Review Reports

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Overall Evaluation

This publication presents a narrative and mechanistic analysis that introduces the notion of a “salivary redoxome” as a hypothesis-generating research framework to investigate redox-related cardiac electrical vulnerability in sudden unexplained cardiac death (SUCD). The resubmission is far better than previous versions in terms of concept development, internal coherence, and the repeated, appropriate caveats that the proposed framework is not intended for clinical screening or prediction.

The topic is pertinent to Medicina and addresses a significant void in our comprehension at the intersection of redox biology, electrophysiology, and noninvasive biomarker research. The manuscript is scientifically sound, well-referenced, and generally well written. However, the manuscript still faces some conceptual, methodological, and presentational issues that limit its impact and require resolution before acceptance.


Major concerns:
1. Epidemiological claims (Section 4.1)

Some epidemiological claims (such as trends in incidence, comparisons across regions, and reasons for the rise in SCD among young adults) are made with greater certainty than the evidence supports. Certain causal attributions (e.g., air pollution, psychological stress) are conjectural in the context of SUCD and must be distinctly identified as associative or hypothetical.

Suggestion: Make epidemiological language clearer, cut down on guesses about causes, and make it clear that SCD trends are different from SUCD-specific information.

2. Saliva–Heart Biological

Even though there are some connections between saliva and overall body oxidative markers, the study does not fully investigate how the redox state in the mouth relates to heart electrical activity. The possible
The potential for confounding originating from the oral cavity (such as periodontal inflammation and microbiome impacts) is recognized but not thoroughly examined.

Suggestion: Include a brief segment that talks about

Why redox indicators in saliva can indicate systemic electrophysiological vulnerability rather than just local oral processes. What biological premises underpin this extrapolation?

3. The Methodology Section

The search approach is presented only briefly. A single author selected the studies, though no reason was given. There is no flowchart or numerical summary of the included studies.
This restriction limits openness even for a narrative evaluation.

Suggestion: The authors should do the following without turning their paper into a systematic review:

Add a short table or paragraph that summarizes the search (databases, number of articles that were screened or included).
Could you please provide a reason for selecting only one reviewer? Clearly, no effort was taken to be complete.

4. Figures: While they are useful for understanding the concepts, they lack scientific rigor.

Figure 1 is descriptive but doesn't help with analysis too much.

Figure 2, based on previous research, supports generic SCD mechanisms but does not advance the salivary redox hypothesis.

Suggestion: Think about changing or adding a schematic drawing that directly connects:
The concept clearly characterizes the relationship between salivary redox indicators, systemic oxidative balance, electrophysiological substrates, and arrhythmic susceptibility.

5. The innovation claim needs to be framed more clearly. The new thing is mostly the integration, not the finding of biomarkers. Some of the assertions in the "Statement of Novelty" could sound stronger than they really are.

Suggestion: Change the way you think about novelty: put ideas together.
In light of a research gap, a testable framework is proposed, not a new biomarker technique.

Small concerns

Language and Style

There are still some small grammatical errors.
Some repetition, especially in the disclaimers, should be reduced.

Words

Use "young individuals" or specify an age range.
Don't switch between "Western," "European," and "North American" groups without a good reason.

Sources

Overall, strong.
If you can, consider adding at least one SUCD review focused on electrophysiology.
Always explain all abbreviations the first time they are used (some appear later).

 

Author Response

We sincerely thank the Reviewer for the careful re-evaluation of our revised manuscript and for the constructive and balanced feedback. We greatly appreciate the positive assessment of the improved conceptual development, internal coherence, and the clear positioning of the proposed salivary redoxome as a hypothesis-generating, non-clinical research framework. The Reviewer’s detailed comments have been invaluable in further refining the epidemiological framing, methodological transparency, biological plausibility, and presentation of the manuscript. We have addressed each concern in detail below.

Major Concerns

1. Epidemiological Claims (Section 4.1)

Reviewer comment:
Some epidemiological statements are presented with excessive certainty, and causal attributions (e.g., air pollution, psychological stress) are speculative in the context of SUCD.

Response:
We agree with this comment and have revised Section 4.1 to improve precision and clarity. Epidemiological language has been softened throughout to clearly distinguish associative observations from causal inference. We now explicitly differentiate between general SCD trends and SUCD-specific evidence, emphasizing that many population-level observations are derived from broader SCD datasets rather than SUCD-focused analyses. Environmental and psychosocial factors (e.g., air pollution, psychological stress) are now consistently described as associative or hypothetical contributors, and their relevance to SUCD is framed cautiously and explicitly as indirect.

2. Saliva–Heart Biological Relationship

Reviewer comment:
The biological rationale linking salivary redox status to cardiac electrical vulnerability is insufficiently developed, and oral confounders are not thoroughly examined.

Response:
We appreciate this important conceptual point. The manuscript has been revised to more clearly articulate the biological premises underpinning the proposed extrapolation. We now emphasize that salivary redox biomarkers are not intended to reflect myocardial-specific processes, but rather to serve as non-invasive indicators of systemic redox balance, which may influence electrophysiological vulnerability indirectly.

In addition, we have expanded discussion of oral and systemic confounders, including periodontal inflammation, microbiome influences, lifestyle factors, and pre-analytical variability. These limitations are now more explicitly integrated into the relevant sections and framed as key challenges for future validation studies rather than overlooked variables.

3. Methodology Section

Reviewer comment:
The search strategy lacks transparency, the use of a single reviewer is not justified, and no numerical summary of study selection is provided.

Response:
We agree that greater transparency is warranted, even for a narrative review. The Methodology section has been revised to address these points without converting the manuscript into a systematic review. Specifically:

• A numerical summary of the literature search has been added, including databases searched, numbers of records screened, full-text articles reviewed, and studies included.
• The single-reviewer approach is now explicitly justified as a deliberate choice to maintain conceptual consistency in the qualitative integration of mechanistic evidence, rather than to achieve exhaustive coverage.
• We clearly acknowledge that the review was not intended to be comprehensive, and that no formal quality-scoring or risk-of-bias assessment was performed, which is now stated explicitly as a limitation of the exploratory design.

4. Figures

Reviewer comment:
The figures are descriptive and do not sufficiently advance the salivary redox hypothesis.

Response:
We agree and have revised the visual framework accordingly. A new conceptual schematic (Figure 3) has been added to directly illustrate the proposed relationships among salivary redox biomarkers, systemic redox balance, electrophysiological substrates, and arrhythmic susceptibility. The figure is explicitly framed as hypothesis-generating and non-predictive, and the accompanying legend has been strengthened to clarify its conceptual intent. Existing figures are now more clearly contextualized to avoid overinterpretation.

5. Framing of Innovation and Novelty

Reviewer comment:
Novelty lies primarily in integration rather than biomarker discovery, and some claims appear overstated.

Response:
We fully agree. The Statement of Contribution and related sections have been revised to emphasize that the primary innovation of this work lies in conceptual integration across redox biology, electrophysiology, and salivary biomarker research. Any language that could be interpreted as proposing a new biomarker or clinical application has been softened or removed. The manuscript now consistently presents the salivary redoxome as a testable, hypothesis-generating research framework, rather than a validated diagnostic approach.

Additional sources:

In response to Reviewer 1’s suggestion, we have expanded the reference list to include more relevant studies focusing on electrophysiology, sudden unexplained cardiac death, and salivary biomarkers.

Response to Minor Concerns

Language and Style

Minor grammatical errors have been corrected, and repetitive disclaimers—while retaining appropriate caution—have been reduced to improve clarity and flow.

Terminology

Terminology has been standardized throughout the manuscript. Age ranges are now specified consistently, and geographically imprecise terms (e.g., “Western”) have been removed in favor of European or North American descriptors aligned with the source data.

Sources and Abbreviations

An additional SUCD review with an electrophysiological focus has been incorporated where appropriate. All abbreviations are now defined at first mention.

Closing Statement

We are grateful to the Reviewer for the thoughtful and constructive feedback. The revisions undertaken have strengthened the manuscript’s scientific rigor, conceptual clarity, and transparency while preserving its exploratory and hypothesis-generating nature. We believe the revised version more clearly communicates the intended contribution and addresses all raised concerns.

