Influence of Liver Condition and Copper on Selective Parameters of Post-Mortem Dog Tissue Samples

Round 1

Reviewer 1 Report

Major comments

1) Experimental Design.   To this reviewer, the study is not a classical  toxicology or pharmacology investigation (ie animals are not free of disease-control groups) as such please clarify and expand in much greater details on the selection process of dogs. Some of the selections process could lead to confound data, please see below

-It is unclear to this reviewer, the rationale for choosing (randomly selecting) animals for the control and experimental groups.  What were the criteria for selection - it appears that quality of life associated with or with out liver pathology was the critical indicator. Was the choice of dogs under the sole discretion of the attending veterinarian, were defined selection criteria provided to the veterinarian for choice of dogs to incorporate into the study and so forth.

- Please comment on the potential of confounding effects of data- several control and liver pathology dogs had similar underlying conditions. The control group included dogs with disease that could impacted the liver, as example cardiac disease (congestive heart failure) can be associated with chronic hepatic  liver pathology (chronic passive congestion), similarly enteritis if associated with bacterial infection  (unknown from the study) can be associated with embolic bacterial hepatitis. The liver injury group also contain dogs with cardiac and possibly enteritis (chronic diarrhea) disease.  Collectively this could affect the data .

-To ensure ethical use of animals-Was instructional animal care approval for animal use obtained for the study, or was the attending veterinarian allowed (ie client approval) to incorporate euthanized dogs data into the study. Please add a statement reflecting this comment to the manuscript

2) Evaluation of Histology. It excellent to have a trained veterinary pathologist evacuate the slides. Was the evaluation- scoring system, described in any previously published study (if so please provide a references). Was numerical values (0-3) provided for the scoring of each histological parameter- this could help with providing a ‘total score, and provides more information for the study. As an example how is mild vs moderate vs severe inflammation characterized.  This reviewer recommends- adding a more detailed histological description and table of the histological scoring (if possible). As well ( if the authors have the images) providing represented histological images (histological micrographs) of mild vs, moderate, vs severe liver lesions (inflammation), as would be helpful for the readers to visualize the tissue injury

3) Figure 1- this reviewer is unsure of the added value of figure 1, and why 1-oleoyl glycerol (18:1) was provided as the test lipid. Please add some detail for the choice of the 1-oleoyl glycerol (18:1) as the indicator lipid.  

4) Line 218. Please reword- Comparison of the study of histological lesion with the results of the current study is quite confusing, in particular comparing 24(101) dog study with the current study of 5 dogs and the rational for addition of beagles (ie control group) to the study.  

5) Conclusion Line 315. The copper accumulation was not related to either liver pathological condition or increase in liver biomarkers. This is an important observation and crux of the manuscript. This could be more clearly stated and elaborated within the discussion. To this reviewer that information was not clearly delineated within the discussion and perhaps could be adjusted.

 

Minor comments

Added units of measurement. The figure and tables should have units of measurement added to the figure legend and table column descriptors

Some section were unclear as written, please review the wording, grammar and description with the manuscript, to ensure easier following for the readers.

Please provide a statement on the general location of the veterinarians involved in the study. (ie Manhattan KS, vs the State of Kansas vs the Mid West, vs USA-

Line 226 and other locations of the manuscript- please define lipid super pathway

Line 207- upper limit of literature? Please clarify upper limits of copper levels within normal limits?

Line 87- please define ICP-OE

Author Response

Comments and Suggestions for Authors

Major comments

1) Experimental Design.  To this reviewer, the study is not a classical  toxicology or pharmacology investigation (ie animals are not free of disease-control groups) as such please clarify and expand in much greater details on the selection process of dogs. Some of the selections process could lead to confound data, please see below

-It is unclear to this reviewer, the rationale for choosing (randomly selecting) animals for the control and experimental groups.  What were the criteria for selection - it appears that quality of life associated with or with out liver pathology was the critical indicator. Was the choice of dogs under the sole discretion of the attending veterinarian, were defined selection criteria provided to the veterinarian for choice of dogs to incorporate into the study and so forth. 

A: Blood samples were collected ante-mortem with analysis completed and logged into the database for later retrieval. All liver samples were collected from post-mortem animals which were retrieved from the bioarchive for analyses in the conduct of this research. These samples and raw data were banked at least a full year prior to the initiation of this retrospective study. No Veterinarian had a role in selecting animals or tissues for this study. Further, samples were selected according to breed predisposition for liver disease and according to their actual liver condition found by histopathologic evaluation. We have rearranged the paragraph in the methods to better clarify and reflect this. 

