Why Skin Carotenoid Measurements Cannot Serve as a Proxy for Macular Pigment Optical Density (MPOD): A Biochemical, Anatomical, Optical, and Statistical Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review focuses on the association between macular and skin carotenoids. This is crucial since skin can be measured considerably quicker and simpler than retina if they have a strong correlation. However, the predominant carotenoids in the skin are lycopene and beta carotene, accounting for 60-70% of total carotenoid content. Only two carotenoids, lutein, and zeaxanthin, are selectively accumulated in the retina and constitute 100% of the total retina carotenoid content. Both lutein and zeaxanthin are present in human skin, but in much smaller, trace amounts compared to carotenes like beta-carotene and lycopene. Additionally, the third macular carotenoid, namely meso-zeaxanthin, is not present in the skin.

Unfortunately, the article requires thorough rewriting; the paper is unclearly written and cannot be recommended for publication in its current state. The manuscript is not well-structured. The large number of sections makes reading difficult and hard to follow. A limited bibliography (14 citations) reflects the poor quality of the literature review. The bibliography does not have the correct order (line: 58). It should be in the order the citations appear in the text, but it's not.

Comments:

Table 1:

(1) The layout of the table to me is strange. It would have been much better if it had not had text at the bottom (lines 45-53).

(2) Analysis of the content of carotenoids in human skin and retina using high-pressure liquid chromatography gives information about the concentration of these pigments. This information will help the reader understand the difference in the concentration of individual carotenoids in the skin and in the macula of the eye.

(3) Phytoene and phytofluene are present in skin. They could not be detected in the SC in vivo by optical noninvasive methods but should be included in the table.

Sec. 2.3.1 In my opinion, carotenoids serve similar fundamental functions in the skin and retina. These pigments are lipid-soluble antioxidants. They are singlet oxygen quenchers and could neutralize free radicals. Both carotenes and xanthophylls are also capable of quenching excited triplet states of singlet oxygen photosensitizers.  Macular and skin carotenoids also provide photoprotection by absorbing harmful short-wavelength light.  Of course, they also have tissue-specific roles in the retina and in the skin.

Sec.3.3 “Melanin Distribution: Uniform in Skin vs. Concentrated in the Fovea” and Sec.5.1 „Melanin Distribution: Uniform in Skin vs. Highly Concentrated in the Fovea” are similar.

Sec.3.4  „Hemoglobin and Vascular Influences” and Sec. 5.2. „Hemoglobin: Dermal Vasculature vs. Choroidal Vasculature”  are similar.

Sec. 6.1 Not all articles relating to this topic have been cited:

https://doi.org/10.1080/1028415X.2024.2370175

https://doi.org/10.1167/iovs.13-11891

 

The correlation between serum and skin carotenoid levels/the correlation with serum carotenoid concentration and MPOD should be mentioned and discussed.

 

This paper has a few errors (e.g. line: 29 incorrectly placed punctuation mark ” .:” ; line 912 misplaced irrelevant text “Check year if needed” ).

Author Response

Response to Reviewer 1

General Comment:

“This review focuses on the association between macular and skin carotenoids…”

Response:
The reviewer correctly notes that skin measurements are simpler than retinal imaging. However, the central thesis of the manuscript is precisely that this convenience does not justify surrogate use, because the biological, optical, and temporal systems are fundamentally different. This is not a limitation of our manuscript; it is the core conclusion supported throughout Sections 2-8.

Reviewer Statement: Predominant Skin vs Retinal Carotenoids

“…the predominant carotenoids in the skin are lycopene and beta-carotene… Only two carotenoids, lutein and zeaxanthin, are selectively accumulated in the retina… meso-zeaxanthin is not present in the skin.”

Response:
I fully agree with this statement, and it is explicitly stated multiple times in the manuscript, including:

• Section 1, 3.1, 7.4, and 8.1, where I emphasize that:
• Skin carotenoid measurements are dominated by β-carotene and lycopene.
• Macular pigment consists exclusively of lutein, zeaxanthin, and meso-zeaxanthin.
• meso-zeaxanthin is not present in skin.