Reviewer 2 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

Major Issues

1. Narrative Review Methodology Lacks Rigor and Transparency

Although the manuscript is clearly framed as a narrative and hypothesis-generating review, the methodological description remains insufficiently rigorous, even by narrative review standards.

• Study selection, screening, and data extraction were performed by a single author, without independent verification or adjudication.
• No structured framework (e.g., SANRA, PRISMA-ScR, or equivalent narrative-review guidance) is referenced or applied.
• The number of retrieved, screened, excluded, and included studies is not reported, nor is there a qualitative justification for why specific mechanistic studies were prioritized over others.
• No formal or semi-formal assessment of study quality, reproducibility, or translational relevance is provided.

 

2. Conceptual Leap from Redox Biology to SUCD Remains Weakly Substantiated

A central limitation is that no direct evidence is presented linking salivary oxidative stress markers to SUCD, or even to validated electrophysiological risk markers in healthy populations.

• The mechanistic discussion convincingly links oxidative stress to arrhythmogenesis at the cellular and tissue level, particularly via ion-channel modulation, calcium handling, and CaMKII signaling.
• However, the transition from myocardial redox signaling to salivary biomarker readouts remains largely inferential.
• Correlations between salivary and plasma oxidative markers are cited, but organ-specific relevance (i.e., myocardium vs. systemic oxidative tone) is not critically examined.
• The manuscript occasionally risks implying that systemic redox imbalance captured in saliva may reflect cardiac-specific electrical vulnerability, a claim that is biologically plausible but currently unproven.

3. Insufficient Critical Appraisal of Biomarker Specificity and Confounding

The manuscript catalogs multiple salivary biomarkers (SOD, CAT, GPx, MDA, TAC), but the critical appraisal of their limitations remains superficial.

Key issues that require deeper discussion include:

• Lack of disease specificity for oxidative stress markers across inflammatory, metabolic, infectious, and psychological conditions.
• High intra-individual and inter-individual variability driven by diet, circadian rhythm, physical activity, oral health, and stress.
• The difficulty of disentangling acute oxidative responses from chronic redox imbalance relevant to arrhythmogenic remodeling.

 

4. Redundancy and Overlength Reduce Scientific Clarity

There is substantial redundancy across the Introduction, Background, Discussion, and Conclusion sections.

• Epidemiological data and conceptual framing are reiterated multiple times with minimal new insight.
• Repeated emphasis on the “conceptual” and “non-clinical” nature of the approach, while appropriate, contributes to unnecessary length.
• Several mechanistic sections overlap in content without advancing the argument.

This redundancy dilutes the manuscript’s impact and obscures its core contribution.

 

 

Author Response

We sincerely thank the Reviewer for the careful and critical evaluation of our manuscript and for the detailed, constructive comments. We appreciate the Reviewer’s recognition of the hypothesis-generating nature of the work and the thoughtful critique aimed at strengthening methodological transparency, conceptual rigor, and scientific clarity. We have revised the manuscript accordingly and address each major concern in detail below.

Major Issues

1. Narrative Review Methodology Lacks Rigor and Transparency

Reviewer comment:
The methodological description is insufficiently rigorous, including single-author screening, lack of a structured framework, absence of numerical reporting, and no assessment of study quality or translational relevance.

Response:
We thank the Reviewer for this important comment. The manuscript has been revised to improve methodological clarity and transparency while maintaining its intended narrative and hypothesis-generating design.

In the revised version, we now provide a clearer description of the literature search strategy, including the databases searched, time frame, and a numerical summary of records screened, reviewed in full text, and included. The review is explicitly framed as a selective narrative synthesis, with studies prioritized based on mechanistic plausibility, relevance to cardiac electrophysiology and arrhythmogenesis, and translational significance, rather than exhaustive coverage.

Formal structured review frameworks (e.g., SANRA, PRISMA-ScR) and formal quality or risk-of-bias assessments were not applied, as the aim of this work was not systematic mapping or scoping of the literature, but conceptual integration of mechanistic and translational evidence. We now explicitly acknowledge this as a limitation inherent to the exploratory, hypothesis-generating nature of the review.

2. Conceptual Leap from Redox Biology to SUCD

Reviewer comment:
The transition from myocardial redox signaling to salivary biomarker readouts remains largely inferential, with no direct evidence linking salivary oxidative markers to SUCD or validated electrophysiological risk markers.