- Please comment on the potential of confounding effects of data- several control and liver pathology dogs had similar underlying conditions. The control group included dogs with disease that could impacted the liver, as example cardiac disease (congestive heart failure) can be associated with chronic hepatic  liver pathology (chronic passive congestion), similarly enteritis if associated with bacterial infection  (unknown from the study) can be associated with embolic bacterial hepatitis. The liver injury group also contain dogs with cardiac and possibly enteritis (chronic diarrhea) disease.  Collectively this could affect the data .

A: Yes, we agree. Many of these animals had underlying conditions that affected the liver and other organ-systems in the body. Of course, at the later stages of life there can be many underlying conditions which are not evident upon clinical examination; but, which upon necropsy would reveal another potential terminal disease. This is why we chose an “indicator” to classify the post-mortem samples within each group – the graded liver histopathology condition. We also described other diseases which were present in each animal under M&M and Discussion. We added this clarification on the first paragraph of the discussion section to better address the potential confounding effects from other terminal conditions. 

-To ensure ethical use of animals-Was instructional animal care approval for animal use obtained for the study, or was the attending veterinarian allowed (ie client approval) to incorporate euthanized dogs data into the study. Please add a statement reflecting this comment to the manuscript.

A: Yes, an IACUC statement with its protocol number was included under “2.1 Animals”.

2) Evaluation of Histology. It excellent to have a trained veterinary pathologist evacuate the slides. Was the evaluation- scoring system, described in any previously published study (if so please provide a references). Was numerical values (0-3) provided for the scoring of each histological parameter- this could help with providing a ‘total score, and provides more information for the study. As an example how is mild vs moderate vs severe inflammation characterized.  This reviewer recommends- adding a more detailed histological description and table of the histological scoring (if possible). As well ( if the authors have the images) providing represented histological images (histological micrographs) of mild vs, moderate, vs severe liver lesions (inflammation), as would be helpful for the readers to visualize the tissue injury

A: The scoring was based on a 10-point scale and the pathologists experience. No prior scoring system from published work was used as a reference. This has been addressed under “2.3 Liver pathology classification”. Unfortunately, it was not possible to collect example images from the histopathology.

3) Figure 1- this reviewer is unsure of the added value of figure 1, and why 1-oleoyl glycerol (18:1) was provided as the test lipid. Please add some detail for the choice of the 1-oleoyl glycerol (18:1) as the indicator lipid. 

A: This figure was added simply as an example for visual representation of the correlations of copper concentration (natural log transformed) with various lipid metabolic end-products. Of the 500 analytes, the 1-oleoyl glycerol had the highest correlation to liver copper. For the correlation one can observe that most data points were within a relatively tight range with the exception of two outliers with very high liver copper. We have revised the figure legend to include units and clarification of the transformation, and provided the following text:

L307-309(with track changes): An example is illustrated in Figure 1, which shows the highest correlation in the study between copper and a molecule from the lipid metabolism (1-oleoyl-glycerol).

4) Line 218. Please reword- Comparison of the study of histological lesion with the results of the current study is quite confusing, in particular comparing 24(101) dog study with the current study of 5 dogs and the rational for addition of beagles (ie control group) to the study. 

A: Reworded to provide more clarity. We also rearranged the first 3 paragraphs of the discussion to provide a more concise flow to the discussion points being made. In essence the second part of the paragraph identified by the reviewer fits better with the lead-in to the discussion.

**5) Conclusion Line 315. The copper accumulation was not related to either liver pathological condition or increase in liver biomarkers. This is an important observation and crux of the manuscript. This could be more clearly stated and elaborated within the discussion. To this reviewer that information was not clearly delineated within the discussion and perhaps could be adjusted. 

 A: We appreciate the comment. Please read L402-412 (with track changes) in the discussion:

From the dog samples selected for this study, only 6 had copper concentrations above 400 ppm on a dry weight basis, which is considered above the upper limit according to Fieten et al., 2014 4. However, dog samples with highest hepatic copper concentrations (1,020 and 2,768 ppm) did not have abnormal liver pathology, and the other animals with increased copper were included in the mild and moderate groups. This happened because copper was measured by ICP-OES instead of being visualized in the slides under the microscope as others have previously reported. For example, in a retrospective study by Poldervaart et al.2the authors reported that 24 of 101 dogs with liver disease in their population died from chronic hepatitis associated with copper storage. They determined copper accumulation using a staining technique to grade and stratify dogs to condition rather than the actual measurement of copper via ICP-OES used in our work. 