This biochemical mismatch is one of the primary arguments explaining why skin carotenoids cannot serve as a proxy for MPOD, not a point of disagreement.

Reviewer Claim: “Thorough rewriting required / unclear / not well structured”

Response:
I respectfully disagree with this assessment.

The manuscript is deliberately structured by domain (biochemical, anatomical, optical, physiological, temporal, statistical), which reflects the multidimensional nature of biomarker validation. This structure is common in mechanistic review articles addressing surrogate biomarker validity.

To improve readability, I have:

• Removed all figures (as agreed during revision),
• Eliminated redundancy where noted,
• Corrected minor typographical issues.

However, the number of sections reflects conceptual separation, not poor organization.

Bibliography Size (14 citations)

“A limited bibliography reflects poor quality”

Response:
The manuscript intentionally focuses on foundational, high-impact studies that directly measure skin carotenoids, macular pigment, or both. This is a mechanistic review, not a narrative survey. Citation count alone is not a valid indicator of review quality. That said, I have added the two suggested references where appropriate.

Reference Order Issue

Response:
Thank you for noting this. The reference order has been corrected to follow the order of appearance in the text.

 

Table 1 Comments

(1) Layout

I have revised the table formatting to improve clarity.

(2) Suggestion to include HPLC concentrations

While HPLC provides absolute concentrations, the manuscript specifically addresses non-invasive optical biomarkers used in clinical and population settings. Including ex vivo HPLC concentrations would not change the biological argument and may distract from the focus on surrogate validity.

(3) Phytoene / phytofluene

Phytoene and phytofluene are indeed present in skin, but they are optically silent for all non-invasive methods discussed and are irrelevant to MPOD. Their exclusion is intentional and scientifically appropriate for a manuscript focused on optical biomarkers and surrogate validity.

 

Section 2.3.1: “Carotenoids serve similar functions”

Response:
I agree that carotenoids share general antioxidant properties across tissues. However, the manuscript explicitly distinguishes:

• General antioxidant activity (skin)
• Highly specialized optical and neural functions (macula), including:
• Blue-light filtration,
• Spatially localized protection,
• Neural tissue preservation.

This distinction is clearly articulated in Sections 2.3, 7.1, and 8.1, and is central to the conclusion that functional overlap does not imply biomarker interchangeability.

 

Redundancy Between Sections (Melanin / Hemoglobin)

Response:
I appreciate this observation. Some conceptual overlap existed to allow independent reading of sections. I have now reduced redundancy and streamlined these discussions to improve flow.

 

Missing Citations Suggested

https://doi.org/10.1080/1028415X.2024.2370175
https://doi.org/10.1167/iovs.13-11891

Response:
Thank you. These references have now been added and discussed in Section 6 where correlations between skin, serum, and retinal carotenoids are addressed.

 

Serum vs Skin vs MPOD Correlations

Response:

I have now explicitly discussed:

• Serum–skin correlations,
• Serum–MPOD correlations,
• Why serum itself is not a surrogate for MPOD.

This discussion reinforces, rather than weakens, the conclusion that biological compartmentalization prevents surrogate substitution.

 

Minor Errors / Typos

Response:
All typographical and formatting errors noted by the reviewer have been corrected.

Final Editorial Note:

The reviewer’s opening biochemical statements align closely with the manuscript’s central conclusions. I believe that the remaining criticisms stem from a misunderstanding of the manuscript’s purpose: it is not to explore whether skin and macular carotenoids are related in a general sense, but to rigorously demonstrate why they cannot be used interchangeably as biomarkers.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript provides an extensive and rigorously argued review explaining why skin carotenoid measurements cannot be used as a proxy for macular pigment optical density (MPOD). The work is comprehensive and demonstrates deep expertise in optical physiology, carotenoid biochemistry, statistical interpretation, and retinal imaging methodologies.
Overall, the manuscript is scientifically strong, but several revisions are required to improve its quality.

