Neutrophil-mediated oxidative stress and albumin structural damage predict COVID-19-associated mortality

  1. Mohamed A Badawy
  2. Basma A Yasseen
  3. Riem M El-Messiery
  4. Engy A Abdel-Rahman
  5. Aya A Elkhodiry
  6. Azza G Kamel
  7. Hajar El-sayed
  8. Asmaa M Shedra
  9. Rehab Hamdy
  10. Mona Zidan
  11. Diaa Al-Raawi
  12. Mahmoud Hammad
  13. Nahla Elsharkawy
  14. Mohamed El Ansary
  15. Ahmed Al-Halfawy
  16. Alaa Elhadad
  17. Ashraf Hatem
  18. Sherif Abouelnaga
  19. Laura L Dugan
  20. Sameh Saad Ali

  • Curated by eLife

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    Evaluation Summary:

    Severe COVID-19 is characterised by a high neutrophil to lymphocyte ratio and an underlying state of oxidative stress in blood has also been hypothesized. In the study presented here for the first time provide evidence that hydrogen peroxide generated by the neutrophil specific enzyme myeloperoxidase is not only accumulated in plasma but also perpetrates structural damage to the strong and weak lipid binding sites on albumin, a key antioxidant in blood plasma, using SLFAs and EPR spectroscopy. This study is timely and relevant in deciding treatment of severe COVID-19 patients with human serum albumin and possibly supplementation of FDA approved antioxidants like glutathione. The main strengths of the manuscript are the novelty. The main weak point is the need to study more patients.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Human serum albumin (HSA) is the frontline antioxidant protein in blood with established anti-inflammatory and anticoagulation functions. Here, we report that COVID-19-induced oxidative stress inflicts structural damages to HSA and is linked with mortality outcome in critically ill patients. We recruited 39 patients who were followed up for a median of 12.5 days (1–35 days), among them 23 had died. Analyzing blood samples from patients and healthy individuals (n=11), we provide evidence that neutrophils are major sources of oxidative stress in blood and that hydrogen peroxide is highly accumulated in plasmas of non-survivors. We then analyzed electron paramagnetic resonance spectra of spin-labeled fatty acids (SLFAs) bound with HSA in whole blood of control, survivor, and non-survivor subjects (n=10–11). Non-survivors’ HSA showed dramatically reduced protein packing order parameter, faster SLFA correlational rotational time, and smaller S/W ratio (strong-binding/weak-binding sites within HSA), all reflecting remarkably fluid protein microenvironments. Following loading/unloading of 16-DSA, we show that the transport function of HSA may be impaired in severe patients. Stratified at the means, Kaplan–Meier survival analysis indicated that lower values of S/W ratio and accumulated H 2 O 2 in plasma significantly predicted in-hospital mortality (S/W≤0.15, 81.8% (18/22) vs. S/W>0.15, 18.2% (4/22), p=0.023; plasma [H 2 O 2 ]>8.6 μM, 65.2% (15/23) vs. 34.8% (8/23), p=0.043). When we combined these two parameters as the ratio ((S/W)/[H 2 O 2 ]) to derive a risk score, the resultant risk score lower than the mean (<0.019) predicted mortality with high fidelity (95.5% (21/22) vs. 4.5% (1/22), log-rank χ 2 =12.1, p=4.9×10 −4 ). The derived parameters may provide a surrogate marker to assess new candidates for COVID-19 treatments targeting HSA replacements and/or oxidative stress.

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  1. Version published to 10.7554/elife.69417 on eLife
  2. eLife

    Evaluation Summary:

    Severe COVID-19 is characterised by a high neutrophil to lymphocyte ratio and an underlying state of oxidative stress in blood has also been hypothesized. In the study presented here for the first time provide evidence that hydrogen peroxide generated by the neutrophil specific enzyme myeloperoxidase is not only accumulated in plasma but also perpetrates structural damage to the strong and weak lipid binding sites on albumin, a key antioxidant in blood plasma, using SLFAs and EPR spectroscopy. This study is timely and relevant in deciding treatment of severe COVID-19 patients with human serum albumin and possibly supplementation of FDA approved antioxidants like glutathione. The main strengths of the manuscript are the novelty. The main weak point is the need to study more patients.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    Activated neutrophils are known to contribute to inflammation and oxidative stress by generating reactive oxygen species (ROS) via NADPH oxidase and superoxide dismutase (SOD) during infections. The ROS can not only damage pathogens but also host blood plasma proteins and lipids. Severe COVID-19 is characterised by a high neutrophil to lymphocyte ratio and an underlying state of oxidative stress in blood has also been hypothesized. In the study presented here for the first time provide evidence that hydrogen peroxide generated by the neutrophil specific enzyme myeloperoxidase is not only accumulated in plasma but also perpetrates structural damage to the strong and weak lipid binding sites on albumin, a key antioxidant in blood plasma, using SLFAs and EPR spectroscopy. This study is timely and relevant in deciding treatment of severe COVID-19 patients with human serum albumin and possibly supplementation of FDA approved antioxidants like glutathione.

  4. eLife

    Reviewer #2 (Public Review):

    In this manuscript the authors performed a prospective study in 25 COVID-19 hospitalized patients and try to associate oxidative stress and hypoalbuminemia with COVID-19 mortality. Although it is an interesting study, the small sample size does not allow safe conclusions. I doubt which may be the clinical impact of this study on developing a better treatment of patients with COVID-19.

  5. Version published to 10.1101/2021.04.01.21254767v2 on medRxiv
  6. ScreenIT

    SciScore for 10.1101/2021.04.01.21254767: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementConsent: Written informed consents were obtained from participants in accordance with the principles of the Declaration of Helsinki.
    IRB: For COVID-19 and control blood/plasma collection, Children’s Cancer Hospital’s Institutional Review Board (IRB) has evaluated the study design and protocol, IRB number 31-2020 issued on July 6, 2020.
    RandomizationNo randomization was done and clinical data were made available after EPR data collection and analysis have been performed.
    Blindingnot detected.
    Power AnalysisNo power analysis was done.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Suspended cells were incubated with combinations of anti-human monoclonal antibodies for subset identification as follows: CD-42b-PE (Beckman Coulter life sciences, IM1417U) for platelets, CD14-PC7 (Beckman Coulter life sciences, A22331) for monocytes, CD66b-APC-Alexa Fluor 750 for neutrophils (Beckman Coulter life sciences, B08756) and CD3-ECD (Beckman Coulter life sciences, IM2705U) for lymphocytes cells were incubated for 30 minutes in the dark at room temperature.
    anti-human monoclonal
    suggested: (Beckman Coulter Cat# IM2705U, RRID:AB_130860)
    Suspended cells were incubated with DCF (20 μM) and combinations of monoclonal antibodies; CD-42b-PE (Platelets), CD14-PC7 (monocytes), CD66b-APC-Alexa Fluor 750 (neutrophils), and CD3-ECD (lymphocytes) for 30 minutes at room temperature in the dark.
    CD3-ECD
    suggested: None
    Software and Algorithms
    SentencesResources
    A number of 20,000 events were acquired and analyzed using CytExpert software to determine the percent and mean fluorescence intensities (MFI) of analyzed cell subsets.
    CytExpert
    suggested: (CytExpert Software, RRID:SCR_017217)
    Images were processed to quantify fluorescence intensity using Gen5 software package 3.08.
    Gen5
    suggested: (Gen5, RRID:SCR_017317)
    Statistical analysis: Statistical analysis and data graphing were performed using OriginPro 2017 (OriginLab Corporation, Northampton, USA).
    OriginPro
    suggested: None
    OriginLab Corporation
    suggested: (Origin, RRID:SCR_014212)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  7. Version published to 10.1101/2021.04.01.21254767v1 on medRxiv