-->PONE-D-25-45261-->-->Stage-Specific Associations of Mineralization Markers with CKM Syndrome: Nationwide Survey and Genetic Evidence for Alkaline Phosphatase’s Unique Clinical Role-->-->PLOS One

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Dear the Editor of the PLOS ONE Journal,

Thank you very much for providing me with this opportunity. I would also like to sincerely acknowledge the authors for their valuable contributions to the scientific community and for advancing knowledge in their respective field.

Manuscript Number: PONE-D-25-45261

Article Type: Research Article

Title: Stage-Specific Associations of Mineralization Markers with CKM Syndrome: Nationwide Survey and Genetic Evidence for Alkaline Phosphatase’s Unique Clinical Role

• My overall evaluation of the manuscript is that the manuscript addresses an important and timely topic: the role of mineralization markers (ALP, calcium, and phosphorus) across stages of Cardiovascular–Kidney–Metabolic (CKM) syndrome using a large, nationally representative dataset (NHANES), complemented by Mendelian randomization analyses. The combination of stage-specific epidemiological analysis, mortality outcomes, and genetic causal inference is a notable strength. The study is generally well designed and statistically sophisticated, and the findings particularly the consistent association of ALP with CKM stages and mortality are potentially clinically relevant. However, several methodological clarifications, interpretational refinements, and presentation improvements are required to strengthen the scientific validity and transparency of the work.

Major Comments

Conceptual clarity of CKM staging and causal interpretation

• The manuscript applies modified AHA CKM staging using NHANES variables, which is reasonable; however, CKM stages are composite constructs incorporating outcomes (CVD, CKD, diabetes) that are also biologically linked to ALP, calcium, and phosphorus. This creates a risk of circularity or over interpretation, particularly when associations are described as “risk” of CKM stages in a cross-sectional design. What I recommend is that the authors are expected to clarify throughout manuscript that the primary analyses are associational, not predictive and replace phrasing such as “risk of CKM stage” with “likelihood of being classified into CKM stage” where appropriate and explicitly discuss potential bidirectional relationships between ALP and CKM components in the discussion.

Study participants

• Participants under 20 years of age or those who were pregnant were excluded. Additionally, individuals with insufficient data to evaluate CKM staging were excluded. According to WHO, age of 18 years and above is considered as an adult, what is your justification to exclude participants under 20 years old? In addition, individuals with insufficient data were excluded from the study. What percentage or which data level was considered insufficient?

Cross-sectional design vs. causal inference

• While the Mendelian randomization (MR) analysis strengthens causal inference for ALP CVD and CKD ALP relationships, the NHANES analyses remain cross-sectional. Some interpretations in the discussion imply disease progression or temporality that cannot be established. Therefore, clearly distinguish between cross-sectional NHANES findings (association), prospective mortality analyses (temporal but limited), and MR findings (genetic causality) and avoid implying that ALP “drives progression” of CKM stages without longitudinal CKM staging data.

Use and interpretation of ALP as a non-specific biomarker

• Total serum ALP includes hepatic, bone, intestinal, and placental isoenzymes, yet the manuscript interprets ALP largely as a calcification/mineralization marker. Liver disease, cholestasis, and subclinical hepatic dysfunction may confound associations. Thus, explicitly acknowledge the non-specific nature of total ALP and discuss potential confounding from liver-related ALP sources. If available, clarify whether AST/ALT or liver disease variables were considered or why they were not included.

Calcium findings: inconsistency across stages and outcomes

• Calcium shows positive association with advanced CKM stages, non-linear associations in RCS models, and inverse association with all-cause mortality. These findings appear biologically inconsistent and require clearer explanation. Therefore, expand discussion on tight homeostatic regulation of serum calcium, influence of albumin (uncorrected calcium limitation), CKD-related hypocalcemia vs. CVD-related hypercalcemia and clarify whether albumin-corrected calcium was considered and justify its exclusion.