The manuscript is overly long and would benefit from significant condensation.
While the arguments are thorough, the review spans an unusually large number of pages and repeats concepts across sections.
Many sections reiterate that skin carotenoids and MPOD differ biologically, optically, anatomically, and statistically.
Condensing the text by ~25–30% would greatly increase readability without losing content.
Consider merging Sections 3–5, which contain overlapping optical/anatomical explanations.

The Abstract is currently dense and lacks supportive numerical metrics. 
 -Include representative correlation ranges (e.g., r = 0.02–0.66).
 -Add specific numerical distinctions between carotenoid types or temporal differences.
-Reduce abstract length by removing methodological detail.

The manuscript includes optical engineering details (e.g., photon-path modeling, wavelength-dependent attenuation, tissue-scattering profiles) that could be moved to supplementary material. 
Add schematic figures or summary tables for non-specialists.
Rephrase complex passages to be more accessible to nutrition and clinical readers.

Lack of a graphical summary or comparison table
Given the complexity of the manuscript, a summary figure or table contrasting Skin Carotenoids vs. MPOD across: biochemistry, transport mechanisms, optical environment, tissue location, 
temporal kinetics, dietary responsiveness, would greatly improve understanding.

Statistical arguments require clearer referencing.
The manuscript cites large cohort studies but does not provide: author names, publication years, study details, exact MPOD and skin carotenoid measurement methods. 
Create a dedicated table summarizing all reported correlation values (r) including: study ID, population size, age group, measurement technique, reported correlation.

Each major section ends with an extensive conclusion. Many could be shortened to 3–5 concise bullet points summarizing key take-home messages.

Author Response

Response to Reviewer 2

I sincerely thank the reviewer for their thoughtful, detailed, and constructive evaluation of our manuscript. I greatly appreciate the reviewer’s recognition of the scientific rigor, depth of expertise, and comprehensive scope of the review. The comments have been highly valuable in improving clarity, readability, and presentation. Below, I address each point in detail.

 

General Comment: Manuscript Length and Repetition

Reviewer comment:
The manuscript is overly long and would benefit from significant condensation. Several concepts are repeated across sections.

Response:
I agree that improving conciseness will enhance readability. In the revised manuscript, I have systematically reduced redundancy and condensed overlapping explanations, particularly in sections discussing optical, anatomical, and physiological distinctions. This was accomplished by tightening language, improving cross-referencing between sections, and shortening section-level summaries, while preserving the conceptual clarity necessary for a multidisciplinary audience. The overall length has been reduced without compromising scientific content or argumentation.

 

Section Organization (Merging Sections 3-5)

Reviewer comment:
Consider merging Sections 3–5 due to overlapping content.

Response:
I appreciate this suggestion. Rather than fully merging these sections, I have streamlined overlapping content and improved structural flow while preserving conceptual separation between biochemical, optical, and anatomical domains. I believe maintaining these distinctions is important for clarity, as each domain addresses a different mechanistic reason for non-equivalence between skin carotenoids and MPOD.

 

Abstract Density and Numerical Metrics

Reviewer comment:
The Abstract is dense and lacks supportive numerical metrics.

Response:
I agree. The Abstract has been revised to be more concise and now includes representative quantitative information, including reported correlation ranges (e.g., near-zero to moderate values reported in the literature) and clear temporal contrasts between skin carotenoid kinetics (weeks) and MPOD response (months). Methodological detail has been reduced to focus on core conclusions.

 

Technical Optical Details

Reviewer comment:
Optical engineering details could be moved to supplementary material or simplified.

Response:
I have condensed highly technical optical descriptions, retaining only those elements essential for understanding why skin-based optical measurements cannot replicate retinal pigment assessment. The revised text is more accessible to nutrition and clinical readers while maintaining scientific accuracy.

 

Lack of a Graphical Summary or Comparison Table

Reviewer comment:
Add a summary figure or comparison table contrasting skin carotenoids and MPOD.

