Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the submitted paper the authors prove in  a North-European clinical population that more frequent and serious exacerbations and higher concentrations of inflammatory markers are associated with VitD deficiency in chronic pulmonary obstructive disease patients.

The authors themselves admit that interconnection between VitD plasma levels and COPD has been thoroughly studied by several earlier publications which limits the novelty of the paper.

Frequency and severity of acute exacerbations  increased with VitD deficiency (Jiang Y et al Clin Therap 225;47:44).  VitD deficiency is frequent in COPD patients, minor variabilities of the VitD binding gene found (Janssen W et al Thorax 2010;65:216). A protective effect of increasing 25-OHD levels (up to sufficient levels) against airway obstruction has been detected by Han Z et al in COPD patients (Sci Rep 2025;15:7474). Lower levels of VitD  plasma levels were associated with more frequent acute exacerbations of the disease, a practically identical with the submitted paper   observation (Onur T et al Asia Pac J Clin Nutr 2025;34:724). VitD deficiency was associated with  more serious acute exacerbations of COPD  (Rus LA et al. Medicina 2025;61:979; Zhou L et al. Intern J Chronic Obstr Pulm  Dis 2025;20:171.).  Preventing infections during acute exacerbations seems to be one important component of defensive effect of sufficient VitD plasma levels according to Sun M et al. (Intern J Chronic Obstr Pulm  Dis 2025;20:2023).  A detailed chapter deals with the role of VitD in COPD in the review paper by Simon P et al. (Nutrients 205;17:1149). No positive effects however, on either exacerbation or on functional lung parameters could be found with VitD supplementation  (Hua Y et al. Biochem Med (Zagreb) 2023;33:030703;Gold DR eta al. J Nutr 2025;155:1417.). And also a recent Cochrane review explicitly denied that VitD therapy could provide any protection against the acute flare-ups of chronic obstructive pulmonary disease (Cochrane Database Syst Rev 2024(9):CD013284). Severe VitD deficiency however was strongly associated with hospitalization for exacerbation of COPD in the previous year(Li B et al. Intern J Chronic Obstr Pulm  Dis 2024;19:1471).

Even a cellular pathomechanism has been spotted for VitD deficiency in airway epithelial cells (Stapleton EM et al. Am J Physiol Cell Physiol 2024;326:C540.).

The paper should be better positioned in the context of these published studies and debates, focusing around its real merits, namely further proving the existence of an association between VitD deficiency and seriousness of exacerbations of COPD in a North-European  population and its connection with more serious systemic inflammation.

As a limitation it has to be stated that still not a causal effect between the two factors  has been proven. In fact, a common socio-economic status and life-style (less fish and VitD enriched food consumed, less outdoor activity) can be common causes for both.

The language of the paper is a correct scientific English. The paper is logically built, Tables, Figures are professionally organized.

Some minor remarks

Abstract, Methods, 5^th line, Mere  FEV₁ measurements are not called dynamic lung study nowadays

Introduction, 5^th line, instead lack of vit D, insert, deficiency

Chapter 2.1, 7^th line, values just below 100% of FEV₁ are considered pathological?

Chapter 2.2 1^st line, at inclusion into the study?  During a bout of exacerbation?  Or independently of it.

Chapter 2.3  FEV₁/CV (Tiffeneau index) is an outdated technique to examine airway obstruction. For a more sophisticated examination peak flow, airway resistance during normal and forced breathing with and without provocation and bronchodilators are now generally applied.

Table 1. COPD medications. More serious forms of exacerbations were not offered different therapies?

 

 

Author Response

Dear Reviewer,

We sincerely thank you for your extremely helpful and constructive comments, which have significantly improved the quality and clarity of our manuscript. All of your considerations have been carefully addressed and implemented as detailed below.

1. Positioning of the study within recent literature

A new paragraph has been added to the Discussion section incorporating recent publications (2024–2025), including four of the recommended references:

“Recent publications from 2024–2025 further support the association between vitamin D deficiency and increased COPD exacerbations (33,34). However, evidence regarding causality and therapeutic benefit remains conflicting, as a recent Cochrane review failed to demonstrate consistent protective effects of vitamin D supplementation (35). Severe vitamin D deficiency, however, was strongly associated with hospitalization for exacerbation of COPD in the previous year (36). In contrast to previous heterogeneous COPD populations, the present study exclusively investigated patients classified as GOLD group E.”

This addition better positions our study within the current debate and clarifies its specific contribution.

