyperechogenic, diffuse inhomogeneous aspects. However, patients with NASH and T2DM demonstrated enlarged left lobe, right lobe, portal vein, and spleen diameters.

Furthermore, notable variations were observed through laboratory assessments. Hematological and biochemical indicators like Hb, triglycerides, ALT, AST, and GGT exhibited significantly higher levels in patients with NASH and T2DM.

Regarding the analysis of inflammatory parameters, significantly elevated serum ferritin and NPAR were detected in the NASH and T2DM group compared to those with HS and T2DM.

These outcomes hold promise for the future, prompting the need for more expansive studies to determine the predictive potential of inflammatory parameters and their integration into medical practice.

 

7. There was no study of the time-dependent progression of HS. Why?

Thank you for your question. This study had a retrospective design, utilizing pre-existing data from observation records. Our future objective is to conduct a prospective study, tracking the progression of NAFLD in T2DM patients over time. By closely monitoring the transition from hepatic steatosis (HS) to non-alcoholic steatohepatitis (NASH), we aim to identify the precise point of transformation. This approach will allow us to more accurately identify the prognostic factors associated with this condition.

 

8. The serum ferritin and neutrophil percentage-to-albumin ratio (NPAR) as important biomarkers require reference(s).

We have incorporated additional references regarding serum ferritin and NPAR levels, both in the Introduction (lines 93-109) and the Discussion (lines 613-621 and 640-657) sections.

 

We greatly appreciate your guidance and sincerely hope that our revisions meet your expectations.

Thank you once again for your valuable feedback!

Kind regards,

Prof Biciusca

 

 

Reviewer 2 Report

The Manuscript: „A current approach to non-alcoholic steatohepatitis in type 2 diabetes mellitus patients’’ by Sorina I. Stan and colleagues attempts to identify surrogate biomarkers that would differentiate the two forms of liver disease in NAFLD (HS from NASH), with emphasis on inflammatory factors. There are already established biomarkers, such as ALT and AST that are helpful in differentiating these two forms of liver diseases. The present study reported statistically significantly higher serum ferritin and the neutrophil percentage-to-albumin ratio (NPAR) values in NASH patients compared to HS patients. It would be interesting to measure these parameters in some more patients to validate these results. The authors have written the manuscript nicely with convincing methodology supported by promising results. After going through the manuscript, I have following comments for the authors:

1.     With 59 patients included in the study, I doubt that the sample size required to interpret the statistical results was somewhat small. Was a sample size analysis performed and was the sample size power sufficient based on the study’s expected effect size, desired statistical power, and significance level? Was there any dropout of the participants?

2.     Was there a statistical difference or similarity in values of serum ferritin and NPAR among males and females?

3.     The tables and figures need to be improved. Avoid making tables with multi coloured cells (rows and columns) for better optic.

Moderate English correction needed.

Author Response

Dear Reviewer,

Thank you for your comment and questions!

I appreciate your efforts to improve the quality and content of our article.

Next, I will try to answer the questions you asked me.

 

1. With 59 patients included in the study, I doubt that the sample size required to interpret the statistical results was somewhat small. Was a sample size analysis performed and was the sample size power sufficient based on the study’s expected effect size, desired statistical power, and significance level? Was there any dropout of the participants?

While the sample size of 59 patients was small, it provided a sufficient foundation for initial insights into the potential utilization of blood biomarkers in NAFLD diagnosis. However, the study possesses certain limitations due to its retrospective nature and single-center execution, further aggravated by restricted admissions during the COVID-19 pandemic. As such, subsequent investigations are necessary to validate the biomarkers' role. As a future direction of exploration, we propose broadening the patient cohort and extending paraclinical assessments.

Throughout the study, there were no patient withdrawals. Patient data were initially recorded using Microsoft Excel software, with subsequent data processing conducted using the free statistical and graphical analysis software, R-Studio. Descriptive statistics were computed using both Excel and R-Studio and normality testing was performed through the Shapiro-Wilk and Kolmogorov-Smirnov tests.

Spearman's method was employed for correlation and covariance analysis, while the comparison of group differences utilized the t-Student and Mann-Whitney U-Test. The interpretation of statistical test results adhered to the following criteria: p<0.05 indicated a statistically significant difference, p<0.01 denoted a highly significant difference, and p<0.001 signified an exceptionally high level of statistical significance.

 

2. Was there a statistical difference or similarity in values of serum ferritin and NPAR among males and females?

The primary objective of this study was to identify clinical and laboratory parameters exclusively associated with an unfavorable progression of NAFLD. The study's findings indicated a statistically significant correlation between two non-invasive markers, ferritin and NPAR, and the adverse outcome of hepatic steatosis (HS) progressing to non-alcoholic steatohepatitis (NASH). The potential diagnostic and prognostic significance of these inflammatory markers in NAFLD will be assessed in a forthcoming study.

The statistical differences uncovered in this study will be calculated in a subsequent publication, offering a more comprehensive examination of how inflammatory markers relate to demographic, clinical, and laboratory parameters between the two patient groups.

To provide further insight, we calculated the values of these tests for both markers, revealing the following outcomes:

1. For NPAR, among women in the NASH and T2DM group, the mean value was 1280.75±156.519, slightly lower but not statistically significant when compared to the mean value of the HS and T2DM group (1360.42±228.38). In men, the mean NPAR value in the NASH and T2DM group was 1701.90±798.32, lower but not significantly different from the HS and T2DM group (1278.78±235.89).
2. Regarding ferritin, in the female group with NASH and T2DM, the mean value was 133.25±23.69 ng/dL, slightly lower and not statistically significant when compared to the HS and T2DM group (147.14±174.58 ng/dL). In men, the mean ferritin value in the NASH and T2DM group was 314.63±61.52 ng/dL, significantly lower (p=0.001) compared to the HS and T2DM group (222.82±71.97 ng/dL).

Considering these gender-specific results, our future research will focus on a comparative analysis, stratified by sex, to provide a more comprehensive understanding of this subject matter.

 

3. The tables and figures need to be improved. Avoid making tables with multi-colored cells (rows and columns) for better optics.

We apologize for the blurriness and the nuances that arose in the tables, all these changes appeared after uploading the text into the magazine template. We have rectified these matters.

 

Thank you once again for your valuable feedback!

Kind regards,

Prof Biciusca.

 

Round 2

Reviewer 1 Report

It has been revised according to comments.