The Relative Bioavailability of Lutein and Zeaxanthin in the Presence of Omega-3 Supplements and Their Effect on Oxidative Stress Levels in Humans: A Pilot Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Methodology
The following points should be more explicitly highlighted as methodological limitations:
Very small sample size, no pre-calculated study power. Differences in baseline values: baseline serum L+Z (Day 7) is more than twice as high in the control group (2.23 vs 1.20 µg/mL), and FMI differs significantly between groups; this complicates the interpretation of iAUC and group comparisons.

Discussion
In the opening paragraphs of the discussion, it should be clearly stated that no key between-group difference in AUC/iAUC was statistically significant (p>0.05) and that "biological responsiveness" is observed descriptively, within the pilot design.
Correlations L+Z/MDA and adherence/MDA (r≈0.53–0.55) should be presented as hypothesis-generating, with an emphasis on multiple tests and small n; avoid causal interpretation ("may have experienced a more pronounced short‑term oxidative flux" is quite strong).

Conclusion 
Remind that the study is a pilot, with a small sample and a short duration.
Clearly state that omega‑3 co‑supplementation did not show a clear additional effect on MDA or other oxidative biomarkers.

Author Response

Comments from Reviewer 1:

The following points should be more explicitly highlighted as methodological limitations:
Very small sample size, no pre-calculated study power. Differences in baseline values: baseline serum L+Z (Day 7) is more than twice as high in the control group (2.23 vs 1.20 µg/mL), and FMI differs significantly between groups; this complicates the interpretation of iAUC and group comparisons.

Reviewer Comment Response:

Thank you for this insightful comment. We agree that these points represent important methodological limitations and have now explicitly acknowledged them in the revised manuscript (see Discussion, Lines 473  - 481).

Additionally, the relatively small sample size and absence of an a priori power calculation reflect the exploratory (pilot) nature of this study and necessitate cautious interpretation of the findings.

Baseline imbalances between groups were also observed. Specifically, serum L+Z concentrations at Day 7 were higher in the control group compared to the intervention group (2.23 vs 1.20 µg/mL), and FMI differed significantly between groups (control, 8.30 vs intervention, 6.21kg/m2, p=0.038). These differences may confound the interpretation of the iAUC and between-group comparisons.

 

Hence, we have explicitly noted these limitations in the Discussion and refined our interpretation to avoid overstatement of group effects, focusing instead on within-group trends and the exploratory nature of the findings.

Discussion
In the opening paragraphs of the discussion, it should be clearly stated that no key between-group difference in AUC/iAUC was statistically significant (p>0.05) and that "biological responsiveness" is observed descriptively, within the pilot design.
Correlations L+Z/MDA and adherence/MDA (r≈0.53–0.55) should be presented as hypothesis-generating, with an emphasis on multiple tests and small n; avoid causal interpretation ("may have experienced a more pronounced short‑term oxidative flux" is quite strong).

Reviewer Comment Response:

Thank you for this important and constructive suggestion. We agree that the interpretation of our findings should more clearly reflect the exploratory nature of the study.

In the revised manuscript, we have explicitly stated in the opening paragraphs of the Discussion that no between-group differences in AUC or iAUC reached statistical significance (p > 0.05). We have clarified that the observed patterns of “biological responsiveness” are descriptive and should be interpreted within the context of a pilot study design (See Discussion, Lines 352 – 361).

We have also revised the presentation of the correlations between L+Z/MDA and adherence/MDA (r ≈ 0.53–0.55). These are now described as hypothesis-generating findings, with explicit acknowledgement of the small sample size and not adjusted for multiple comparison (See Discussion, Line 425 – 428).

Furthermore, we have softened the language in the Discussion to avoid causal or overly strong interpretations. For example, statements such as “may have experienced a more pronounced short-term oxidative flux” have been revised to more cautious, descriptive phrasing (See Discussion, Lines 448 – 450).

Conclusion 
Remind that the study is a pilot, with a small sample and a short duration.
Clearly state that omega‑3 co‑supplementation did not show a clear additional effect on MDA or other oxidative biomarkers.

Reviewer Comment Response:

Thank you for this helpful suggestion. In the revised manuscript, we have explicitly reiterated that this study is a pilot investigation with a small sample size and short intervention duration. This clarification has been incorporated in the conclusion. We have also clearly stated that omega-3 co-supplementation did not demonstrate a significant or consistent additional effect on MDA or other oxidative stress biomarkers. The interpretation has been revised to avoid overstatement and to reflect the absence of clear evidence for an added benefit within the current study design.

These revisions have been incorporated in the conclusion section (Lines 488 – 498).

Reviewer 2 Report

Comments and Suggestions for Authors

This was an interesting article that has important health implications for vision related problems such as age-related macular degeneration. Please consider the following suggestions for improvement:

Abstract

This is strictly personal preference, but I think three letter abbreviations for Lutein (Lut) + Zeaxanthin (Zea) would look better and be easier to read. I also recommend ARMD for age-related macular degeneration (instead of AMD). Also, you may want to consider not abbreviating Lutein and Zeaxanthin in any of the text and just using their full names throughout, unless you need to abbreviate them for a table or figure.

In line 18 and 31 you have used the full name for lutein and zeaxanthin, but since you have previously defined the abbreviations in the abstract, you should use the abbreviations here, too. (Or just don't use the abbreviations at all!)

Keywords – need to add some

Introduction

In line 59, rather than say “of these compounds (L+Z)”, you can just say “L+Z”.

In line 60, use the abbreviations for lutein and zeaxanthin since you have already defined them previously. Or use full names throughout.

In line 69, use the abbreviation for lutein since you have previously defined it.

In line 75, can you please mention if the omega-3 intake was through diet, supplements, or both for this referenced study? In this same line, you do not need the word “Also” since it is followed by “in addition”.

In line 78, remove the word “very” since “very variable” doesn’t sound right. You could either leave it as just “variable” or change it something like “notably variable”.

In line 83 in this sentence: The hypothesis was that omega-3-fatty acids supplement with L+Z would be more bioavailable compared to taking L+Z supplements alone, I recommend the following rewording: The hypothesis was that the omega-3-fatty acid supplement combined with L+Z would be more bioavailable compared to taking L+Z supplements alone. 

Methods

In line 113, where it says “venous blood samples (20 ml) before and after the wash-out”, was blood taken right after the wash out and before the intervention, or after the intervention, or both? This is explained later in line 141 but you may want to add more detail here (near line 113) as well.

Did the instructions for taking the supplement include time of day? Did they take it during a meal?

Lines 153-154 – you have repeated this phrase twice: the collected blood samples were centrifuged at 1000 x g 154 for 15 minutes at room temperature to separate serum

In line 156, remove the ( symbol at the beginning.

In line 175, I suggest defining the MDA abbreviation in the next line and not the subtitle heading.

In line 180, please provide the model and manufacturer of the spectrophotometer.

Can you provide more information (in addition to the citation) about the kit-provided formula? Perhaps show the formula?

Results

In line 205, change while to white.

Lines 205-206 – “56.3% of 205 the subjects were never married while 43.8% were married.” I’m not sure this information is useful for this study.

In line 207, do you mean to say “waist circumference”?

Remove the unnecessary ( symbol in line 208.

In Table 1, I assume you meant to write “Men” and not M95. For all units with m squared, the 2 needs to be a superscript. I suggest using a,b,c,d or symbols instead of numbers for your footnotes since this may be confusing with the type of units shown.

For column 1 in Tables 1 and 2, some abbreviations should be defined in the footnotes.

Lines 266-67 are a repeat of the previous two lines.

Line 285-287 appear to be footnotes that have lost their table.

Most of the tables have mean and SEM written in one format, but Table 9 shows it in a different format. I suggest using the same format throughout the paper.

Discussion

In lines 373-382, consider combining some of the shorter paragraphs into a larger more cohesive paragraph.

Overall, the discussion could be written with a better flow and organization. In some places it reads as if items were cut and pasted in without proper transitions.

Consider adding a strengths and weakness section.

Somewhere in the discussion or conclusion (or both), the authors should remind the reader of the main hypothesis and the final outcome related to the hypothesis.

Author Response

Comments for Reviewer 2

This was an interesting article that has important health implications for vision related problems such as age-related macular degeneration. Please consider the following suggestions for improvement:

Abstract

This is strictly personal preference, but I think three letter abbreviations for Lutein (Lut) + Zeaxanthin (Zea) would look better and be easier to read. I also recommend ARMD for age-related macular degeneration (instead of AMD). Also, you may want to consider not abbreviating Lutein and Zeaxanthin in any of the text and just using their full names throughout, unless you need to abbreviate them for a table or figure.

In line 18 and 31 you have used the full name for lutein and zeaxanthin, but since you have previously defined the abbreviations in the abstract, you should use the abbreviations here, too. (Or just don't use the abbreviations at all!)

Reviewer Comment Response:

We thank the reviewer for this helpful suggestion. For clarity and consistency with commonly used terminology in the field, we have retained the abbreviation “L+Z” throughout the manuscript, as this format is widely used in the literature to denote combined lutein and zeaxanthin. We believe this notation also improves readability when referring to the combined biomarker.

Regarding age-related macular degeneration, we have retained the abbreviation “AMD”, as this is the most commonly used term in the current literature.

However, we appreciate the reviewer’s suggestion and have ensured that all abbreviations are clearly defined at first use and used consistently throughout the manuscript to enhance readability. Further, we have reviewed the manuscript to ensure that the use of abbreviations does not compromise readability.

