Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a meta-analysis aimed at determining whether levels of copper, ceruloplasmin, zinc, and manganese in the brain, cerebrospinal fluid (CSF), serum/plasma, whole blood, and hair are associated with the risk of Parkinson’s disease (PD). The authors concluded that patients with PD exhibit decreased copper concentrations in the substantia nigra and other brain regions, a trend toward increased CSF copper and decreased serum/plasma copper levels, reduced serum/plasma ceruloplasmin levels, decreased zinc levels in serum/plasma with increased zinc levels in whole blood and hair, and increased manganese levels in hair.

Although these findings are potentially important, many readers of this manuscript—including neurologists, neuropathologists, and neuroscientists—would be particularly interested in how these alterations relate to the known neuropathological changes and molecular mechanisms underlying PD pathogenesis. In addition, the clinical implications of these findings should be discussed in greater depth, including whether changes in metal concentrations in blood or CSF could serve as diagnostic biomarkers or reflect disease activity or progression. Expanding the Discussion section to address these aspects would substantially enhance the translational relevance and impact of the study.

Author Response

REVIEWER # 1

This manuscript presents a meta-analysis aimed at determining whether levels of copper, ceruloplasmin, zinc, and manganese in the brain, cerebrospinal fluid (CSF), serum/plasma, whole blood, and hair are associated with the risk of Parkinson’s disease (PD). The authors concluded that patients with PD exhibit decreased copper concentrations in the substantia nigra and other brain regions, a trend toward increased CSF copper and decreased serum/plasma copper levels, reduced serum/plasma ceruloplasmin levels, decreased zinc OKmanganese levels in hair.

Although these findings are potentially important, many readers of this manuscript—including neurologists, neuropathologists, and neuroscientists—would be particularly interested in how these alterations relate to the known neuropathological changes and molecular mechanisms underlying PD pathogenesis. In addition, the clinical implications of these findings should be discussed in greater depth, including whether changes in metal concentrations in blood or CSF could serve as diagnostic biomarkers or reflect disease activity or progression. Expanding the Discussion section to address these aspects would substantially enhance the translational relevance and impact of the study.

Ok. We have incorporated several new paragraphs into the Discussion to further clarify the implications of our findings for the pathogenesis of Parkinson’s disease. All modifications have been highlighted in red.

Reviewer 2 Report

Comments and Suggestions for Authors

This review/meta-analysis evaluates studies describing transition metals (copper, zinc, and manganese) and ceruloplasmin as the potential risk factor for PD. The authors utilize both common and random effects models to aggregate the data, demonstrating abnormal levels of transition metals and ceruloplasmin in samples from PD compared to controls. This review/meta-analysis uses a sufficient number of studies, which may provide a useful reference for searching meaningful biomarker(s) for PD.

Comments:

1. “Core Collection - databases from 1966 to November 29, 2025”. The author should consider performing a sub-group analysis (i.e. detection method) to validate that data collected close to 1966 won’t introduce inaccurate analysis.
2. Table 2 requires further optimization. It would be ideal to use symbols in the “main result” section to illustrate key findings of various papers (i.e. using +, ++ or +++ to illustrate the levels of transition metals).
3. What are the advantages and disadvantages of the various analytical methods used in the studies? Please include an additional table to discuss these methods. Did they perform absolute quantification or relative quantification? Also, the authors indicated that some studies were excluded due to the lack of a control group. It would be great if the authors can address how control groups might influence the measurement of metal levels when using different analytical methods.

Author Response

REVIEWER # 2

This review/meta-analysis evaluates studies describing transition metals (copper, zinc, and manganese) and ceruloplasmin as the potential risk factor for PD. The authors utilize both common and random effects models to aggregate the data, demonstrating abnormal levels of transition metals and ceruloplasmin in samples from PD compared to controls. This review/meta-analysis uses a sufficient number of studies, which may provide a useful reference for searching meaningful biomarker(s) for PD.

Comments:

1. “Core Collection - databases from 1966 to November 29, 2025”. The author should consider performing a sub-group analysis (i.e. detection method) to validate that data collected close to 1966 won’t introduce inaccurate analysis.

We appreciate the reviewer’s suggestion to perform a subgroup analysis based in the detection method, particularly to evaluate whether data collected closer to 1966 might introduce bias. The results of this subgroup analysis excluding those works published before 1985 should be as follows:

 

Copper concentrations in CSF.

Overall comparisons, difference of means (95% C.I.); P: -6.41 (-12.11, -0.70); P = 0.0279

Excluding Campanella et al., 1973 [23]: −7.10 (−12.61; −1.59); P = 0.0116

In both scenarios, the comparison is statistically significant.

 

Copper concentrations in blood/plasma.

Overall comparisons, difference of means (95% C.I.); P:  -2.56 (-4.69, -0.43); P = 0.0183

Excluding studies previous to 1985: −6.76 (−8.47; −5.05); P < 0.0001

In both scenarios, the comparison is statistically significant.

 

Ceruloplasmin concentrations in serum/plasma

Overall comparisons, difference of means (95% C.I.); P: -4.90 (-5.90, -3.90); P < 0.0001.

Excluding Campanella et al., 1973 [23]: −4.21 (−5.19; −3.22); P < 0.0001

In both scenarios, the comparison is statistically significant.

 

Manganese concentrations in serum/plasma

Overall comparisons, difference of means (95% C.I.); P:  -0.08 (-0.16, -0.01); P = 0.0355

Excluding Kanabrocki et al., 1967 [38]: −0.11 (−0.20; −0.02); P = 0.0117.

In both scenarios, the comparison is statistically significant.

 

We have introduced a brief comment in the discussion regarding this issue

 

 

1. Table 2 requires further optimization. It would be ideal to use symbols in the “main result” section to illustrate key findings of various papers (i.e. using +, ++ or +++ to illustrate the levels of transition metals).

OK, Done

1. What are the advantages and disadvantages of the various analytical methods used in the studies? Please include an additional table to discuss these methods. Did they perform absolute quantification or relative quantification? Also, the authors indicated that some studies were excluded due to the lack of a control group. It would be great if the authors can address how control groups might influence the measurement of metal levels when using different analytical methods. 

OK, we have added a Table as indicated to discuss these methods and explained the influence of lack of a control group at the end of the Discussion.

 

The changes we have made are highlighted in red colour

Reviewer 3 Report

Comments and Suggestions for Authors

This is an interesting, in depth review and meta-analysis on copper, ceruloplasmin, zinc, manganese and relationship to the risk for Parkinson’s disease. I have only minor recommendations that are below given.

It has been reported that significant amounts of Fe, Cu and Zn are bound to neuromelanin in neurons of human putamen , premotor cortex, cerebellum, and substantia nigra in normal subjects. This should be mentioned (Zecca et al PNAS 2008).

Brain aging is the major risk factor for PD, AD and other neurodegenerative diseases. The age related changes of Cu and Zn in regions like in locus coeruleus and substantia nigra during aging should be considered, since these regions have the highest neuronal loss in PD. There is an age related decrease of Cu in locus coeruleus. These regions contain the highest amount of neuromelanin that is bound to these metals (Zecca et al PNAS 2004).

I kindly suggest authors to cite the above aspects in Discussion about the ability of neuromelanin to accumulate Fe, Cu and Zn.

Author Response

REVIEWER # 3

This is an interesting, in depth review and meta-analysis on copper, ceruloplasmin, zinc, manganese and relationship to the risk for Parkinson’s disease. I have only minor recommendations that are below given.

It has been reported that significant amounts of Fe, Cu and Zn are bound to neuromelanin in neurons of human putamen , premotor cortex, cerebellum, and substantia nigra in normal subjects. This should be mentioned (Zecca et al PNAS 2008).

Brain aging is the major risk factor for PD, AD and other neurodegenerative diseases. The age related changes of Cu and Zn in regions like in locus coeruleus and substantia nigra during aging should be considered, since these regions have the highest neuronal loss in PD. There is an age related decrease of Cu in locus coeruleus. These regions contain the highest amount of neuromelanin that is bound to these metals (Zecca et al PNAS 2004).

I kindly suggest authors to cite the above aspects in Discussion about the ability of neuromelanin to accumulate Fe, Cu and Zn.    

We have addressed the points raised by the reviewer in the Discussion and incorporated the two corresponding citations into the reference list. All modifications have been highlighted in red.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

he manuscript has been revised appropriately in accordance with my suggestions.