 

Minor comments

Added units of measurement. The figure and tables should have units of measurement added to the figure legend and table column descriptors.

A: All the parameters were natural logs and initial data were in ppm.

Some section were unclear as written, please review the wording, grammar and description with the manuscript, to ensure easier following for the readers.

A: We have attempted to review once more to enhance the clarity and conciseness of the paper. The corrections were intermittent and(or) addressed the other reviewers comments. Hopefully this revised version reads more clearly. Thank you for the input.

Please provide a statement on the general location of the veterinarians involved in the study. (ie Manhattan KS, vs the State of Kansas vs the Mid West, vs USA-

A: The [Veterinary] Pathology was conducted at Kansas State University. Clinics were all within the Mid West. We have added on L 129-130 (with track changes): All liver sampleswerefrom previously deceased dogs in which tissues and medical histories were retrieved from either the Hill’s bioarchive (internal) or from external private Veterinary clinics(within the Mid West, U.S.A.).

Line 226 and other locations of the manuscript- please define lipid super pathway

A: This term was intended to provide a general grouping for the lipid metabolites identified in the study. However, since this is somewhat arbitrary and not a clearly defined term in nutritional biochemistry and metabolism, we revised in each case to terms such as lipid metabolic pathway and similar.

Line 207- upper limit of literature? Please clarify upper limits of copper levels within normal limits? 

A: Yes, thank you for your comment. The upper limit was 400 ppm according to Fieten et al 2014 (reference 4). This paragraph has been reworded.

Line 87- please define ICP-OE

A: Thank you for your note. We have added the definition:

L187-188 (with track changes): Copper concentration in the liver was determined by ICP-OES(inductively coupled plasma- optical emission spectrometry; Optima 4300 DV, PerkinElmer instruments, Waltham, MA).

Reviewer 2 Report

Hello,

Thank you for the article submission.

I am sorry to have to suggest that major revision is needed. These are some initial comments:-

1) Basic sentence structure needs to be improved in order to meet minimal acceptable scientific standards used in journal publication eg the opening sentence, line 10 of the article does not read well. There are numerous other examples. CBC is Complete Blood Count not Cell blood count, line 15 etc

2) Materials and Methods, line 67 - No mention is made of why these dogs were euthanized. As a result, "Normal" cannot be extrapolated to be "control". 

3) Sample analysis - Blood chemistry and CBC were measured immediately after death?, line 82. Do you mean that the relevant samples were taken prior to the administration of the Euthanasia agent? If so, how long before were these samples taken and how were they stored prior to testing?

4) There are major concerns with the Liver pathology classification, eg line 89 - Mild, moderate and severe are lesion modifiers and cannot of themselves be used as parameters to compare Liver pathology based on breed, age, gender etc in Table 1 or plasma metabolites, Table 3. This unfortunately precludes any interpretation of subsequent sections. If classification of mild, moderate and severe has to be used, proper definitions or explanations are required and the classification requires significant revision.

There was no definition of 'normal' liver or liver neoplasia. No gross descriptions were provided - how much of the liver was affected by cancerous lesions? 

Atelectasis, line 96, is collapse of the lung and is used to describe a pulmonary lesion, not liver pathology.

Line 97, primary diagnoses included arthritis, cardiac disease, cushing's disease, renal disease etc...these are examples of systemic diseases that would affect blood chemistry and CBC's etc. Without liver tissue controls; because the liver is very vascular and without measurements of these parameters from other tissues (heart, kidney etc), in these patients it is difficult if not impossible to draw conclusions.

Necrosis, line 101 is one classification that would have a major effect on measurements of metabolites. Hepatic samples were taken post mortem - how long after death? Autolysis of the liver would need to be considered as this process as well would impact these results. These important variables did not appear to be considered.

"Cortical" is not used in classifying liver tumors as the liver does not have a cortex or medulla. Pheochromocytoma is an adrenal medullary neoplasm. 

Please address these initial comments and consider revision. 

Thanks very much.

Author Response

Hello,

Thank you for the article submission.

I am sorry to have to suggest that major revision is needed. These are some initial comments:-

1) Basic sentence structure needs to be improved in order to meet minimal acceptable scientific standards used in journal publication eg the opening sentence, line 10 of the article does not read well. There are numerous other examples. CBC is Complete Blood Count not Cell blood count, line 15 etc

A: Thank you for your suggestions. We have corrected L15 to “complete blood count”. The “Simple Summary” has been revised.