Response:
We fully agree with this assessment and have revised the manuscript to more explicitly emphasize this limitation and clarify conceptual boundaries.

Revisions include:

• Clear and repeated statements that no direct clinical evidence currently links salivary oxidative stress markers to SUCD or to validated electrophysiological risk markers in healthy populations.
• Explicit differentiation between systemic redox balance and myocardial redox signaling, emphasizing that salivary biomarkers are proposed as indirect, non-specific indicators of global physiological redox tone rather than cardiac-specific readouts.
• Strengthened language throughout the manuscript to avoid any implication that salivary redox profiles reflect cardiac-specific electrical vulnerability, framing the relationship as biologically plausible but currently unproven.
• Alignment of the text with the newly added conceptual schematic (Figure 3), which illustrates an indirect, systemic pathway linking redox balance to electrophysiological susceptibility in a hypothesis-generating manner.

These revisions aim to prevent overinterpretation and clearly distinguish mechanistic plausibility from clinical validation.

3. Insufficient Critical Appraisal of Biomarker Specificity and Confounding

Reviewer comment:
The critical discussion of salivary biomarker limitations remains superficial, particularly regarding non-specificity and confounding.

Response:
We agree and have expanded the critical appraisal of biomarker limitations and confounders.

The revised manuscript now more explicitly discusses:

• The lack of disease specificity of oxidative stress markers across inflammatory, metabolic, infectious, and psychological conditions.
• High intra-individual and inter-individual variability driven by diet, circadian rhythm, physical activity, oral health, and psychosocial stress.
• The challenge of distinguishing acute oxidative responses from chronic redox imbalance, which is more likely to be relevant to arrhythmogenic remodeling.
• The need for standardized sampling protocols and repeated measurements in future studies to improve interpretability.

These limitations are now more clearly integrated into the relevant sections and explicitly framed as barriers to immediate translational application.

4. Redundancy and Overlength Reduce Scientific Clarity

Reviewer comment:
Substantial redundancy across sections reduces clarity and dilutes the manuscript’s core contribution.

Response:
We acknowledge this concern and have revised the manuscript to improve conciseness and focus. Specifically:

• Repeated epidemiological descriptions have been condensed.
• Overlapping conceptual statements and repeated disclaimers regarding the non-clinical nature of the framework have been reduced while retaining appropriate caution.
• Mechanistic sections were streamlined to minimize overlap and improve argumentative progression.

These changes were made to enhance readability and highlight the manuscript’s integrative contribution more clearly.

Closing Statement

We thank the Reviewer again for the thoughtful and rigorous critique. The revisions undertaken have strengthened the manuscript’s methodological transparency, conceptual precision, and critical balance, while preserving its hypothesis-generating intent. We believe the revised version more clearly delineates the scope, limitations, and intended contribution of the proposed framework.

Round 2

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Every request was fulfilled right away, and the article has become more influential.  

Reviewer 2 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

no comments

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

1. Manuscript Summary
A conceptual and mechanistic narrative review proposes salivary oxidative stress indicators as non-invasive methods for early risk identification of Sudden Unexplained Cardiac Death (SUCD) in young, seemingly healthy persons. Redox imbalance, ROS-mediated arrhythmogenesis, electrophysiologic remodeling, and saliva-based biomarkers, including SOD, CAT, GPx, MDA, and TAC, are reviewed. They propose the "Salivary Redoxome" as a future integrated biosensing platform. The review examines molecular pathways, epidemiology, analytical considerations, and biosensors to support saliva as an SUCD risk biomarker.

2. Key Strengths
1. Hot and relevant subject
In young people, traditional diagnostic modalities (ECG, echocardiography, genetics) often fail to identify subclinical susceptibility; therefore, molecular screening tools for SUCD are needed.
2. Mechanism-driven story
The academically sound sections on oxidative regulation of ion channels, CaMKII-mediated calcium leakage, and mitochondrial failure (pp. 5–7) are nicely integrated with arrhythmogenic substrate literature.
3. Translational idea “Salivary Redoxome.”
The paper presents a novel biomarker panel and biosensor technology that aligns with digital and preventive cardiology trends.
4. Correct clinical and epidemiologic context
Epidemiology (pp. 4–5) accurately explains incidence (1–2/100,000), male preponderance, athletic risk, and SUCD's diagnostic blind spot.

3. Major Issues (Revise Significantly)
1. The Medicina narrative review approach is inadequate.
The Methods section (p. 3) does not meet MDPI narrative or scoping review standards.
Issue: Sparse description of search technique.
No information on study selection, number of included studies, exclusion flow, or priority criteria.
• No difference between mechanistic, translational, or clinical evidence.
Recommendation:
Structure the Methods section as a narrative review:
• Information about databases, search dates, and Boolean strategies.
• Inclusion/exclusion criteria.
The inclusion of animal, cellular, or human investigations.
• Managing duplicate reports.
• Assessment strategy for mechanistic validity.
2. The review exaggerates clinical applicability without proof.
The text suggests salivary biomarkers may be helpful to for screening SUCD, but no research has been conducted on them.
• No direct correlation between salivary redox state and electrophysiologic indicators (QT dispersion, PVC load, HRV changes) in healthy kids.
• SUCD has a low prevalence, reducing its prognostic utility.
Recommendation
Authors must acknowledge the conceptual character of the proposal and mention that screening is speculative until prospective cohorts are done.
3. Evidence is not critically assessed.
In the biomarker section (p. 7), salivary SOD, CAT, GPx, and MDA correlate with serum equivalents, although no meta-analysis is provided.
• No mention of inter-assay variability, intra-individual variance, or diagnostic accuracy measures (AUC, sensitivity/specificity).
• No comparison with established cardiac biomarkers (troponin, BNP) or autonomic signs.
Recommendation
Evaluate the strength of evidence regarding the saliva-blood link.
• Oxidative biomarker specificity.
• Oral cavity irritation might confuse.
• Insufficient long-term cardiology data.

4. SUCD pathophysiology is oversimplified.
The text (p. 6–7) and Figure 3 suggest a straight path from oxidative imbalance to SUCD.
Several factors, including genetic ion-channelopathies, subtle structural cardiomyopathies, and autonomic triggers, cause SUCD.
• Lifestyle and environmental factors
Recommendation
Include the complexity of SUCD processes and the avoidance of oxidative stress.

5. No summary table or organized discussion
High-quality narrative reviews often include: • Comparative biomarker table (mechanism, clinical evidence, limits).
• Summary of research on salivary redox indicators in cardiovascular settings.
• Proposed “Salivary Redoxome” workflow visual model.

6. Citation errors and gaps
Citations are needed for mechanistic statements like ROS and electrophysiologic heterogeneity (page 5, lines 174–184).
Some epidemiological assertions need systematic review, proof, or citations.
4. Minor Issues
1. English form and syntax
Though comprehensible, the work needs expert polishing.
Examples: • “we are trying to explore” → “this review explores” • “depending on future funding” should be omitted (non-scientific language).
• Long paragraphs need separation for reading.
2. Author figures require citation or justification.
Figures must be credited as “created with BioRender” or similar.
3. Speculation overuse
Replace revolution, real-time population screening, and paradigm change with neutral scientific language.
4. Keywords
Some terms are redundant (oxidative stress, redox imbalance). Think about reorganising.
5. Abbreviations
First-use abbreviations (TAC, SUCD, ROS) should be defined.

Author Response

We sincerely thank the reviewer for the detailed, balanced, and constructive evaluation of our manuscript. We appreciate the positive assessment of the novelty, mechanistic rationale, and translational perspective of the proposed “Salivary Redoxome” concept. The reviewer’s comments helped us significantly improve methodological clarity, scientific caution, and presentation quality. Our responses are provided below.

1. Narrative Review Methodology

Reviewer comment:
The Methods section does not meet MDPI standards for a narrative or scoping review.

Response:
We agree with this comment. The Methods section has been revised and expanded to clearly present the study as a narrative and mechanistic literature review, not a systematic review. We now provide detailed information on databases searched, search period, Boolean strategies, inclusion and exclusion criteria, handling of duplicate records, and prioritization of mechanistic and translational relevance. We also clearly state that human, animal, and cellular studies were included and qualitatively assessed based on biological plausibility rather than quantitative synthesis.

Changes made:

• Expanded and restructured the Methods section.
• Clearly labeled the review as narrative and conceptual.

2. Overstatement of Clinical Applicability

Reviewer comment:
The manuscript exaggerates the clinical applicability of salivary biomarkers without direct evidence.

Response:
We fully agree. The manuscript has been carefully revised to explicitly acknowledge the conceptual and hypothesis-generating nature of the proposed approach. We now clearly state that salivary redox biomarkers are not validated screening tools for SUCD and that any screening application remains speculative until confirmed by prospective cohort studies. The low prevalence of SUCD and the absence of direct clinical correlations are now clearly emphasized.

Changes made:

• Revised Abstract, Aim, Discussion, and Conclusion to reduce clinical overstatement.
• Clearly framed screening as a future research objective only.

3. Critical Assessment of Biomarker Evidence

Reviewer comment:
The strength of evidence linking salivary and systemic biomarkers is not sufficiently evaluated.

Response:
We agree. The biomarker section has been revised to include a more critical discussion of evidence strength and limitations. We now explicitly address inter-assay variability, intra-individual variability, lack of diagnostic accuracy metrics, and the influence of oral cavity conditions. We also clarify that salivary oxidative biomarkers cannot currently be compared with established cardiac biomarkers such as troponin or BNP in terms of prognostic performance.

Changes made:

• Strengthened critical discussion of biomarker specificity and limitations.
• Added caution regarding saliva–blood correlation interpretation.

4. Oversimplification of SUCD Pathophysiology

Reviewer comment:
The manuscript presents an oversimplified pathway from oxidative stress to SUCD.

Response:
We agree with this concern. The revised manuscript now emphasizes the multifactorial and complex nature of SUCD, involving genetic ion-channelopathies, subtle cardiomyopathies, autonomic imbalance, and environmental and lifestyle triggers. Oxidative stress is now clearly described as a modulatory factor, not a single causal pathway. In addition, Figure 3, which may have contributed to oversimplification, has been removed, and the complexity is addressed in the text.

Changes made:

• Removed Figure 3.
• Expanded textual discussion of SUCD complexity.

5. Summary Tables and Visual Models

Reviewer comment:
The review lacks summary tables and organized visual models.

Response:
We appreciate this suggestion. A comparative table summarizing key salivary redox biomarkers, their mechanistic relevance, available evidence, and limitations has been retained and clarified. However, due to the absence of direct clinical SUCD studies, additional clinical summary tables or workflow models were not added to avoid speculative interpretation. This limitation is now clearly acknowledged.

Changes made:

• Clarified rationale for included and excluded tables.
• Highlighted lack of SUCD-specific clinical data as a research gap.

6. Citations and Reference Gaps

Reviewer comment:
Some mechanistic and epidemiological statements require additional citations.

Response:
We agree. The manuscript has been revised to add appropriate citations for mechanistic statements related to ROS-mediated electrophysiologic heterogeneity and for epidemiological assertions where needed.

Changes made:

• Added missing and supporting references.

Minor Issues

English language and style:
The manuscript has undergone language editing to improve clarity, sentence structure, and tone. Informal or speculative phrases have been replaced with neutral scientific language, and long paragraphs have been divided.

Figures:
All figures are now properly described and justified. Author-created figures are clearly identified in the legends.

Speculation:
Overly strong terms such as “revolution” or “population screening” have been replaced with cautious, neutral wording.

Keywords and abbreviations:
Redundant keywords were revised, and all abbreviations are now defined at first use.

Final Statement

The manuscript has been substantially revised to improve methodological transparency, critical assessment of evidence, conceptual clarity, and scientific caution, while preserving its innovative objective of exploring a novel molecular research gap linking redox biology with sudden unexplained cardiac death. We thank the reviewer for their valuable input, which significantly strengthened the quality of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

In the review entitled “Salivary Redox Biomarkers as a Non-Invasive Screening Tool for Early Detection of Sudden Unexplained Cardiac Death Risk in Young Healthy Individuals: A Mechanistic and Conceptual Literature Review”, Kamar et al sought to explore the emerging role of salivary redox biomarkers as non-invasive indicators of early cardiac vulnerability leading to sudden unexplained cardiac death.