2. Clarification of causality and study limitations

A new paragraph has been added to the Limitations section:

“Taken together with recent literature, the present study does not aim to establish causality but rather to reinforce, in a well-defined North-European high-risk COPD population, the robustness of the association between severe vitamin D deficiency, systemic inflammation and exacerbation burden.”

Additionally, the following statement has been added at the end of the Limitations section:

“Finally, vitamin D deficiency may represent a marker of reduced outdoor activity, nutritional habits, or socio-economic determinants rather than a direct mechanistic driver. Despite strong associative evidence, the most recent Cochrane review (2024) concluded that vitamin D supplementation does not consistently prevent acute COPD exacerbations (35).”

These additions clarify the observational nature of the study and acknowledge potential residual confounding.

3. Addition of a mechanistic paragraph

A new paragraph has been added at the end of the Discussion section addressing a potential epithelial mechanism:

“Recent studies suggest that vitamin D deficiency may also impair airway epithelial hydration. Calcitriol deficiency increases epithelial sodium channel (ENaC) activity, enhancing sodium and water absorption from the airway surface liquid. Reduced surface hydration may compromise mucociliary clearance and innate immune defense, promoting mucus plugging and increasing susceptibility to exacerbations (46).”

This addition strengthens the biological plausibility of the observed association.

4. Minor revisions and technical clarifications

All minor remarks have been carefully addressed.

i. Terminology (“dynamic lung study”)
Replaced with: “spirometric lung function parameters.”

ii. Terminology (“lack of vitamin D”)
Replaced with: “vitamin D deficiency.”

iii. Timing of blood sampling (Chapter 2.2)
Clarified as follows:
“Venous peripheral blood samples were collected at study inclusion during a clinically stable phase of COPD, in the absence of acute exacerbation…”

This is consistent with the previously stated exclusion criterion:
“COPD exacerbation or respiratory infection within two weeks prior to inclusion.”

iv. Spirometry and FEV₁/FVC ratio
Airflow limitation in the present study was assessed using post-bronchodilator spirometry in accordance with GOLD recommendations, where an FEV₁/FVC ratio <0.70 remains the established diagnostic criterion for COPD. The term “dynamic spirometry” has been replaced by “spirometry” to avoid ambiguity.

v. COPD medications (Table 1)
All included patients fulfilled the criteria for GOLD group E and were therefore treated according to contemporary guideline-recommended therapy for frequent exacerbators. As a result, maintenance pharmacotherapy did not significantly differ between vitamin D groups. A clarifying sentence has been added to the Results section:

“Since all included patients belonged to GOLD group E and were treated according to contemporary guidelines, no significant differences in maintenance pharmacotherapy were observed between vitamin D groups.”

We again thank you for the insightful and constructive feedback, which has substantially strengthened the manuscript.

Sincerely,

Apostolos Sioutas

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors This manuscript aims to determine the relationship between vitamin D deficiency, inflammation, and exacerbation frequency in COPD patients belonging to the GOLD E group. The analysis of patients with frequent exacerbations is more valuable compared to studies involving heterogeneous COPD populations. The study design is good, the methods are well described, and the results are consistent. The work contributes a novel element by demonstrating a very strong correlation between 25(OH)D levels and exacerbation frequency in the homogeneous GOLD E group (minimized heterogeneity).   Notes:   1. Abstract. Please specify in the "Methods" section that the study is cross-sectional in nature.   2. Methods. Please consider shortening the data in the Data Handling and Data Protection section.   3. Please specify whether hs-CRP was determined in the same analytical run for all samples.   4. Results. In Table 1: Please clarify the values ​​and range (Age, BMI, WBC, SAT, FEV), e.g., age, whether it is AM (SD) or Median (IQR). For age, Median and IQR are correct, etc.   5. Please add the exact median and IQR values ​​for hs-CRP and the number of exacerbations in the text of the results (not just in the figures).   6. Discussion: Please consider shortening the section regarding recruitment difficulties in RCTs (PRECOVID) to balance the interpretation of your results with the literature.   7. The authors correctly identify the limitations resulting from the cross-sectional nature of the study. However, in several places (particularly in the abstract and conclusion), the wording suggests a potential clinical benefit of vitamin D supplementation. It is worth clearly separating the obtained correlation from the interpretation of causality to avoid overinterpretation/ erroneous conclusions.   8. Although the authors mention seasonality and variability in vitamin D levels in the limitations, they should add information about the month of sample collection (if available) or explicitly highlight this factor as a potential source of bias in the results or methods sections.   9. The obtained correlation coefficient of r = −0.74 is quite high. The authors should comment on possible confounding factors (e.g., general health status, physical activity) and consider whether the correlation is partially amplified by intervening variables (e.g., disease severity).    