2) Materials and Methods, line 67 - No mention is made of why these dogs were euthanized. As a result, "Normal" cannot be extrapolated to be "control". 

A: The dogs were euthanized for a variety of reasons all related to normal end-of-life decisions regarding health and quality of life as part of the consult between owner and clinician. It is assumed as such that there was some underlying health issue associated with the dogs prior to euthanasia that may have confounded the study. Thus, there was no “control” group in this experiment as that would have required either a longitudinal study, or sacrifice of otherwise normal healthy animals which was not an acceptable experimental approach for the study sponsor or authors. However, those dogs that were deemed to fit into the category of “NORMAL” would serve as the reference for dogs not presenting with liver storage disease. We have rephrased in the manuscript that there was no true control under section 2.3: There was not a true control in this study because all samples were from previously deceased dogs which had some type of terminal health condition.  

3) Sample analysis - Blood chemistry and CBC were measured immediately after death?, line 82. Do you mean that the relevant samples were taken prior to the administration of the Euthanasia agent? If so, how long before were these samples taken and how were they stored prior to testing?

A: Blood samples were collected just prior to euthansia - minutes at most - so yes euthasol was administered AFTER blood collection. This section has been clarified in the manuscript. After collection they were taken over to the Clinical Lab and analyzed. Blood chemistry was determined on COBAS 6000 C501 centrifuged for 10 min @ 3000 RPM - room temp. The CBC's were analyzed on a SYSMEX XT2000i after rocking (modest shaking; 1-2 minutes) then immediately measured. 

4) There are major concerns with the Liver pathology classification, eg line 89 - Mild, moderate and severe are lesion modifiers and cannot of themselves be used as parameters to compare Liver pathology based on breed, age, gender etc in Table 1 or plasma metabolites, Table 3. This unfortunately precludes any interpretation of subsequent sections. If classification of mild, moderate and severe has to be used, proper definitions or explanations are requiredand the classification requires significant revision.

A: Similar question of Reviewer 1. This has been addressed to provide additional clarity to the groupings. The separation into categories was done by a pathologist and based on experience and on a 10-point scale, which was included under 2.3 Liver Pathology Condition. We have attempted to locate images to include in this work, but the slides were not available to us. We have, however, added a description of what was considered mild, moderate, and severe under section 2.3. 

There was no definition of 'normal' liver or liver neoplasia. No gross descriptions were provided - how much of the liver was affected by cancerous lesions?

A: Unfortunately, it was not possible to do a gross evaluation of the liver. Whole organs are not preserved. Rather, the bioarchive contains samples of thousands of organ sub-samples as a bank for future research, and it is a standing routine to perform CBC and chemistry before euthanasia that match with the tissue samples. We added on L203-205 (with track changes): The extent of liver neoplasia could not be captured because the study was limited to using slides of previously collected dog liver samples. Thus, there was no gross pathology evaluation of each subject.

Atelectasis, line 96, is collapse of the lung and is used to describe a pulmonary lesion, not liver pathology.

A: This has been removed and corrected. 

Line 97, primary diagnoses included arthritis, cardiac disease, cushing's disease, renal disease etc...these are examples of systemic diseases that would affect blood chemistry and CBC's etc. Without liver tissue controls; because the liver is very vascular and without measurements of these parameters from other tissues (heart, kidney etc), in these patients it is difficult if not impossible to draw conclusions.

A: We concur that other underlying conditions could cloud or confound our conclusions, but in a tissue retrospective study this is an assumption going in. We have attempted to acknowledge this point in the discussion. However, that this was not a controlled longitudinal survival study does not negate the value of the information that we can glean from these tissues. We hope our explanation in the discussion is satisfactory to address this point.

Necrosis, line 101 is one classification that would have a major effect on measurements of metabolites. Hepatic samples were taken post mortem - how long after death? Autolysis of the liver would need to be considered as this process as well would impact these results. These important variables did not appear to be considered.

A: The time is near immediate and has been updated in the materials and methods 2.2 As sample analysis:

L184-185 (with track changes): Liver biopsies were harvested at necropsy to fit into 1.8 ml cryovials. These were immediately flash frozen in liquid nitrogen and stored at -80 °C until analysis.  

"Cortical" is not used in classifying liver tumors as the liver does not have a cortex or medulla. Pheochromocytoma is an adrenal medullary neoplasm. 

