Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The proposed manuscript “Advanced glycation end products as mediators in the association between pulse wave velocity and glycated haemoglobin in healthy adults: results of the cross-sectional EVasCu study” greatly contributes to the study of cardiovascular disease and may have an impact in clinical studies and public health. All categories such as sex and smoking status, weight were taken into account and were well investigated. The manuscript is technically sound, presented in an intelligible fashion, and written in good-quality English. Statistical analysis was well selected and performed. Impact of the study was well highlighted. I did not find any crucial errors within the proposed manuscript. I believe that the study met the Biomedicines journal requirements and will be of interest to the journal audience. I have three very minor comments to authors:

1) Line 44: just “sex” should be the factor that affects arterial stiffness (instead of “male sex”)

2) The authors used p ≤ 0.05 as a boundary for statistical significance, which is correct. Statistical tests are usually categorized as p ≤ *0.05; **0.01; ***0.001. Maybe this categorization would be more helpful. 

3) What software did the authors use for generation of Scatterplot? Add this information to the manuscript.

After considering the overall quality of the manuscript, I suggest its acceptance. 

 

Author Response

Comments 1: [The proposed manuscript “Advanced glycation end products as mediators in the association between pulse wave velocity and glycated haemoglobin in healthy adults: results of the cross-sectional EVasCu study” greatly contributes to the study of cardiovascular disease and may have an impact in clinical studies and public health. All categories such as sex and smoking status, weight were taken into account and were well investigated. The manuscript is technically sound, presented in an intelligible fashion, and written in good-quality English. Statistical analysis was well selected and performed. Impact of the study was well highlighted. I did not find any crucial errors within the proposed manuscript. I believe that the study met the Biomedicines journal requirements and will be of interest to the journal audience. I have three very minor comments to authors:]

Response 1: [We would like to express our gratitude to the reviewer for taking the time to assess our article and for providing constructive feedback.]

 

Comments 2: [Line 44: just “sex” should be the factor that affects arterial stiffness (instead of “male sex”)]

Response 2: [Thank you for the reviewer's comment, we have substituted ‘sex’ as a factor, instead of ‘male sex’ in line 43.]

 

Comments 3: [The authors used p ≤ 0.05 as a boundary for statistical significance, which is correct. Statistical tests are usually categorized as p ≤ *0.05; **0.01; ***0.001. Maybe this categorization would be more helpful.]

Response 3: [Thank you for the reviewer's comment, we have categorised statistical significance according to the classification suggested in Figure 2, Table 1, Table 2, Table 3, Supplementary Table S3 and Table S4.]

 

Comments 4: [What software did the authors use for generation of Scatterplot? Add this information to the manuscript.]

Response 4: [Thank you for the reviewer's comment, we have included in the methodology the software used to generate the scatterplot.

Lines 253-254: “STATA 15.1 software was used to generate the LOESS scatterplot”.]

 

Comments 5: [After considering the overall quality of the manuscript, I suggest its acceptance.]

Response 5: [We thank the reviewer´s opinion on our article and for considering it for acceptance.]

 

Reviewer 2 Report

Comments and Suggestions for Authors

This article titled “Advanced glycation end products as mediators in the associa- 2tion between pulse wave velocity and glycated haemoglobin in 3 healthy adults: results of the cross-sectional EVasCu study”explores the relationship between glycosylated hemoglobin and aortic pulse wave velocity in subjects and analyzes its internal mechanisms. The study has some practical significance, but there are problems and shortcomings in the introduction of the method part of the article, which may affect the overall quality of the study, and the detailed comments from the reviewers are as follows:

1.     The authors did not provide information on the gender ratio of participants in the dataset.

2.     The specific meaning and calculation method of the EVA index mentioned by the author in the sample calculation section need to be clarified so that readers can understand its statistical significance.

3.     Authors are advised to be more specific about exclusion criteria, such as which medications would cause exclusion, and the specific type of cardiovascular disease.

4.     Please provide the specific methods and steps for the a-PWV evaluation, as well as the detailed parameters of the non-invasive oscillatory measurement technology - nique mobile-o-graph® (IEM GmbH).

5.     The authors are advised to describe the specific method and procedure for the determination of HbA1c by HPLC, as well as the detailed parameters of the AD-118 AMS A1c analyzer HA-8180 V. It is also introduced that venous blood collection after 12 hours of fasting should detail the specific time and method of collection.

6.     The authors mention the avoidance of skin defects and the choice of a darkroom for measurements, please provide specific guidelines or criteria for how to do it.

7.     The selected covariates (age, gender, smoking status, etc.) should be based on previous research and scientific literature, please provide specific references or evidence.

8.     Socioeconomic status as a covariate may have an important impact, but please elaborate on its measurement methods and criteria.

9.     The method of processing and measurement of blood samples is clearly described, but please ensure that the system provided (e.g., Roche Diagnostics COBAS C711 system) and reagents (e.g., hexokinase, chemiluminescent particle immunoassay) are accurate and widely accepted.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Comments 1: [This article titled “Advanced glycation end products as mediators in the associa- 2tion between pulse wave velocity and glycated haemoglobin in 3 healthy adults: results of the cross-sectional EVasCu study”explores the relationship between glycosylated hemoglobin and aortic pulse wave velocity in subjects and analyzes its internal mechanisms. The study has some practical significance, but there are problems and shortcomings in the introduction of the method part of the article, which may affect the overall quality of the study, and the detailed comments from the reviewers are as follows:]

Response 1: [We would like to thank the reviewer for taking the time to review our article and provide constructive comments.]

 

Comments 2: [The authors did not provide information on the gender ratio of participants in the dataset.]

Response 2: [Thank you for the reviewer's comment. We have added the absolute number next to the percentages to complete the information in Table 1 and to be able to estimate the gender ratio of the participants in the dataset.]

 

Comments 3: [The specific meaning and calculation method of the EVA index mentioned by the author in the sample calculation section need to be clarified so that readers can understand its statistical significance.]

Response 3: [Thank you for the reviewer's comment. We have expanded the section on sample size calculation, providing information on the meaning of the EVA index, which was previously not explained and could lead to confusion with other terms. We have also clarified the calculation of sample size, specifying the importance of exceeding the sample size calculated by Epidat.

Lines 104-113: “The sample size was calculated via Epidat software, which yielded a sample size of 355 healthy participants, an estimated effect of 1, an alpha risk of 0.05 and an absolute precision level of 0.04 to detect a statistically significant result for the Early Vascular Aging Index (EVA Index) [29]. The EVA Index is used to assess vascular health and identify signs of accelerated arterial aging, making it clinically relevant in the early detection of cardiovascular risk factors. The effect size of 1 was determined on the basis of previous studies to ensure that the calculation had sufficient statistical power to allow the study to detect significant differences between individuals. Finally, 390 participants were enrolled, a number that exceeded the sample size originally calculated to allow for potential dropouts or study variability.”]

 

Comments 4: [Authors are advised to be more specific about exclusion criteria, such as which medications would cause exclusion, and the specific type of cardiovascular disease.]

Response 4: [Thank you for the reviewer's comment. We agree with the reviewer's comment and have incorporated the exclusion criteria.

Lines 117-120: “Subjects enrolled in other trials of any intervention, those receiving pharmacological treatment (such as hypoglycaemic drugs, lipid-lowering drugs, antihypertensive drugs, etc.), or those with a previous diagnosis of CVD (such as diabetes, arterial hypertension, myocardial infarction, or stroke) were excluded from the study.”]

 

Comments 5: [Please provide the specific methods and steps for the a-PWV evaluation, as well as the detailed parameters of the non-invasive oscillatory measurement technology - nique mobile-o-graph® (IEM GmbH).]

Response 5: [Thank you for the reviewer's comment. In the methodology section (2.3.), we have provided more information on the assessment of a-PWV, as well as on the parameters provided by the Mobil-o-graph oscillatory device.

Lines 124-139: “Arterial stiffness was assessed by a-PWV via the non-invasive oscillometric technique Mobil-O-Graph® (IEM GmbH). This method has been shown to provide values with an acceptable level of accuracy compared with intra-aortic measurements [30]. The validated ARCSolver algorithm (Austrian Institute of Technology GmbH, Vienna Austria) was used [31,32]. Measurements were performed in a quiet environment after a five-minute rest period, during which the participant was asked to avoid caffeine, smoking or physical activity for at least 30 minutes prior to measurement. With the participant seated with their legs uncrossed and feet flat on the floor, a cuff appropriate for the size of the non-dominant arm was placed approximately 2 cm above the elbow. The arterial symbol was placed over the brachial artery, and the pressure tube was passed unbent behind the neck. After entering the patient's age, sex, weight, height and smoking habits into the software, the participant was instructed not to speak during the measurement. The device inflates the cuff and records the oscillations of the brachial pulse wave, providing pulse wave analysis, along with measurements of brachial blood pressure parameters, central blood pressure, augmentation pressure, the augmentation index, peripheral vascular resistance, and cardiac output.”]

 

Comments 6: [The authors are advised to describe the specific method and procedure for the determination of HbA1c by HPLC, as well as the detailed parameters of the AD-118 AMS A1c analyzer HA-8180 V. It is also introduced that venous blood collection after 12 hours of fasting should detail the specific time and method of collection.]

Response 6: [Thank you for the reviewer's comment. The specific procedure as well as the detailed parameters of the analyser were performed in the hospital laboratory, we have added this information in the new version of the article. The time and specific method of venous blood collection have been detailed in this new version.

Lines 141-149: “Venous blood samples were taken from participants between 8:00 and 9:00 am. The participants were instructed to fast overnight, with no food or caloric drinks for 12 hours prior to blood extraction. Venipuncture was performed via the antecubital vein of each participant. Blood samples were collected and stored according to established protocols and then sent to the laboratory of the Virgen de La Luz Hospital in Cuenca for subsequent analysis. The samples were handled in a controlled manner to maintain their integrity and obtain accurate laboratory results. HbA1c was determined by high-performance liquid chromatography using the ADAMS A1c analyser HA-8180 V from A. Menarini Diagnostics®.”]

 

Comments 7: [The authors mention the avoidance of skin defects and the choice of a darkroom for measurements, please provide specific guidelines or criteria for how to do it.]

Response 7: [Thank you for the reviewer's comment. More information on skin care and dark room selection was provided as suggested by the reviewer.

Lines 162-168: “The participants were asked not to use any skin creams prior to the measurement to avoid interference with the results [34]. In cases where creams were used, the skin of the forearm was cleaned with soap and water and dried before measurement. In addition, the light in the room was controlled, and a dim room was chosen for all measurements. The amount of light in the room was controlled to avoid interference from any external light source, both natural and artificial.”]

 

Comments 8: [The selected covariates (age, gender, smoking status, etc.) should be based on previous research and scientific literature, please provide specific references or evidence.]

Response 8: [Thank you for the reviewer's comment. Specific references to previous research on possible covariates were provided (Line 172).]

 

Comments 9: [Socioeconomic status as a covariate may have an important impact, but please elaborate on its measurement methods and criteria.]

Response 9: [Thank you for the reviewer's comment. Due to a lack of data to calculate socioeconomic status from the available data, education level, which is recognised as an important socioeconomic determinant of cardiovascular risk, was used instead. Information has been added to clarify this.

Lines 197-201: “Educational level was measured via a self-report questionnaire with 6 response options: i) unable to read or write; ii) incomplete primary education; iii) primary education; iv) secondary education; v) professional education/higher education; and vi) university education. Educational level has been recognised as a socioeconomic determinant of CV risk [38] and was therefore included as a covariate.”]

 

Comments 10: [The method of processing and measurement of blood samples is clearly described, but please ensure that the system provided (e.g., Roche Diagnostics COBAS C711 system) and reagents (e.g., hexokinase, chemiluminescent particle immunoassay) are accurate and widely accepted.]

Response 10: [Thank you for the reviewer's comment. Blood samples were analysed at the reference hospital in Cuenca (Virgen de La Luz Hospital), using an accurate and widely accepted system and reagents. Information on the management of the analysis of blood samples by the hospital laboratory was added to the manuscript.

Lines 182-184: “Blood samples were collected and stored according to established protocols and then sent to the laboratory of the Virgen de La Luz Hospital in Cuenca for subsequent analysis.”]

 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors need to work on the paper more diligently to make it useful for the readers and scientific community.

1.      The title could be more concise while retaining essential details. Consider rephrasing to something like, "Role of Advanced Glycation End Products in Mediating Pulse Wave Velocity and Glycated Hemoglobin in Healthy Adults."

2.      Expand on the novelty of the study. While it mentions that this is the first study to explore the mediation effect of SAF in this relationship, consider providing more context about prior research gaps.

3.      Discuss potential biases introduced by the recruitment method (e.g., advertisement through social networks). Did this method result in a representative sample of the target population?

4.      Some confounding variables, such as dietary habits and physical activity, were only partially addressed.

5.      Add more details about its SAF validation and reproducibility across different populations.

6.      Provide a more explicit rationale for choosing specific models (e.g., unadjusted vs. adjusted). Were other statistical approaches considered, such as structural equation modeling, to strengthen the mediation analysis?

7.      Elaborate on the practical implications of using SAF as a biomarker in healthy adults. How feasible is its adoption in routine clinical practice?

8.      Expand on the limitations due to the study population being predominantly healthy European adults. Could ethnic or genetic factors influence SAF and its relationship with HbA1c and a-PWV?

9.      Figure 2 is not upto the mark

10.   The plagiarism percentage i.e 25% is very high.

Author Response

Comments 1: [The authors need to work on the paper more diligently to make it useful for the readers and scientific community.]

Response 1: [Thank you for your feedback. We appreciate your commitment to ensuring the quality and relevance of the manuscript. In response to your comments, we have undertaken a comprehensive review of the manuscript to improve its clarity, scientific rigour, and usefulness to readers and the wider scientific community.]

 

Comments 2: [The title could be more concise while retaining essential details. Consider rephrasing to something like, "Role of Advanced Glycation End Products in Mediating Pulse Wave Velocity and Glycated Hemoglobin in Healthy Adults."]

Response 2: [Thank you for the reviewer's comment. As suggested, we have changed the title to a version similar to the one provided by the reviewer.

“Role of Advanced Glycation End Products in Mediating Glycated Haemoglobin and Pulse Wave Velocity in Healthy Adults”.]

 

Comments 3: [Expand on the novelty of the study. While it mentions that this is the first study to explore the mediation effect of SAF in this relationship, consider providing more context about prior research gaps.]

Response 3: [Thank you for the reviewer's comment. A suggested, the information regarding the novelty of the study has been expanded, considering previous gaps in knowledge.

Lines 58-66: “Several studies have reported a significant association between PWV and HbA1c across different demographic groups, including the Spanish population [13,16,17]. However, important gaps in the understanding of this association remain, as the underlying biological mechanisms are not fully understood. Several pathophysiological pathways associated with HbA1c, such as arterial wall thickness, advanced protein glycation, stress and/or chronic inflammation, influence arterial stiffness [6,18,19]. In particular, advanced protein glycation and the subsequent accumulation of advanced glycation end products (AGEs) may play key mediating roles in this relationship [6], although this interpretation is speculative and needs to be confirmed.”

Lines 79-91: “To our knowledge, there are no reports exploring the possible mediating role of SAF in the relationship between HbA1c and a-PWV. Although previous studies have speculated on this possibility[6], research has been limited to exploring bivariate relationships between parameters without considering all three simultaneously [6,17,20,21]. Mediation analysis provides information on the association between two factors, in this case, HbA1c and a-PWV, and the extent to which a third variable, SAF, mediates the main association. Furthermore, the study of this relationship in subjects without diagnosed pathologies could be highly important since it could identify direct and intermediate or mediating factors at an early stage, contributing to improving the design of preventive strategies for cardiovascular health. Because of the need to rationally substantiate the mechanism of arterial stiffness, this study was conducted with the objective of assessing the associations among the variables HbA1c, a-PWV and SAF, as well as the ability of the SAF to mediate the main association between HbA1c and a-PWV in healthy adults.”]

 

Comments 4: [Discuss potential biases introduced by the recruitment method (e.g., advertisement through social networks). Did this method result in a representative sample of the target population?]

Response 4: [Thank you for the reviewer's comment. The possible bias due to the recruitment method was added as a limitation, as suggested by the reviewer.

Lines 446-448: “Third, the recruitment method may have influenced the composition of the sample, limiting the representativeness of the study population in relation to the target population.”]

 

Comments 5: [Some confounding variables, such as dietary habits and physical activity, were only partially addressed.]

Response 5: [Thank you for the reviewer's comment. The discussion of the article was restructured to emphasise why ethnicity, diet or physical activity were not included as covariates in our study. In addition, the lack of inclusion of potential confounding variables was included as a limitation of our study.

Lines 405-424: “The results of this study should be interpreted with caution, as some potentially relevant covariates were not included in the analysis, which may affect the generalisability of the findings. Ethnicity and genetic factors may influence SAF [25], but our study consisted of a healthy population of European origin, so the inclusion of this variable was not considered due to the homogeneity of the sample. Previous studies have reported that diet and food preparation may influence some variables, especially AGEs [54–56]. In our study, this information was collected via the Mediterranean Diet Adherence Screener questionnaire [57]; however, there could be recall bias, in addition to the questionnaire’s inability to assess in depth the cooking, frequency, and quantity of food consumed. The level of physical activity could be another possible confounding factor in the main association [58]. In our study, physical condition was assessed via the International Fitness Scale [59], which is subjective in nature and depends on self-perception when self-reported. Physical activity was also assessed via wearable activity trackers (Xiaomi bands), but the rate of data loss for this variable was significant. These variables were not included in the study as potential confounders, as the DAG plot indicated that they were not included according to the MSAS. In addition, the adjusted model was analysed via multiple linear regression, which revealed that there was no collinearity between the variables and that 90% of the variability in a-PWV was explained by the covariates selected by the DAG (MSAS). This suggests that the selected variables adequately capture the relevant factors for the model.”

Lines 453-455: “Sixth, another limitation of our study could be the non-inclusion of confounding variables, such as ethnicity, genetic factors, diet or physical activity, which could bias the model.”]

 

Comments 6: [Add more details about its SAF validation and reproducibility across different populations.]

Response 6: [Thank you for the reviewer's comment. As suggested, more details on the validation and reproducibility of the SAF in different populations have been included

Lines 72-78:“ Skin autofluorescence (SAF) is a non-invasive, rapid, and simple technique for the direct measurement of AGE accumulation that has been widely validated and reproduced in different populations, including healthy individuals, patients with diabetes, and different ethnic groups [20,24,25]. Despite its proven accuracy and efficacy, it is important to consider specific factors that may influence the test results, particularly age, which has been shown to significantly affect SAF measurements ​[24].”]

 

Comments 7: [Provide a more explicit rationale for choosing specific models (e.g., unadjusted vs. adjusted). Were other statistical approaches considered, such as structural equation modeling, to strengthen the mediation analysis?]

Response 7: [Thank you for the reviewer's comment. A mediation analysis was performed because the aim of the study was to evaluate how SAF could explain part of the association between HbA1c and a-PWV. This association is widely known, however, the role of SAF in this relationship has not been evaluated so far. To do this, it is important to explore how the model might change when adjusting for confounding variables. Structural equation modelling is a powerful option for modelling complex associations, but this approach was not considered due to the small sample size, the simplicity of the model, and the multiple analyses performed (analysis of covariance, multiple regression, adjusted and unadjusted mediation, use of bootstrapping). A more explicit explanation of the choice of adjusted and unadjusted models has been provided in the study methodology.

Lines 235-238: “Mediation analyses were performed both with the unadjusted model to assess the direct and indirect relationships between the three variables of interest and with the adjusted MSAS model to ensure more accurate and robust estimates.”]

 

Comments 8: [Elaborate on the practical implications of using SAF as a biomarker in healthy adults. How feasible is its adoption in routine clinical practice?]

Response 8: [Thank you for the reviewer's comment. The practical implications of using SAF as a biomarker in healthy adults have been expanded and its use in consultation has been presented as feasible due to the speed and simplicity of the test.

Lines 436-440: “In addition, it could improve primary care in general by facilitating decision making, reducing the burden on the healthcare system and promoting personalised medicine. However, although it is a simple and rapid test, cost‒benefit studies are needed to assess its introduction into routine clinical practice.”]

 

Comments 9: [Expand on the limitations due to the study population being predominantly healthy European adults. Could ethnic or genetic factors influence SAF and its relationship with HbA1c and a-PWV?]

Response 9: [Thank you for the reviewer's comment. As suggested, the study limitations have been expanded to include the influence of ethnicity on SAF and its relationship with HbA1c and a-PWV.

Lines 405-409:“ The results of this study should be interpreted with caution, as some potentially relevant covariates were not included in the analysis, which may affect the generalisability of the findings. Ethnicity and genetic factors may influence SAF [25], but our study consisted of a healthy population of European origin, so the inclusion of this variable was not considered due to the homogeneity of the sample.”

Line 453-455: “Sixth, another limitation of our study could be the non-inclusion of confounding variables, such as ethnicity, genetic factors, diet or physical activity, which could bias the model.”]

 

Comments 10: [Figure 2 is not upto the mark.]

Response 10: [Thank you for the reviewer's comment. Figure 2 has been improved, providing a higher quality image.]

 

Comments 11: [The plagiarism percentage i.e 25% is very high.]

Response 11: [Thank you for the reviewer's comment. The fact that a larger number of studies have been previously published on the EVasCu study is a point of discrepancy in terms of plagiarism rate, as a significant part of the methodology is similar. However, it should be noted that the remaining sections of the study are completely different. In an effort to mitigate the above concerns, changes have been made to the methodology, including changes in wording, with the objective of reducing the plagiarism rate.]

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

All my questions have been well addressed, now I recommend to accept. 

Reviewer 3 Report

Comments and Suggestions for Authors

Authors have addressed/answered all my queries and i am satisfied with the revised manuscript.