Additionally, we have revised the manuscript to ensure consistent use of terminology throughout. As “lutein + zeaxanthin (L+Z)” is defined at first mention, we now use the abbreviation “L+Z” consistently in the text, including in lines 18 and 31.

 

Keywords

Thanks for this suggestion. The following keywords have now been included – Lutein; Zeaxanthin; Omega-3 fatty acids; Oxidative stress; Malondialdehyde (MDA); Antioxidants; Pilot study; Bioavailability; Inflammation; Human clinical trial; Nutritional supplementation;

 

Introduction

In line 59, rather than say “of these compounds (L+Z)”, you can just say “L+Z”.

Reviewer Comment Response:

Thank you for this helpful suggestion. We agree that the phrasing can be simplified for clarity. Accordingly, we have revised line 59 to read “L+Z” instead of “of these compounds (L+Z)”.

In line 60, use the abbreviations for lutein and zeaxanthin since you have already defined them previously. Or use full names throughout.

Reviewer Comment Response:

Thank you for this helpful suggestion. We agree that consistency in terminology improves readability. Accordingly, we have revised line 60 to use the abbreviations for lutein and zeaxanthin, consistent with their earlier definition.

In line 69, use the abbreviation for lutein since you have previously defined it.

Reviewer Comment Response:

Thank you for this helpful suggestion. We agree that consistent use of abbreviations improves clarity. Accordingly, we have revised line 69 to use the abbreviation for lutein, consistent with its earlier definition in the manuscript.

 

In line 75, can you please mention if the omega-3 intake was through diet, supplements, or both for this referenced study? In this same line, you do not need the word “Also” since it is followed by “in addition”.

Reviewer Comment Response:

Thank you for this helpful suggestion. We agree that additional detail improves clarity. Accordingly, we have revised line 75 to specify whether omega-3 intake in the referenced study was derived from diet, supplements, or both.

We have also removed the word “Also” to avoid redundancy, as it was followed by “in addition.”

 

In line 78, remove the word “very” since “very variable” doesn’t sound right. You could either leave it as just “variable” or change it something like “notably variable”.

Reviewer Comment Response:

Thank you for this helpful suggestion. We agree that the phrasing can be improved for clarity. Accordingly, we have removed the word “very” and revised the text to read ‘variable’

 

In line 83 in this sentence: The hypothesis was that omega-3-fatty acids supplement with L+Z would be more bioavailable compared to taking L+Z supplements alone, I recommend the following rewording: The hypothesis was that the omega-3-fatty acid supplement combined with L+Z would be more bioavailable compared to taking L+Z supplements alone. 

Reviewer Comment Response:

Thank you for this helpful suggestion. We agree that the revised wording improves clarity and readability. Accordingly, we have adopted the suggested phrasing in line 83.

 

Methods

In line 113, where it says “venous blood samples (20 ml) before and after the wash-out”, was blood taken right after the wash out and before the intervention, or after the intervention, or both? This is explained later in line 141 but you may want to add more detail here (near line 113) as well.

Reviewer Comment Response:

Thank you for this helpful observation. We agree that the timing of blood sample collection should be clarified earlier in the manuscript for improved readability.

Accordingly, we have revised line 113 to specify that venous blood samples were collected before (baseline) and after the wash-out (pre-intervention) and following the intervention.

Did the instructions for taking the supplement include time of day? Did they take it during a meal? –

Reviewer Comment Response:

Thank you for this important comment. We agree that details regarding supplement administration are essential for reproducibility and clarity.

Accordingly, we have revised the Methods section to specify how the participants were instructed to take the supplement:

The subjects were provided with instructions on the number of capsules to take daily (the control group consumed 1 capsule of the L+Z supplement daily, while the intervention group consumed L+Z (1 capsule/day) plus Omega-3 supplements (1 capsule/day). Adherence to both the LCOD diet and supplements protocols was monitored. (See Method Section, Lines 141 – 144).

Lines 153-154 – you have repeated this phrase twice: the collected blood samples were centrifuged at 1000 x g 154 for 15 minutes at room temperature to separate serum

Reviewer Comment Response:

Thank you for highlighting this repetition. We agree that this was redundant. Accordingly, we have removed the duplicated phrase in lines 187 - 188 to improve clarity and readability.

In line 156, remove the ( symbol at the beginning.

Reviewer Comment Response:

Thank you for pointing this out. We agree that this was a typographical error. Accordingly, we have removed the extraneous “(” symbol at the beginning of line 189.

In line 175, I suggest defining the MDA abbreviation in the next line and not the subtitle heading.