A: This has been corrected. Thank you,

Please address these initial comments and consider revision. 

 

Thanks very much.

 

Reviewer 3 Report

The manuscript investigates the relationship between hepatic copper concentrations and liver pathology in 55 dogs submitted to necropsy. Ta data presented in the paper are not innovative, and particularly pathological studies have great limitations and errors and this makes the results obtained difficult to evaluate. Confirming the low attention in the results presentation the liver copper concentrations detected are presented in Table 1 and in the text without reference to the measurement unit. Furthermore several typing errors are present throughout the text.  In conclusion, I believe that this manuscript is not to be accepted for publication and that it can only be re-evaluated after a  deep and thorough review, particularly of the part relating to pathological investigations.

Author Response

Comments and Suggestions for Authors

The manuscript investigates the relationship between hepatic copper concentrations and liver pathology in 55 dogs submitted to necropsy. Ta data presented in the paper are not innovative, and particularly pathological studies have great limitations and errors and this makes the results obtained difficult to evaluate.

While the approach may not have been new to the area of science, the information gleaned did provide some new insights into this condition. Specifically, there were numerous metabolites that have rarely been described in the literature previously, such as 2-aminoheptanoate. This study does differ from previous work in the comprehensive nature of the compounds measured (>500 metabolites) from post-mortem samples.

Confirming the low attention in the results presentation the liver copper concentrations detected are presented in Table 1 and in the text without reference to the measurement unit.

All the parameters were measured in ppm or similar and converted to natural log. 

Furthermore, several typing errors are present throughout the text. 

We have attempted to address all grammar, spelling, and punctuation and provide clarity in a few sections that may not have been clear. We have also made significant changes to the manuscript according to comments from the other reviewers. The pathologist who evaluated the liver histopathology for this study has also been consulted. She reviewed all the slides that were questionable and applied corrections.

In conclusion, I believe that this manuscript is not to be accepted for publication and that it can only be re-evaluated after a  deep and thorough review, particularly of the part relating to pathological investigations.

We believe that the manuscript is much improved and that you may reconsider it for publication. Thank you.

 

Round 2

Reviewer 1 Report

All previous reviewer comments were adequately addressed and as such the manuscript is ready for publication.

One comment the liver pathology description is quite long and perhaps could be shortened to a more concise form .This  'point'  however, is minor and does  not warranted any revisions

 

Author Response

All previous reviewer comments were adequately addressed and as such the manuscript is ready for publication.

One comment the liver pathology description is quite long and perhaps could be shortened to a more concise form .This  'point'  however, is minor and does  not warranted any revisions

Thank you for your reply.

Reviewer 2 Report

Hello,

Please consider addressing these issues:-

1) "This study offers further insight regarding changes in metabolism due to hepatic erosion."  

What is hepatic erosion? No such entity exists. I suggest you have the pathologist who did the histopathology review this paper one more time.

2) "Further, liver neoplasia had an impact on nucleotide, energy and amino acid metabolism. This suggests rapid cell division and increase in metabolism and energy expenditure."

The second sentence is obvious. Please edit these 2 sentences to convey the point you wish to make.

3) There remains occasional issues with sentence structure in the manuscript. Would be best to correct these.

Thanks,

Author Response

1) "This study offers further insight regarding changes in metabolism due to hepatic erosion."  

What is hepatic erosion? No such entity exists. I suggest you have the pathologist who did the histopathology review this paper one more time.

This was a term chosen by us and not the pathologist. We have replaced it with “hepatic tissue damage”.

2) "Further, liver neoplasia had an impact on nucleotide, energy and amino acid metabolism. This suggests rapid cell division and increase in metabolism and energy expenditure."

The second sentence is obvious. Please edit these 2 sentences to convey the point you wish to make.

We have replaced it by: Further, dogs with liver neoplasia had increased nucleotide, energy and amino acid metabolism, which suggest rapid cell division and increase in metabolism and energy expenditure. 

3) There remains occasional issues with sentence structure in the manuscript. Would be best to correct these.

Thank you for the note, we have revised the paper one last time and made more changes.

Reviewer 3 Report

The revision performed by the Authors has largely solved the critical points highlighted in the first review. In conclusion, I believe that the manuscript can be considered for publication in the journal.

Author Response

The revision performed by the Authors has largely solved the critical points highlighted in the first review. In conclusion, I believe that the manuscript can be considered for publication in the journal.

Thank you!