Correlation of ammonia and blood laboratory parameters with hepatic encephalopathy: A systematic review and meta-analysis

Ali Sepehrinezhad; Negin Ghiyasi Moghaddam; Navidreza Shayan; Sajad  Sahab Negah

doi:10.1371/journal.pone.0307899

Peer Review History

Original SubmissionMarch 14, 2024
10 May 2024 Decision Letter - Peter Starkel, Editor PONE-D-24-09594Correlation of Ammonia and Blood Laboratory Parameters with Hepatic Encephalopathy: A Systematic Review and Meta-AnalysisPLOS ONE Dear Dr. Sahab Negah, 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. In particular reviewer 2 raised serious concerns regarding several methodological aspects. Those concerns need to be thoroughly addressed and the required changes and adaptations need to be made to the manuscript. This likely requires additional work and potentially re-analysis of the data. 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To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ Additional Editor Comments: In particular reviewer 2 raised serious concerns regarding several methodological aspects. Those concerns need to be thoroughly addressed and the required changes and adaptations need to be made to the manuscript. This likely requires additional work and potentially re-analysis of the data. All changes must be clearly highlighted in the paper and a complete and detailed point by point reply explaining how the criticism has been addressed needs to be provided if the authors opt for revision of their paper. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: No ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: No ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Hepatic encephalopathy seen in liver cirrhosis is thought to involve multiple factors, with ammonia believed to play a central role. However, there are reports suggesting that the pathophysiology of hepatic encephalopathy cannot be accurately assessed based solely on blood ammonia levels. The authors conducted a meta-analysis of 79 studies, investigating not only the correlation between hepatic encephalopathy and blood ammonia levels in liver cirrhosis but also its association with creatinine, albumin, sodium, IL-6, and TNF-α concentrations. As a result, cases with hepatic encephalopathy showed significantly elevated levels of ammonia, creatinine, IL-6, and TNF-α, while albumin and sodium levels were significantly lower. Therefore, the authors concluded that relying solely on targeting ammonia in the treatment of hepatic encephalopathy might not be sufficient and suggested a new strategy involving addressing inflammation, creatinine, albumin, and sodium concentrations. This conclusion is supported by a comprehensive examination of numerous cases using appropriate methods. However, there are several points that warrant further discussion. The authors also note significant variability in ammonia levels. One possible cause is the variation in ammonia reference values among studies. In multicenter studies involving ammonia levels, some use ratios to the upper limit of the reference value rather than raw data. It would be beneficial to address how this aspect is evaluated. Blood ammonia levels are not necessarily an appropriate indicator for evaluating hepatic encephalopathy, partly because many of the examined ammonia values are from venous blood samples. Venous blood ammonia, being detoxified through the glutamine synthesis pathway in skeletal muscles, tends to be lower than arterial blood levels. However, since the ammonia entering the brain is transported by arterial blood, venous blood ammonia levels may not accurately reflect the concentration of ammonia flowing into the brain. This issue also warrants discussion in the analysis. Reviewer #2: In this paper, Sepehrinezhad A and coll. attempted to synthetize the correlation of blood ammonia with hepatic encephalopathy in a meta-analysis. I have major concerns about the methodology of this paper, particularly regarding the literature search, the criteria for inclusion and exclusion of studies which are missing and the statistical analyses. Key search terms must be noted in the main text and must be combined within each database (not done), abstracts from liver congresses are usually screened (not done), literature search must be updated in April 2024 and some studies were not identified. In order to reduce risk of bias, strict criteria for inclusion and exclusion of studies must be clearly defined prior to the literature search. Also, aims of the study and endpoints are missing in the text. The selection of the studies for inclusion in the metaanalysis includes usually 4 processes which is mandatory to preserve: identification, screening, eligibility (missing) and inclusion (Fig 1). Regarding statistical analysis, I2 alone is not enough to assess heterogeneity between studies. Moreover, in cases of moderate or high heterogeneity, the methodological section of each study is usually re-reviewed to determine whether any discrepancy could be identified, and sensitivity analyses excluding the discrepant study is classically performed (not done). Therefore, because the methodology and statistical analyses were not complete and rigorous, I recommend rejecting this article. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0307899.r001