Reviewer Comment Response:

Thank you for this helpful suggestion. We agree that defining abbreviations within the main text rather than in a subtitle improves clarity. Accordingly, we have revised the manuscript to define MDA in the first instance within the text following the subtitle (See, Method section Line 209).

 

In line 180, please provide the model and manufacturer of the spectrophotometer.

Reviewer Comment Response:

Thank you for this important suggestion. We agree that providing detailed instrumentation information improves reproducibility. Accordingly, we have revised line 180 to include the model and manufacturer of the spectrophotometer used in the analysis ( See Method section,  Lines 213  – 214)

Can you provide more information (in addition to the citation) about the kit-provided formula? Perhaps show the formula?

Reviewer Comment Response:

Thank you for this valuable suggestion. We agree that providing additional detail regarding the kit-based calculations improves transparency and reproducibility.

Accordingly, we have expanded the Methods section to include a brief description of the formula provided by the assay kit manufacturer. Where appropriate, we have also included the formula used to calculate [e.g., MDA concentration], in addition to the original citation (See Method section, Lines 214  – 225).

 

Results

In line 205, change while to white.

Reviewer Comment Response:

Thank you for pointing this out. We agree that this was a typographical error. Accordingly, we have corrected “white” to “while” in line 205.

 

Lines 205-206 – “56.3% of 205 the subjects were never married while 43.8% were married.” I’m not sure this information is useful for this study.

Reviewer Comment Response:

Thank you for this helpful observation. We agree that this information is not essential to the interpretation of the study outcomes. Accordingly, we have removed the statement regarding marital status from lines 205–206 = lines 250 - 251 to improve conciseness and maintain focus on relevant participant characteristics.

 

In line 207, do you mean to say “waist circumference”?

Reviewer Comment Response:

Thank you for this helpful observation. Yes, we intended to refer to “waist circumference.” We have now corrected this in line 207 = line 233 for clarity and accuracy.

Remove the unnecessary ( symbol in line 208.

Reviewer Comment Response:

Thank you for pointing this out. We agree that this was a typographical error. Accordingly, we have removed the unnecessary “(” symbol in line 208 = line 210.

In Table 1, I assume you meant to write “Men” and not M95. For all units with m squared, the 2 needs to be a superscript. I suggest using a,b,c,d or symbols instead of numbers for your footnotes since this may be confusing with the type of units shown.

Reviewer Comment Response:

Thank you for these helpful observations on Table 1.

We have made the following revisions to improve clarity and consistency:

• Corrected the typographical error “M95” to “Men”.
• Revised all units involving squared terms to ensure that “m²” is presented with the appropriate superscript formatting.
• Updated the table footnotes to use alphabetical markers (a, b, c, d) instead of numerical notation to avoid potential confusion with unit expressions.

These changes have been incorporated into Table 1 in the revised manuscript.

 

Discussion

In lines 373-382, consider combining some of the shorter paragraphs into a larger more cohesive paragraph.

Reviewer Comment Response:

Thank you for this helpful suggestion. We agree that combining shorter paragraphs improves readability and overall flow. Accordingly, we have revised lines 373 - 382 by consolidating the shorter paragraphs into a more cohesive and integrated discussion.

Rather than relying solely on single time-point comparisons, this study employed interval-specific analyses using incremental area under the curve (iAUC) to better capture biological responsiveness. While total serum L+Z concentrations, assessed by AUC, were comparable between groups across the study duration, iAUC analysis revealed day-dependent differences that were not evident using AUC alone. See lines 357  - 361.

 

Overall, the discussion could be written with a better flow and organization. In some places it reads as if items were cut and pasted in without proper transitions.

Reviewer Comment Response:

Thank you for this valuable feedback. We agree that the flow and organisation of the Discussion can be improved.

In response, we have carefully revised the section to enhance logical progression and coherence. Specifically, we have improved transitions between paragraphs, reorganised related ideas for better continuity, and refined the wording to ensure a more cohesive narrative throughout.

We believe these revisions have strengthened the readability and clarity of the Discussion.

 

Consider adding a strengths and weakness section.

Reviewer Comment Response:

Thank you for this suggestion. These aspects have now been addressed within the Discussion section (see final paragraphs lines 426 - 485). We believe this integrated approach avoids repetition while still providing a clear and balanced evaluation of the study’s strengths and limitations.

 

Somewhere in the discussion or conclusion (or both), the authors should remind the reader of the main hypothesis and the final outcome related to the hypothesis.

Reviewer Comment Response:

Thank you for this valuable suggestion. We agree that reiterating the main hypothesis alongside the study outcomes improves clarity and strengthens the overall narrative.

Accordingly, we have revised the Conclusion to explicitly restate the primary hypothesis and clearly summarise the findings in relation to it. We have also ensured that the conclusions are appropriately aligned with the pilot nature of the study and the observed results (See Conclusion section, Lines 489 – 496).