Review Reports - Mitochondrial DNA Haplogroups Influence Oxidative Stress Profiles and Susceptibility to Metabolic Syndrome in an Asian Population

Round 1

Reviewer 1 Report

The assessment of oxidative stress markers (TBARS and free thiols) in conjunction with mitochondrial DNA haplogroup analysis is scientifically relevant, as mitochondrial genetic variation directly modulates the efficiency of oxidative phosphorylation and, consequently, the production of reactive oxygen species (ROS)—a key mechanistic link in the relationship with the metabolic outcomes under investigation.

In the discussion section (lines 289–291), the authors refer to recent advances in sequencing technologies that have enabled the identification of new genetic variants and their association with various diseases. However, this approach creates a methodological expectation that is not supported by the experimental design described in the "Materials and Methods" section; the latter employs PCR followed by multiplex genotyping or xMAP (Luminex)-based immunoassay, rather than actual sequencing. We recommend that the authors revise the rationale presented in the Introduction to clearly distinguish between (a) the general context of the field, where sequencing has facilitated the discovery of disease-associated variants, and (b) the specific objective of the present study, which appears to be the validation or screening of previously reported variants using rapid, cost-effective probe-based methods.

Author Response

Reviewer 1

Author's Reply to the Review Report

Comments for Authors

Major comments

Detailed comments

Response:

We thank the reviewer for this important and constructive comment. We agree that the original wording could inadvertently imply that high-throughput sequencing was performed in the present study. We have therefore revised the Introduction, Materials and Methods, and opening paragraph of the Discussion to clearly distinguish the broader role of sequencing technologies in the discovery and characterization of mtDNA variants from the targeted genotyping strategy used in our study.

Specifically, our study did not perform whole-mitochondrial-genome sequencing or aim to identify novel mtDNA variants. Instead, we used a targeted multiplex PCR and probe-based Luminex genotyping approach to classify previously established mitochondrial haplogroups based on selected haplogroup-defining mtSNPs. Forty mtSNPs were selected based on the MITOMAP database and previously established phylogenetic information for Chinese and Japanese populations, allowing classification of the major mtDNA haplogroups and relevant sub-haplogroups in our Taiwanese cohort.

We have revised the Introduction to clarify that advances in sequencing technologies provide the broader scientific context for the characterization of mitochondrial genetic variation, whereas the present study used a targeted, rapid, and cost-effective genotyping strategy for large-scale classification of established mtDNA haplogroups. We have also revised the Materials and Methods to explicitly state that our approach was based on multiplex PCR and probe-based Luminex detection rather than mitochondrial genome sequencing. Finally, the corresponding statement at the beginning of the Discussion has been revised to avoid any implication that sequencing was performed in the present study.

We believe these revisions have clarified the methodological scope and rationale of the study and have addressed the reviewer’s conce

Author Response File: Author Response.docx

Reviewer 2 Report

This is an interesting study investigating the association between mitochondrial DNA (mtDNA) haplogroups, oxidative stress biomarkers (TBARS and free thiols), and metabolic syndrome in,486 Korean adults. The paper is well written, with a lot of interesting data, but there are several recommendations that should be considered.
1.
Please describe in more detail the experimental procedures such as serum processing and storage conditions, sample volume, assay protocol, reaction conditions, number of technical replicates, and analytical quality control.
2.
Please list the reagent manufacturers (DTNB, TEPP) in the Material and Method section, as well as the spectrophotometer model used in the study.
3.
The authors did not define the units for thiols. It is unclear whether they are mmol/L or something else.
4.
Please clarify the statistical analysis section by indicating the level of statistical significance.
5.
It is stated that participants were recruited during routine health check-ups. However, the inclusion and exclusion criteria are not sufficiently described, especially regarding conditions or medications that may affect oxidative stress biomarkers (e.g., acute inflammatory diseases, chronic kidney or liver disease, malignancy, antioxidant supplementation, or other relevant comorbidities).
6.
The panel of oxidative stress biomarkers assessed in this study is relatively limited. Analysis of TBARS and free thiols solely cannot provide the clear status of redox state. Please include additional markers of oxidative stress and antioxidant defense system (e.g., SOD, CAT, glutathione, superoxide anion radical, hydrogen peroxide, and nitrites).
7.
Please update references list with more recent studies. Several key references cited in the Introduction section are relatively old.
8.
The discussion would benefit from a more comprehensive mechanistic interpretation of the observed associations between mitochondrial DNA haplogroups and oxidative stress biomarkers.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

Author's Reply to the Review Report

Comments for Authors

Major comments

Detailed comments

Reviewer Comment 1:
Please describe in more detail the experimental procedures such as serum processing and storage conditions, sample volume, assay protocol, reaction conditions, number of technical replicates, and analytical quality control.

Response:
Thank you for this valuable comment. We have substantially expanded the description of the serum free thiol and TBARS assays in the revised Materials and Methods section. Specifically, we have added details regarding serum collection, clotting and centrifugation conditions, serum aliquoting and storage at −80 °C, and the single-thaw procedure. For the serum free thiol assay, we have clarified the sample dilution, sample volume, buffer conditions, DTNB concentration, incubation time, measurement wavelengths, calculation method, reagent blanks, technical replicates, and intra-assay coefficient of variation (CV). For the TBARS assay, we have added the serum volume, reagent volumes and concentrations, reaction volume, heating conditions, extraction and centrifugation procedures, detection wavelength, TEPP-based calibration curve, technical replicates, reagent blanks, and intra-assay CV.

These additions provide greater methodological transparency and directly address the reviewer’s concerns regarding sample processing, assay conditions, reproducibility, and analytical quality control. The revised text has been incorporated into the Materials and Methods section.

Reviewer Comment 2:
Please list the reagent manufacturers (DTNB, TEPP) in the Material and Method section, as well as the spectrophotometer model used in the study.

Response:

Thank you for this helpful suggestion. We have revised the Materials and Methods section to provide the requested information. The manufacturer of DTNB and TEPP (Sigma-Aldrich, St. Louis, MO, USA) has now been specified. We have also identified the instruments used for absorbance measurements, including the Varioskan microplate reader (Thermo Scientific, Breda, The Netherlands) for the serum free thiol assay and the DU-640 spectrophotometer (Beckman Coulter, USA) for the TBARS assay. These details have been added to improve the reproducibility and methodological clarity of the study.

Reviewer Comment 3:
The authors did not define the units for thiols. It is unclear whether they are mmol/L or something else.

Response:

Thank you for pointing this out. We apologize for the lack of clarity regarding the units of the thiol measurements. We have clarified that both plasma TBARS and thiol concentrations are expressed in µmol/L. The units have been explicitly added to the Methods section, table titles, and figure axis labels, and the terminology has been standardized throughout the manuscript to ensure consistency.

Reviewer Comment 4:
Please clarify the statistical analysis section by indicating the level of statistical significance.

Response:

Thank you for this helpful comment. We have revised Section 2.4 (Statistical Analysis) to explicitly state that a two-sided p value < 0.05 was considered statistically significant. We have also clarified that Bonferroni correction was applied for comparisons involving the nine mtDNA haplogroups, with a corrected significance threshold of p < 0.0056 (0.05/9). This clarification has been added to the Statistical Analysis section of the revised manuscript.

Reviewer Comment 5:
It is stated that participants were recruited during routine health check-ups. However, the inclusion and exclusion criteria are not sufficiently described, especially regarding conditions or medications that may affect oxidative stress biomarkers (e.g., acute inflammatory diseases, chronic kidney or liver disease, malignancy, antioxidant supplementation, or other relevant comorbidities).

Response:

We thank the reviewer for this important comment. We agree that the inclusion and exclusion criteria should be described more clearly, particularly with respect to medical conditions and medications that may influence systemic oxidative stress biomarkers. In the present cross-sectional cohort, participants were recruited from individuals undergoing routine general health examinations at our medical center in Taiwan and were not selectively recruited according to the presence or absence of specific chronic diseases. The study was designed to characterize the associations among mitochondrial DNA haplogroups, metabolic syndrome, and systemic oxidative/antioxidative status in a general health-examination population, thereby reflecting the range of metabolic and health conditions encountered in the community.

Because this was an observational, population-based analysis, we did not exclude participants solely on the basis of common chronic diseases or medication use, including conditions that may potentially influence oxidative stress. Similarly, information on antioxidant supplementation was not systematically incorporated into the present analysis. We acknowledge that these factors may contribute to inter-individual variation in TBARS and thiol concentrations and may represent potential residual confounding.