Author Response

Dear Reviewer,

We sincerely thank you for your careful evaluation of our manuscript and for your constructive and insightful comments, which have helped us to further improve the clarity and scientific rigor of our work. All remarks have been carefully considered and addressed as outlined below.

1. The cross-sectional nature of the study is now clearly specified in the Methods section of the Abstract.

2. The section on Data Handling and Data Protection has been shortened and now reads: “Data Handling and Data Protection. All collected data were pseudonymized and handled in accordance with the General Data Protection Regulation (GDPR). Data were securely stored and accessible only to authorized study personnel.”

3. An additional sentence has been added to section 2.3 to clarify laboratory procedures: “All hs-CRP analyses were performed in the same certified clinical laboratory using standardized methods.”

4. Table 1 has been revised accordingly, and the Statistical Analysis section now clearly states: “Continuous variables are presented as mean ± standard deviation (SD) when normally distributed, and as median with interquartile range (IQR) when normality assumptions were not fulfilled.”

5. The following paragraph has been added to the Results section to provide exact values in the text: “Median hs-CRP was 4.7 mg/L (IQR 1.1–19.0) in the low vitamin D group compared to 1.7 mg/L (IQR 0.8–3.3) in the high vitamin D group. The median number of exacerbations during the preceding year was 5 (IQR 4–7) versus 2 (IQR 2–3), respectively.”

6. The previous paragraph discussing recruitment difficulties in RCTs has been replaced with the following shortened version: “Recruitment challenges in vitamin D supplementation trials have been illustrated in the PRECOVID study, where a large proportion of screened patients were excluded due to ongoing supplementation or sufficient baseline vitamin D levels (40). Such factors may reduce statistical power and limit the ability to detect a treatment effect. These findings highlight the methodological complexity of conducting adequately powered RCTs in contemporary populations with relatively lower prevalence of severe vitamin D deficiency. In the present study, all subjects were recruited between August 2012 and October 2018, a period when vitamin D deficiency still had a relatively high prevalence among Swedish patients with COPD.”

7. The Abstract conclusion has been revised to avoid overinterpretation and now reads: “These findings demonstrate a strong association between low vitamin D levels, systemic inflammation and exacerbation frequency in COPD patients belonging to GOLD group E. However, due to the cross-sectional design, no causal relationship can be inferred, and prospective interventional studies are required to determine whether correction of vitamin D deficiency improves clinical outcomes.”

The final Conclusion section has also been revised and now states: “The results of the present study demonstrate a strong association between low vitamin D levels, systemic inflammation and exacerbation frequency in COPD patients belonging to GOLD group E. However, prospective interventional studies are required to clarify whether correction of vitamin D deficiency improves clinical outcomes.”

8. Although patients were recruited between August 2012 and October 2018, the exact month of blood sampling for individual participants was not systematically documented for analytical purposes. This has now been explicitly clarified in the Methods section and highlighted in the Limitations section as a potential source of bias. In the Methods section it is now stated: “Blood samples were collected between August 2012 and October 2018; however, the exact month of sampling for individual participants was not systematically documented for analytical purposes.” In the Limitations section, the following has been added: “Second, seasonal variation in vitamin D levels was not accounted for. Although patients were recruited between August 2012 and October 2018, the exact month of blood sampling for individual participants was not available for adjustment in the analysis. Given Sweden’s northern latitude and marked seasonal variation in ultraviolet radiation, this may have influenced measured 25(OH)D concentrations and represents a potential source of bias.”

9. A paragraph has been added to the Discussion addressing the relatively strong correlation (r = −0.74) and the possibility of residual confounding. The following text has been included: “The strength of the observed correlation between serum 25(OH)D levels and exacerbation frequency warrants cautious interpretation. Although inclusion of a homogeneous population of frequent exacerbators (GOLD group E) reduces clinical heterogeneity, unmeasured confounding factors such as general health status, physical activity level, nutritional status, outdoor exposure, or residual differences in disease severity may have contributed to the magnitude of the association. Furthermore, intervening variables linked to both vitamin D levels and exacerbation risk could partially amplify the correlation. Due to the cross-sectional design, reverse causation cannot be excluded, and longitudinal studies are required to clarify directionality.”