Revision 1
5 Jul 2024 Author Response To: 5th, July, 2024 To: PLOS ONE, Editor-in-Chief Dear Editor-in-Chief We would like to express our appreciation to the reviewer for his/her insightful comments, which have significantly helped us improve our manuscript. We have incorporated your feedback into our revisions, and we believe that your comments have helped to clarify and strengthen our paper. Please find our responses to your specific comments below (in blue), and note that the revised text in the manuscript is highlighted in blue for your convenience. Reviewer #1: Hepatic encephalopathy seen in liver cirrhosis is thought to involve multiple factors, with ammonia believed to play a central role. However, there are reports suggesting that the pathophysiology of hepatic encephalopathy cannot be accurately assessed based solely on blood ammonia levels. The authors conducted a meta-analysis of 79 studies, investigating not only the correlation between hepatic encephalopathy and blood ammonia levels in liver cirrhosis but also its association with creatinine, albumin, sodium, IL-6, and TNF-α concentrations. As a result, cases with hepatic encephalopathy showed significantly elevated levels of ammonia, creatinine, IL-6, and TNF-α, while albumin and sodium levels were significantly lower. Therefore, the authors concluded that relying solely on targeting ammonia in the treatment of hepatic encephalopathy might not be sufficient and suggested a new strategy involving addressing inflammation, creatinine, albumin, and sodium concentrations. This conclusion is supported by a comprehensive examination of numerous cases using appropriate methods. However, there are several points that warrant further discussion. The authors also note significant variability in ammonia levels. One possible cause is the variation in ammonia reference values among studies. In multicenter studies involving ammonia levels, some use ratios to the upper limit of the reference value rather than raw data. It would be beneficial to address how this aspect is evaluated. Blood ammonia levels are not necessarily an appropriate indicator for evaluating hepatic encephalopathy, partly because many of the examined ammonia values are from venous blood samples. Venous blood ammonia, being detoxified through the glutamine synthesis pathway in skeletal muscles, tends to be lower than arterial blood levels. However, since the ammonia entering the brain is transported by arterial blood, venous blood ammonia levels may not accurately reflect the concentration of ammonia flowing into the brain. This issue also warrants discussion in the analysis. Our response: Thank you for your valuable comment. Based on various clinical studies, it has been observed that both venous and arterial ammonia levels have strong correlations with the severity of hepatic encephalopathy. Some studies even suggest a venous sample is adequate for measuring ammonia levels. Additionally, since arterial ammonia levels are higher than venous levels, it is believed that the venous level can accurately represent the level that may lead to brain injury, as arterial ammonia reaches the brain more quickly. Therefore, venous ammonia levels can be considered as reliable biomarkers. Thus, it appears that our systematic review and meta-analysis can overlook the kind of arterial and venous sampling used for ammonia measurement. Mehmood, M. A., T. Waseem, F. Z. Ahmad, and M. A. Humayun. "Measuring partial pressure of ammonia in arterial or venous blood vs total ammonia levels in hepatic encephalopathy." J. Gastroenterol. Hepatol 2 (2013): 602. Nicolao, Francesca, Cesare Efrati, Andrea Masini, Manuela Merli, Adolfo Francesco Attili, and Oliviero Riggio. "Role of determination of partial pressure of ammonia in cirrhotic patients with and without hepatic encephalopathy." Journal of hepatology 38, no. 4 (2003): 441-446. Ong JP, Aggarwal A, Krieger D, Easley KA, Karafa MT, Van Lente F, Arroliga AC, Mullen KD. Correlation between ammonia levels and the severity of hepatic encephalopathy. Am J Med. 2003 Feb 15;114(3):188-93. doi: 10.1016/s0002-9343(02)01477-8. PMID: 12637132. K.N, Sricharan and Shifali Prabhaker. “Arterial vs Venous Ammonia Levels in Correlation with severity of Hepatic Encephalopathy.” International Journal of Contemporary Medical Research [IJCMR] (2019): n. pag. Reviewer #2: In this paper, Sepehrinezhad A and coll. attempted to synthetize the correlation of blood ammonia with hepatic encephalopathy in a meta-analysis. I have major concerns about the methodology of this paper, particularly regarding the literature search, the criteria for inclusion and exclusion of studies which are missing and the statistical analyses. Key search terms must be noted in the main text and must be combined within each database (not done), abstracts from liver congresses are usually screened (not done), literature search must be updated in April 2024 and some studies were not identified. In order to reduce risk of bias, strict criteria for inclusion and exclusion of studies must be