To address the reviewer’s concern, we have clarified the study population and acknowledged this issue as a limitation of the present study. We have also revised the Discussion to emphasize that the observed associations should be interpreted as associations within a real-world health-examination cohort rather than as evidence independent of all potential clinical or treatment-related confounders. Future prospective studies with detailed assessment of comorbidities, medication use, antioxidant supplementation, dietary habits, and other lifestyle factors will be necessary to further determine the independent relationship between mitochondrial haplogroups, oxidative stress, and metabolic syndrome.

Reviewer Comment 6:
The panel of oxidative stress biomarkers assessed in this study is relatively limited. Analysis of TBARS and free thiols solely cannot provide the clear status of redox state. Please include additional markers of oxidative stress and antioxidant defense system (e.g., SOD, CAT, glutathione, superoxide anion radical, hydrogen peroxide, and nitrites).

Response:

Thank you for this important and constructive suggestion. We agree that assessment of a broader panel of oxidative stress and antioxidant defense biomarkers, including enzymatic antioxidants (e.g., SOD and CAT), glutathione, reactive oxygen species, and nitrite-related parameters, would provide a more comprehensive characterization of systemic redox status.

In the present study, however, our analyses were specifically designed to evaluate two complementary circulating indicators of oxidative/antioxidative status: thiobarbituric acid reactive substances (TBARS), as an index of lipid peroxidation, and serum free thiols, as an indicator of systemic antioxidant/redox-buffering capacity. These biomarkers were selected based on our previous investigations demonstrating their relevance to mitochondrial biology, including their associations with mitochondrial DNA copy number. Thus, although TBARS and thiols do not encompass the entire spectrum of oxidative stress and antioxidant defense pathways, their combined assessment provides informative evidence of the systemic oxidative–antioxidative imbalance relevant to the objectives of the present study.

We acknowledge, however, that the absence of additional parameters such as SOD, CAT, glutathione, superoxide anion, hydrogen peroxide, and nitrite limits the comprehensiveness of our assessment and should be considered when interpreting the findings. Because these measurements were not prospectively included in the present study, additional biomarkers cannot be retrospectively incorporated into the existing dataset. We have therefore explicitly acknowledged this issue as a limitation in the revised Discussion.

Revision made in the manuscript:
We added the following statement to the limitations section of the Discussion:

“Oxidative stress was evaluated using a limited number of biomarkers, and additional markers of mitochondrial function and reactive oxygen species production may provide a more comprehensive assessment.”

We further clarified that future studies incorporating a broader panel of ROS-related and antioxidant defense parameters would be valuable for validating and extending the present findings.

Reviewer Comment 7:
Please update references list with more recent studies. Several key references cited in the Introduction section are relatively old.

Response:

Thank you for this valuable suggestion. We agree that the Introduction and Discussion would benefit from incorporating more recent evidence. Accordingly, we have updated the reference list and supplemented the manuscript with recent studies and reviews published from 2022 to 2026 addressing mitochondrial genetic variation, mitochondrial bioenergetics, oxidative stress, mitochondrial DNA (mtDNA) abnormalities, metabolic syndrome, diabetes, and population-specific effects of mtDNA variants.

In particular, we have incorporated recent evidence concerning the relationships between mtDNA variation and mitochondrial bioenergetics, mtDNA abnormalities and metabolic syndrome, mtDNA copy number and diabetes/metabolic syndrome, oxidized mtDNA and inflammatory signaling, and the population-dependent associations of mtDNA variants with type 2 diabetes. We also included a recent systematic review and meta-analysis addressing mtDNA variants, copy number, and haplogroups in relation to type 2 diabetes across different ethnic populations, which is particularly relevant to our Taiwanese cohort.

We retained several established older references where they provide important foundational evidence, while using the newer literature to update and strengthen the current scientific context. These revisions have been incorporated primarily into the Introduction and Discussion sections, and the corresponding references have been added to the reference list.

Revision made in the manuscript: The Introduction and Discussion have been updated with recent literature concerning mtDNA variation, mitochondrial bioenergetics, oxidative stress, metabolic syndrome, diabetes, and population-specific mitochondrial effects.

Reviewer Comment 8:
The discussion would benefit from a more comprehensive mechanistic interpretation of the observed associations between mitochondrial DNA haplogroups and oxidative stress biomarkers.

Response:

Thank you for this important and constructive comment. We agree that the original Discussion did not sufficiently elaborate on the potential biological mechanisms linking mtDNA haplogroup background with systemic oxidative stress and metabolic abnormalities.

In response, we have substantially expanded the Discussion to provide a more comprehensive and biologically plausible mechanistic framework. Specifically, we now discuss how inherited mtDNA variation may influence mitochondrial oxidative phosphorylation, mitochondrial bioenergetics, substrate utilization, and redox signaling, which could subsequently affect electron leakage and mitochondrial reactive oxygen species (ROS) generation. Increased oxidative stress may promote lipid peroxidation and oxidation of circulating thiol groups, thereby contributing to the higher TBARS and lower thiol concentrations observed in our study.

We have also expanded the discussion of haplogroup B, which showed relatively higher TBARS and lower thiol concentrations and was significantly associated with diabetes and metabolic syndrome in our cohort. We discuss the potential relevance of the T16189C variant and the 9-bp deletion characteristic of haplogroup B, while emphasizing that haplogroups comprise multiple linked variants and that the observed phenotype should not be attributed to a single mtDNA polymorphism without direct functional evidence.

Importantly, we have revised the interpretation to avoid implying a direct causal effect of haplogroup B or T16189C. We now emphasize that the biological effects of mtDNA variation may depend on the broader mitochondrial genetic background, nuclear genetic background, tissue type, environmental exposure, and metabolic state. Thus, the higher oxidative stress observed among haplogroup B carriers is interpreted as an association that is biologically compatible with increased susceptibility to metabolic stress rather than evidence that haplogroup B directly causes oxidative stress or metabolic syndrome.

Furthermore, the revised Discussion now considers the bidirectional relationship between mitochondrial oxidative stress and metabolic syndrome. Metabolic abnormalities such as hyperglycemia, dyslipidemia, obesity, hypertension, and insulin resistance may increase mitochondrial metabolic stress and ROS production, while oxidative stress may in turn impair insulin signaling, mitochondrial function, endothelial homeostasis, and inflammatory regulation. We also discuss the potential involvement of oxidized mtDNA and TLR9-dependent inflammatory signaling as an additional mechanism connecting mitochondrial oxidative stress with chronic low-grade inflammation in metabolic syndrome.

We have additionally clarified the potential significance of circulating thiols as indicators of systemic redox imbalance and discussed the relatively higher thiol concentrations observed in haplogroup D carriers. However, because our study did not directly assess mitochondrial respiration, mitochondrial ROS production, mtDNA copy number, glutathione metabolism, or antioxidant enzyme activity, we have deliberately avoided making definitive mechanistic or causal claims.

Finally, we have strengthened the limitations of our interpretation by explicitly acknowledging that the cross-sectional design cannot establish causality and that TBARS and serum thiols reflect systemic redox status rather than directly measuring mitochondrial ROS production or respiratory-chain function. We therefore present the proposed mechanisms as plausible interpretations that require confirmation by future functional and prospective studies.

Revision made in the manuscript: The Discussion has been substantially expanded to address the potential pathway of:

mtDNA haplogroup background → mitochondrial bioenergetics/oxidative phosphorylation → ROS generation → lipid peroxidation and thiol oxidation → impaired insulin signaling/inflammation → metabolic dysfunction and metabolic syndrome.

We believe that these revisions provide a more comprehensive mechanistic interpretation while maintaining an appropriately cautious interpretation of our cross-sectional findings.

Additional revision

In addition to the specific comments above, we carefully reviewed the terminology used throughout the manuscript to avoid causal overinterpretation. In particular, the previous wording “The causal relationship between specific mtDNA haplogroups, the oxidative/anti-oxidative burden, and the risk of metabolic syndrome” has been revised to:

“Associations between specific mtDNA haplogroups, oxidative/antioxidative burden, and metabolic syndrome.”

Similarly, the phrase “To investigate the causal relationship…” has been revised to “To further investigate the associations between oxidative/antioxidative burden, mtDNA haplogroups, and metabolic syndrome…”

These changes more accurately reflect the observational and cross-sectional nature of our study.

We sincerely appreciate the Reviewer’s thoughtful comments, which have helped us improve the manuscript by strengthening both the updated literature background and the mechanistic interpretation of our findings.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors have responded to all requests, so I propose that the manuscript be accepted in its current form.

The authors have responded to all requests, so I propose that the manuscript be accepted in its current form.The authors have responded to all requests, so I propose that the manuscript be accepted in its current form.