Phosphorus: contradictory associations across analyses

• Phosphorus is associated with stage 3 and 4b in multinomial models, not associated with advanced CKM after adjustment, and strongly associated with mortality. This pattern suggests context-specific effects, primarily driven by CKD severity. Thus, explicitly frame phosphorus as a late-stage CKD marker rather than a general CKM biomarker and emphasize that phosphorus may be more relevant to mortality risk than CKM stage classification.

Mendelian randomization: instrument selection and interpretation

• The MR analysis is a strength, but removing >80% of ALP-associated SNPs raises concerns about instrument strength and selection bias. The MR-Egger results are largely non-significant. The authors are recommended to provide F-statistics or mean F-statistics to demonstrate instrument strength, discuss the discrepancy between IVW and MR-Egger results more cautiously and avoid strong causal language where MR evidence is borderline or inconsistent.

Minor Comments

• Several sentences require grammatical polishing (e.g., “To addressing this gap,” “This was because the relative risk ratios…”).

• Use consistent terminology for: “advanced CKM stages” vs. “advanced CKM”, “CKM-cause mortality” vs. “CKM-related mortality”

• Clearly state whether calcium and phosphorus RRRs are per 1 mmol/L, 1 mg/dL, or per SD increase.

• Ensure consistent use of OR vs. RRR vs. HR.

• Figures showing RCS curves should clearly label reference values.

• Strengths of the Study

• Large, nationally representative sample.

• Comprehensive CKM staging framework.

• Robust survey-weighted statistical methods.

• Integration of mortality analysis.

• Use of Mendelian randomization to explore causality.

• Clear evidence supporting ALP as a consistent CKM-associated biomarker.

Overall Recommendation

Major revision

The manuscript is scientifically valuable and suitable for publication after conceptual clarification, cautious interpretation of causality, and improved discussion of biomarker specificity and biological mechanisms. Addressing the points above will significantly enhance the rigor, clarity, and impact of the study.

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Reviewer #1: Yes:   Dr. Hussen Abdu Muhidin

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-->PONE-D-25-45261R1-->-->Stage-Specific Associations of Mineralization Markers with CKM Syndrome: Nationwide Survey and Genetic Evidence for Alkaline Phosphatase’s Unique Clinical Role-->-->PLOS One

Dear Dr. Shen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jul 03 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you’re ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the ‘Submissions Needing Revision’ folder to locate your manuscript file.

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-->

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Rashmi Supriya

Academic Editor

PLOS One

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Reviewer #2: All comments have been addressed

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: Yes

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-->6. Review Comments to the Author

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Reviewer #2: 1. The claim that ALP is more robust than calcium or phosphorus is not yet methodologically fair. ALP is modeled as quartiles, whereas calcium and phosphorus are modeled as continuous variables in mg/dL. Statistical significance across these different parameterizations is not directly comparable. The conclusion that ALP has a “unique clinical role” may therefore reflect modeling choices rather than true biological superiority. A stronger comparison would analyze all three markers on the same scale, such as z-scores, quartiles, or restricted spline models.

2. Although sensitivity analyses (liver disease exclusion, ALT/AST adjustment, and albumin-corrected calcium) are commendable, residual confounding remains a concern. Key unadjusted factors, such as parathyroid hormone, fibroblast growth factor-23, phosphate binder use, and vitamin D supplement use, directly influence mineral metabolism and CKM progression.

3. The NHANES analysis is based on a multi-ethnic U.S. population including Mexican Americans, Non-Hispanic Blacks, and multiracial groups. However, the MR analysis exclusively uses summary statistics from individuals of European ancestry (UK Biobank and FinnGen). Genetic variants (SNPs) associated with ALP levels can vary significantly across ethnicities. Applying European-derived genetic instruments to interpret findings from a diverse U.S. cohort may limit the validity of the causal inferences made in the conclusion.

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Reviewer #2: No

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Stage-Specific Associations of Mineralization Markers with CKM Syndrome: Nationwide Survey and Genetic Evidence for Alkaline Phosphatase’s Unique Clinical Role

PONE-D-25-45261R2

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