clearly defined prior to the literature search. Also, aims of the study and endpoints are missing in the text. The selection of the studies for inclusion in the metaanalysis includes usually 4 processes which is mandatory to preserve: identification, screening, eligibility (missing) and inclusion (Fig 1). Regarding statistical analysis, I2 alone is not enough to assess heterogeneity between studies. Moreover, in cases of moderate or high heterogeneity, the methodological section of each study is usually re-reviewed to determine whether any discrepancy could be identified, and sensitivity analyses excluding the discrepant study is classically performed (not done). Therefore, because the methodology and statistical analyses were not complete and rigorous, I recommend rejecting this article. Our response: I appreciate your attention to detail and thoughtful critique of the methodology used in this study. As you we have suggested, expanded our literature search to include publications up to June 2024 and have incorporated our key search terms into the main body of the revised manuscript. The primary keyword search pattern for each database is now available in Supplementary Table 2. For the following comment “abstracts from liver congresses are usually screened (not done)”, as we mentioned in the Prisma flowchart, we excluded abstracts from the conference because they didn’t have enough data for concluding as well as there weren’t inclusion criteria for the conference paper. For the comment “In order to reduce risk of bias, strict criteria for inclusion and exclusion of studies must be clearly defined prior to the literature search”, I would like to confirm that we have implemented these measures. We established general exclusion criteria, which included non-English papers, abstracts, and reviews. Additionally, we only included studies that met the following specific criteria: i) studies that compared cirrhosis with hepatic encephalopathy, ii) the use of a valid evaluation method, iii) a valid and clear definition of cases, and iv) the measurement of outcomes using a reproducible and reliable method. To further minimize the risk of bias, we conducted an appraisal utilizing the Joanna Briggs Institute (JBI) critical appraisal checklist for case-control studies. This approach shows that an appraisal checklist can effectively mitigate the risk of bias in studies with poor methodology. In the comment, "The selection of the studies for inclusion in the meta-analysis involves four mandatory processes: identification, screening, eligibility (missing), and inclusion (Fig 1)," the eligibility of studies was assessed at three levels: general criteria, PICO criteria, and outcome measurements. For instance, we considered Population (i.e., cirrhosis and hepatic encephalopathy), Comparison (HE as the case and cirrhosis as the control), and Outcome (e.g., serum biomarkers). Given that our manuscript focused on evaluating observational studies, we intentionally refrained from providing a specific definition of intervention; instead, we exclusively included observational studies. Regarding the comment, "I2 alone is not sufficient to assess heterogeneity between studies in statistical analysis. In cases of moderate or high heterogeneity, the methodological section of each study is usually re-reviewed to identify discrepancies, and sensitivity analyses excluding the discrepant study are typically performed (not done)," we precisely reviewed the methodology of each study and focused on the assessment methods, such as the evaluation of inflammatory markers using the Eliza method. Additionally, we only included similar studies in the sub-analysis that differed in one variable. I believe the major comments on the methodology were due to the concise nature of this part. The manuscript's word limitation was the main challenge we encountered. Following the aforementioned details, we have provided a comprehensive description of the comments in the supplementary document. Sincerely yours Dr. Sajad Sahab Negah, PhD Department of Neuroscience, Faculty of Medicine, Mashhad University of Medical Sciences, Pardis Campus, Azadi Square, Kalantari Blvd., Mashhad, Iran. Tel: +98-51-38002473; Email: sahabnegahs@mums.ac.ir Attachments Attachment Submitted filename: Responses to reviewers.pdf https://doi.org/10.1371/journal.pone.0307899.r002
15 Jul 2024 Decision Letter - Peter Starkel, Editor Correlation of Ammonia and Blood Laboratory Parameters with Hepatic Encephalopathy: A Systematic Review and Meta-Analysis PONE-D-24-09594R1 Dear Dr. Sahab Negah, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Peter Starkel, M.D., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Data have been updated and sufficient clarifications concerning the methods used have been provided. Reviewers' comments: https://doi.org/10.1371/journal.pone.0307899.r003
Formally Accepted
23 Jul 2024 Acceptance Letter - Peter Starkel, Editor PONE-D-24-09594R1 PLOS ONE Dear Dr. Sahab Negah, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr Peter Starkel Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0307899.r004

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