We again thank you for your valuable comments, which have helped us to substantially improve the manuscript.

Sincerely,

Apostolos Sioutas

Reviewer 3 Report

Comments and Suggestions for Authors

The authors conducted a cross-sectional study was conducted on 111 patients with stable COPD. Patients were divided into two groups based on their serum 25(OH)D levels. The results show that patients with low serum 25(OH)D (<50 nmol/L) had significantly higher CAT score and level of serum-high sensitivity (hs)-CRP and exhibited significantly more exac erbations compared to those with higher levels (p >< 0.001, p < 0.001 and p < 0.0001, respectively).

The Major is about the structure and simplicity of the study. The study looks a simple report with one table and two figures where simple statistics have been applied.

 Introduction is also very simple with providing a logic of the study. Many important points have been missed here to explain.

There are no advance scientific findings and neither the sample size has been justified.

Negative and positive control are missing. The presentation of all sections is not scientifically suitable for its publication. Results have been presented in a very simple and poor way.

The discussion section lacks a proper justification. The data is very old which seems without a major objective.

Conclusion is very poor and the study objectives are simple and descriptive.

Author Response

Dear Reviewer,

We sincerely thank you for your thorough evaluation of our manuscript and for your critical comments. We have carefully reconsidered the structure, scientific depth, and overall presentation of the study and have made substantial revisions to improve clarity, methodological transparency, and scientific rigor. We hope that the revised version now better meets your expectations.

1. Regarding the overall structure and simplicity of the study, we have substantially revised the manuscript to improve scientific clarity and presentation. The Introduction has been expanded to better justify the rationale of focusing specifically on GOLD group E patients and to more clearly position the study within the context of previous literature. Recent studies (2024–2025) and mechanistic insights have been incorporated to strengthen the scientific background.

2. Concerning the comment that the study appears simple with limited tables and figures, we would like to emphasize that the study design was intentionally focused and hypothesis-driven. Nevertheless, we have improved the presentation of the Results section by adding detailed descriptive statistics directly in the text (median and IQR values for hs-CRP and exacerbation frequency) and clarifying statistical methods and data distribution. The structure of the manuscript has been refined to enhance scientific clarity and readability.

3. With respect to sample size justification, we acknowledge that a formal power calculation was not performed prior to this cross-sectional analysis. However, the study includes 111 well-characterized patients belonging exclusively to GOLD group E, thereby minimizing heterogeneity and strengthening internal validity. This has now been more clearly emphasized in the revised Discussion.

4. Regarding the absence of negative and positive controls, we respectfully clarify that this is an observational cross-sectional study and not an interventional experimental design. Therefore, classical experimental control groups are not applicable. Instead, patients were stratified based on predefined vitamin D thresholds (<50 nmol/L vs. ≥50 nmol/L), allowing comparison between clinically relevant exposure groups.

5. The Results section has been restructured to improve scientific presentation. Exact statistical values have been added to the text, distribution methods clarified, and laboratory procedures explicitly described. We have also added a detailed paragraph addressing the relatively strong correlation (r = −0.74), including discussion of potential confounding factors and reverse causation.

6. The Discussion section has been expanded and strengthened. Recent literature has been incorporated, mechanistic explanations have been added (including oxidative stress and epithelial ENaC-mediated hydration mechanisms), and the limitations have been more explicitly and critically addressed. We have also clearly separated correlation from causality and removed any wording that could imply therapeutic recommendations.

7. Regarding the age of the data, we clarified that patient recruitment occurred between 2012 and 2018, during a period when vitamin D deficiency prevalence was still high in Sweden. Importantly, the biological measurements were performed using standardized and quality-controlled laboratory methods. The relevance of the findings remains valid, particularly as recent literature continues to demonstrate similar associations.

8. The Conclusion section has been rewritten to avoid overinterpretation. The revised conclusion now clearly states that the findings demonstrate a strong association but do not establish causality, and that prospective interventional studies are required to determine clinical benefit.

We have made extensive structural, methodological, and interpretative revisions throughout the manuscript. We sincerely hope that the current revised version, as provided , now adequately addresses your concerns and meets the scientific standards required for publication.

We are grateful for your critical feedback, which has significantly strengthened the manuscript.

Sincerely,

Apostolos Sioutas

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors did not address my comments

Author Response

English , tables, figures requested changes made.

Author Response File: Author Response.docx