The paper is well written, the topic is novel and intriguing and potential clinical implication might be significant. However, some issues are worthy to be pointed out:

1) In the introduction the authors state that “Sudden unexplained cardiac death in the young…is relatively low compared to adult sudden cardiac death”. Actually, a clear definition of SUCD is lacking, together with age which “young” term refers to. The authors should add definition of SUCD and age at which it occurs.

2) In Figure 1, epidemiological features of SUCD are presented in a sequential modality, although there is not a “cascade” relationship between the items. I suggest to modify the Figure accordingly

3) Some English grammar editing could be useful. The terms “SUCD” and “SCD” are often used interchangeably throughout the paper

4) Figure 3 is quite poor in data and probably could be enriched by detailed presentation of biomarkers playing role in oxidative stress and in redox imbalance, as well as known exact mechanism of interaction with myocardial substrates leading to reentry-circuits

5) By describing existing literature showing potential role of salivary oxidative stress and redox imbalance on cardiac arrhythmogenesis, the authors should specify if previous researches were conducted on experimental materials, animal models or humans: this inferential and mechanistic framework requires to be confirmed by clinical ground. In other words, robust clinical evidences of elevated salivary oxidative stress in patients with SUCD are needed. In the absence of such demonstration, the main message of the paper should be deeply damped to a hypothesis-generating study

6) A Table illustrating published clinical studies reporting data about correlation between levels of oxidative stress biomarkers and clinical outcome would be desirable

7) As stated by the authors, different systemic conditions (i.e., fasting status, smoking, infectious disease) may influence salivary and serum levels of oxidative biomarkers. In light of this view, the authors should explain whether a single sampling of salivary material might be sufficient to predict risk of SUCD or whether it needs to be periodically assessed to follow-up the patient

Comments on the Quality of English Language

Quite good

Author Response

We thank the reviewer for the positive evaluation of the manuscript and for the constructive comments, which helped us improve clarity, consistency, and scientific balance. Our responses are provided below.

1) Definition of SUCD and age range

We agree. The Introduction has been revised to include a clear definition of sudden unexplained cardiac death (SUCD) as sudden death occurring in individuals with no identifiable structural, toxicological, or genetic cause after comprehensive investigation. The term “young” is now clearly defined as individuals ≤35–40 years of age, in accordance with commonly used definitions in the literature.

2) Figure 1 presentation

We agree with the reviewer. Figure 1 has been revised to present epidemiological features as independent parameters rather than as a sequential or causal cascade.

3) English editing and use of SUCD vs SCD

We agree. The manuscript has undergone English language editing to improve grammar and readability. Terminology has been standardized throughout the manuscript, using “SUCD” only for unexplained cases and “SCD” for sudden cardiac death in general.

4) Figure 3

We thank the reviewer for this suggestion. After careful consideration, Figure 3 has been removed from the revised manuscript. The figure was largely conceptual and overlapped with textual explanations. The relevant mechanistic information has been fully integrated into the main text to improve clarity and avoid redundancy.

5) Experimental vs clinical evidence and conceptual nature

We fully agree. The revised manuscript now clearly distinguishes between experimental, animal, and human studies, as well as between saliva-based and blood-based biomarker research. We explicitly acknowledge that robust clinical evidence directly linking salivary oxidative stress biomarkers to SUCD is currently lacking. Accordingly, the manuscript clearly presents this work as a conceptual and hypothesis-generating study aimed at defining a research gap and guiding future clinical validation.

6) Table of clinical studies

We appreciate this suggestion. However, to our knowledge, no published clinical studies have directly evaluated salivary oxidative stress biomarkers in patients with SUCD. Therefore, constructing a SUCD-specific clinical table would be misleading. This limitation is now clearly stated in the manuscript and highlighted as an important research gap.

7) Single vs repeated salivary sampling

We agree that a single salivary measurement is unlikely to be sufficient. The revised manuscript now clearly states that repeated and longitudinal salivary sampling would be required to account for physiological variability and confounding factors such as diet, stress, smoking, and infection.

Final statement

The manuscript has been revised to improve definitions, figure presentation, terminology consistency, methodological transparency, and conceptual balance, while preserving the innovative aim of exploring the link between molecular redox biomarkers and sudden unexplained cardiac death.

Reviewer 3 Report

Comments and Suggestions for Authors

Conceptual Scope: Overreach Between Mechanism and Screening Application

The central premise—that salivary redox biomarkers could serve as an early screening tool for SUCD risk—is intellectually appealing but currently speculative. While oxidative stress is convincingly implicated in arrhythmogenesis, the manuscript does not sufficiently demonstrate:

Why systemic or salivary redox imbalance would precede and specifically predict lethal ventricular arrhythmias, rather than reflect non-specific physiological stress.

How salivary markers would discriminate high-risk individuals from the vast population of healthy young people with transient oxidative fluctuations.

 

Lack of Cardiology-Specific Risk Stratification Context

The review insufficiently integrates existing cardiac risk stratification frameworks, such as:

ECG-based markers (QT dispersion, early repolarization, Brugada patterns),

Genetic channelopathies and molecular autopsy findings,

Imaging-based phenotypes (early cardiomyopathic changes).

 

Methodology: Narrative Review Without Systematic Rigor

Although labeled a literature review, the methodology remains insufficiently rigorous for a mechanistic and translational proposal:

No PRISMA-style framework or flow diagram is provided.

Inclusion/exclusion criteria are broad and loosely defined.

The review does not clearly differentiate human vs animal data, saliva vs serum studies, or cardiac-specific vs systemic disease contexts.

 

 

Biomarker Specificity and Confounding

A major unresolved issue is biological specificity. Salivary oxidative markers are known to be influenced by:

Physical exercise,

Psychological stress,

Diet, sleep deprivation,

Oral and periodontal disease.

 

 

Author Response

We thank the reviewer for the constructive and detailed comments. We agree that caution is required when discussing clinical application; however, we respectfully clarify that the primary purpose of this manuscript is to identify and explore a clear research gap and to propose an innovative mechanistic research direction, rather than to claim an established screening tool.

While oxidative stress is not specific to sudden unexplained cardiac death (SUCD), substantial evidence supports its role as a key molecular modulator of cardiac electrical instability. The manuscript now more clearly explains that salivary redox biomarkers are proposed as integrative indicators of systemic redox vulnerability, which may contribute to arrhythmogenic susceptibility when combined with known cardiac risk substrates. This approach aims to link molecular imbalance with electrical instability, addressing a currently unexplored area in SUCD research.

We emphasize that the innovative aspect of this work lies in connecting molecular redox biology with SUCD vulnerability in young, apparently healthy individuals, a population for which no validated molecular biomarkers currently exist. The manuscript has been revised to highlight this research gap while avoiding overstatement of predictive or diagnostic capability.

Regarding discrimination between high-risk individuals and healthy subjects, we clarify that the proposed value of salivary redox biomarkers is not based on single measurements, but on longitudinal redox profiling and combined biomarker patterns, integrated with ECG findings, genetic background, and clinical context. This reflects modern precision medicine approaches rather than traditional binary screening.

In response to the comment on cardiology risk stratification, the revised manuscript now clearly positions salivary redox biomarkers as a complementary research layer, intended to work alongside established tools such as ECG markers, genetic channelopathies, and imaging-based phenotypes, not as a replacement.

We also acknowledge the methodological limitations of a narrative review. The manuscript has been clearly labeled as a conceptual and mechanistic narrative review, which was necessary due to the absence of direct clinical studies linking salivary redox biomarkers with SUCD. Distinctions between human and experimental studies, saliva-based and blood-based biomarkers, and cardiac-specific versus systemic data have been clarified.

Finally, we fully agree that salivary oxidative markers are influenced by exercise, stress, diet, sleep, and oral health. These confounding factors are now more explicitly discussed, and the manuscript emphasizes the need for strict sampling protocols, oral health assessment, and repeated measurements in future validation studies.

In summary, the manuscript has been revised to preserve the innovative concept, clearly define its research-oriented scope, and explicitly acknowledge its limitations, while maintaining its core objective: to open a new molecular research direction linking redox biology with sudden unexplained cardiac death.