Response:
I agree that a high-level comparison is valuable. In response, I have added a comprehensive summary table contrasting skin carotenoids and MPOD across biochemical composition, transport mechanisms, tissue localization, optical environment, temporal kinetics, and dietary responsiveness. I intentionally used a table rather than figures to avoid copyright constraints and production delays, while still providing clear visual synthesis.

 

Statistical Referencing and Correlation Studies

Reviewer comment:
Statistical arguments require clearer referencing, including study details.

Response:
I have strengthened statistical referencing by explicitly citing representative cohort and supplementation studies, including measurement modalities and reported correlation ranges. The intent of this review is to demonstrate biological and mechanistic non-equivalence, rather than to perform a formal meta-analysis. Accordingly, correlations are discussed as illustrative evidence rather than exhaustive statistical compilation.

 

Section-End Conclusions

Reviewer comment:
Section conclusions are long and repetitive.

Response:
I agree and have revised section conclusions to concise bullet-point summaries (3–5 points each) that highlight key take-home messages without repetition.

 

Overall Assessment

I thank the reviewer again for their insightful feedback. The revisions prompted by these comments have significantly improved the manuscript’s clarity, organization, and accessibility, while preserving its scientific rigor and central conclusions.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is interesting. Several comments can improve the manuscript' views.

 

1. Table 1 should have references and value description is needed. Present, then how many?Accumulated then how long how many?

 

2. It is difficult to understand why the author focused on skin and retina only. How about other tissues and their relationships?

 

3. Furthermore, why carotenoids should be focused in this review article? There are other types of nutrients including polyphenols, minerals, vitamins, and so on.

Author Response

Comment 1: Table 1 references and value description

Reviewer comment:
Table 1 should have references and value description is needed. Present, then how many? Accumulated then how long how many?

Response:
I agree that clearer contextualization strengthens Table 1. In the revised manuscript, I have added explicit literature references to Table 1 and clarified that the table is intended to provide a qualitative comparative overview, not absolute concentration values. As discussed in the text, reported carotenoid levels vary widely depending on analytical method, tissue layer, population, and sampling conditions. Providing single numeric values would therefore risk misinterpretation. I have revised the table caption and accompanying text to clarify these points and to emphasize relative composition, selectivity, and biological relevance rather than absolute quantities.

 

Comment 2: Focus on skin and retina only

Reviewer comment:
It is difficult to understand why the author focused on skin and retina only. How about other tissues and their relationships?

Response:
The focus on skin and retina is intentional and central to the manuscript’s objective. Skin carotenoid measurements are explicitly promoted in the literature and by commercial devices as potential surrogates for retinal carotenoid status, particularly macular pigment optical density (MPOD). This specific claim does not exist for other tissues. Accordingly, the review is designed to critically evaluate this proposed substitution, rather than to provide a general survey of carotenoid distribution across all tissues. I have clarified this rationale in the Introduction to better explain why skin–retina comparisons are uniquely relevant.

 

Comment 3: Why focus on carotenoids rather than other nutrients

Reviewer comment:
Why carotenoids should be focused in this review article? There are other nutrients including polyphenols, minerals, vitamins, etc.

Response:
Carotenoids are the exclusive focus of this review because macular pigment is composed solely of carotenoids, specifically lutein, zeaxanthin, and meso-zeaxanthin. No other nutrient classes contribute to MPOD or share its optical and biological properties. Moreover, skin-based optical measurements are designed to detect carotenoid chromophores, not polyphenols, minerals, or vitamins. Therefore, extending the discussion to other nutrients would fall outside the scope of the scientific question addressed in this manuscript. I have added clarifying language to explicitly state this scope.

 

Overall Response

I appreciate the reviewer’s thoughtful questions, which have helped us clarify scope, intent, and presentation. The manuscript has been revised accordingly to improve clarity while maintaining its focused scientific objective.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the thorough response.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been sufficiently improved. The revisions made by the authors have added significant value to the manuscript, substantially enhancing its clarity and overall quality.

Reviewer 3 Report

Comments and Suggestions for Authors

The comments have been replied.