Reviewer ’s report  
 
Title: Anemia following zinc treatment for  Wilson's disease:a case report and literature review  
 
Version: 0 Date: 19 Jan 2019  
 
Reviewer: Raffaele Iorio  
 
Reviewer's report:  
 
The Authors described a ch ild who developed severe anemia following  zinc monotherapy for 
Wilson's disease. This case is interesting and well compared with other similar cases previously 
reported in literature. Unfortunately, there are many points that have to be clarified:  
a. why this patient was overtreated with zinc? could the Authors provide details about the 
reasons (liver function test, copper and zinc parameters) for which doses of zinc were increased, 
please?  
b. AASLD and EASL guidelines on WD should be considered in relatio n to the zinc doses used 
and to the desired zinc and copper parameters during therapy.  
c. As for the statement "increased 24 -h urinary copper excretion (360 µg, normal: 15 -60 µg)" 
(paragraph Case presentation, lines 6 -7), pediatric diagnostic criteria shou ld be considered 
(Nicastro et al. Re -evaluation of the diagnostic criteria for Wilson disease in children with mild 
liver disease. Hepatology 2010;52(6):1948 -56). 
d. As for the sentence "Since then, she has continued to take zinc at a dose of 240 mg/day, a nd 
strictly avoids foods with high copper levels" (paragraph Case presentation, line 24), according 
to available guidelines, foods with very high concentrations of copper (shellfish, nuts, chocolate, 
mushrooms, and organ meats) generally should be avoided,  during the first year of treatment. 
Especially in WD patients treated with zinc,  which inhibts intestinal copper absorption, dietary 
copper restriction is not mandatory. As reported in ESPGHAN WD guidelines (Socha et al. 
Journal of Pediatric Gastroentero logy and Nutrition: February 2018 - Volume 66 - Issue 2 - p 
334-344), "zinc should not be taken with food because it interferes with its absorption, and 
dietary copper restriction is not recommended because zinc blocks copper absorption from the 
intestinal  track".  
e. As for the lack of response of patient's anemia to blood transfusion (2 units) (in the manuscript 
the Authors stated:  "She received transfusion of 2 units of erythrocyte suspension and continued 
oral zinc at her previous dose. There was no sig nificant improvement in hematological 
parameters"), further details are desirable. Could the Authors clarify the reasons for which the 
patient did not show a favorable (at least transient) response to blood transfusion. Was this 
phenomenon observed in othe r patients reported in literature? Could the Authors give further 
details on the pathogenic mechanisms of copper deficiency anemia? It is very impressive that 
zinc withdrawal (plus 1 unit of blood)  induced an improvement in patient's hemoglobin levels 
within 1 week, from 4 to 6.7 grams (Table 1). This impressive result should be better discussed.  
f. In the Abstract the Authors affirmed "Her hemoglobin was gradually restored to the normal 
range after the adjustment of zinc dose and diet therapy for 4 month s", in the paragraph "Case 
presentation" the following sentence is present: "When zinc was withdrawn for one week, her 
hemoglobin level rose to 6.7 g/dL. She was then discharged and a normal diet was resumed. 
Low-dose zinc was recommended, but the girl's p arents refused the treatment though we told the 
possible flare of liver damage, even acute liver failure after the cessation of zinc treatment".  As 
the Authors  themselves underline, the definitive suspension of therapy in WD  is very hazardous 
and can in duce liver failure.  This point needs to be stressed in Discussion.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
No 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in t he manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
Not relevant to this manuscript  
 
Quality of wr itten English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content o f the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any  non-financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
'I declare that I have no competing interests'  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on  the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license  (http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: Anemia following zinc treatment for  Wilson's disease:a case report and literature review  
 
Version: 0 Date: 19 Jan 2019  
 
Reviewer: Raffaele Iorio  
 
Reviewer's report:  
 
The Authors described a ch ild who developed severe anemia following  zinc monotherapy for 
Wilson's disease. This case is interesting and well compared with other similar cases previously 
reported in literature. Unfortunately, there are many points that have to be clarified:  
a. why this patient was overtreated with zinc? could the Authors provide details about the 
reasons (liver function test, copper and zinc parameters) for which doses of zinc were increased, 
please?  
b. AASLD and EASL guidelines on WD should be considered in relatio n to the zinc doses used 
and to the desired zinc and copper parameters during therapy.  
c. As for the statement "increased 24 -h urinary copper excretion (360 µg, normal: 15 -60 µg)" 
(paragraph Case presentation, lines 6 -7), pediatric diagnostic criteria shou ld be considered 
(Nicastro et al. Re -evaluation of the diagnostic criteria for Wilson disease in children with mild 
liver disease. Hepatology 2010;52(6):1948 -56). 
d. As for the sentence "Since then, she has continued to take zinc at a dose of 240 mg/day, a nd 
strictly avoids foods with high copper levels" (paragraph Case presentation, line 24), according 
to available guidelines, foods with very high concentrations of copper (shellfish, nuts, chocolate, 
mushrooms, and organ meats) generally should be avoided,  during the first year of treatment. 
Especially in WD patients treated with zinc,  which inhibts intestinal copper absorption, dietary 
copper restriction is not mandatory. As reported in ESPGHAN WD guidelines (Socha et al. 
Journal of Pediatric Gastroentero logy and Nutrition: February 2018 - Volume 66 - Issue 2 - p 
334-344), "zinc should not be taken with food because it interferes with its absorption, and 
dietary copper restriction is not recommended because zinc blocks copper absorption from the 
intestinal  track".  
e. As for the lack of response of patient's anemia to blood transfusion (2 units) (in the manuscript 
the Authors stated:  "She received transfusion of 2 units of erythrocyte suspension and continued 
oral zinc at her previous dose. There was no sig nificant improvement in hematological 
parameters"), further details are desirable. Could the Authors clarify the reasons for which the 
patient did not show a favorable (at least transient) response to blood transfusion. Was this 
phenomenon observed in othe r patients reported in literature? Could the Authors give further 
details on the pathogenic mechanisms of copper deficiency anemia? It is very impressive that 
zinc withdrawal (plus 1 unit of blood)  induced an improvement in patient's hemoglobin levels 
within 1 week, from 4 to 6.7 grams (Table 1). This impressive result should be better discussed.  
f. In the Abstract the Authors affirmed "Her hemoglobin was gradually restored to the normal 
range after the adjustment of zinc dose and diet therapy for 4 month s", in the paragraph "Case 
presentation" the following sentence is present: "When zinc was withdrawn for one week, her 
hemoglobin level rose to 6.7 g/dL. She was then discharged and a normal diet was resumed. 
Low-dose zinc was recommended, but the girl's p arents refused the treatment though we told the 
possible flare of liver damage, even acute liver failure after the cessation of zinc treatment".  As 
the Authors  themselves underline, the definitive suspension of therapy in WD  is very hazardous 
and can in duce liver failure.  This point needs to be stressed in Discussion.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
No 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in t he manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
Not relevant to this manuscript  
 
Quality of wr itten English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content o f the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any  non-financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
'I declare that I have no competing interests'  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on  the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license  (http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: Anemia following zinc treatment for  Wilson's disease:a case report and literature review  
 
Version: 0 Date: 19 Jan 2019  
 
Reviewer: Raffaele Iorio  
 
Reviewer's report:  
 
The Authors described a ch ild who developed severe anemia following  zinc monotherapy for 
Wilson's disease. This case is interesting and well compared with other similar cases previously 
reported in literature. Unfortunately, there are many points that have to be clarified:  
a. why this patient was overtreated with zinc? could the Authors provide details about the 
reasons (liver function test, copper and zinc parameters) for which doses of zinc were increased, 
please?  
b. AASLD and EASL guidelines on WD should be considered in relatio n to the zinc doses used 
and to the desired zinc and copper parameters during therapy.  
c. As for the statement "increased 24 -h urinary copper excretion (360 µg, normal: 15 -60 µg)" 
(paragraph Case presentation, lines 6 -7), pediatric diagnostic criteria shou ld be considered 
(Nicastro et al. Re -evaluation of the diagnostic criteria for Wilson disease in children with mild 
liver disease. Hepatology 2010;52(6):1948 -56). 
d. As for the sentence "Since then, she has continued to take zinc at a dose of 240 mg/day, a nd 
strictly avoids foods with high copper levels" (paragraph Case presentation, line 24), according 
to available guidelines, foods with very high concentrations of copper (shellfish, nuts, chocolate, 
mushrooms, and organ meats) generally should be avoided,  during the first year of treatment. 
Especially in WD patients treated with zinc,  which inhibts intestinal copper absorption, dietary 
copper restriction is not mandatory. As reported in ESPGHAN WD guidelines (Socha et al. 
Journal of Pediatric Gastroentero logy and Nutrition: February 2018 - Volume 66 - Issue 2 - p 
334-344), "zinc should not be taken with food because it interferes with its absorption, and 
dietary copper restriction is not recommended because zinc blocks copper absorption from the 
intestinal  track".  
e. As for the lack of response of patient's anemia to blood transfusion (2 units) (in the manuscript 
the Authors stated:  "She received transfusion of 2 units of erythrocyte suspension and continued 
oral zinc at her previous dose. There was no sig nificant improvement in hematological 
parameters"), further details are desirable. Could the Authors clarify the reasons for which the 
patient did not show a favorable (at least transient) response to blood transfusion. Was this 
phenomenon observed in othe r patients reported in literature? Could the Authors give further 
details on the pathogenic mechanisms of copper deficiency anemia? It is very impressive that 
zinc withdrawal (plus 1 unit of blood)  induced an improvement in patient's hemoglobin levels 
within 1 week, from 4 to 6.7 grams (Table 1). This impressive result should be better discussed.  
f. In the Abstract the Authors affirmed "Her hemoglobin was gradually restored to the normal 
range after the adjustment of zinc dose and diet therapy for 4 month s", in the paragraph "Case 
presentation" the following sentence is present: "When zinc was withdrawn for one week, her 
hemoglobin level rose to 6.7 g/dL. She was then discharged and a normal diet was resumed. 
Low-dose zinc was recommended, but the girl's p arents refused the treatment though we told the 
possible flare of liver damage, even acute liver failure after the cessation of zinc treatment".  As 
the Authors  themselves underline, the definitive suspension of therapy in WD  is very hazardous 
and can in duce liver failure.  This point needs to be stressed in Discussion.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
No 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in t he manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
Not relevant to this manuscript  
 
Quality of wr itten English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content o f the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any  non-financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
'I declare that I have no competing interests'  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on  the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license  (http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant