COVID-19 and its clinical severity are associated with alterations of plasma sphingolipids and enzyme activities of sphingomyelinase and ceramidase

  1. Christiane Mühle
  2. Andreas Kremer
  3. Marcel Vetter
  4. Jonas Schmid
  5. Susanne Achenbach
  6. Fabian Schumacher
  7. Bernd Lenz
  8. Céline Cougoule
  9. Nicolas Hoertel
  10. Alexander Carpinteiro
  11. Erich Gulbins
  12. Burkhard Kleuser
  13. Johannes Kornhuber

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Abstract

In the current pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; COVID-19), a better understanding of the underlying mechanisms is essential to reduce morbidity and mortality and treat post-COVID-19 disease. Here, we analyzed alterations of sphingolipids and their metabolizing enzymes in 125 men and 74 women tested positive for SARS-CoV-2 and hospitalized with mild, moderate or severe symptoms or after convalescence.

The activities of acid and neutral sphingomyelinases (ASM, NSM), which hydrolyze sphingomyelin to ceramide, were significantly increased in COVID-19 patients, while the activity of neutral ceramidase (NC), which hydrolyzes ceramide to sphingosine, was reduced. These alterations could each contribute to elevated ceramide levels in patients. Accordingly, liquid chromatography tandem-mass spectrometry (LC-MS/MS) yielded increased levels of ceramides 16:0 and 18:0 with highest levels in severely affected patients and similar effects for dihydroceramides 16:0 and 18:0, whereas levels of (dihydro-)ceramides 24:0 were reduced. Furthermore, sphingomyelin 20:0; 22:0 and 24:0 as substrates of ASM and NSM as well as their dihydrosphingomyelin counterparts were reduced in patients as well as sphingosine-1-phosphate further downstream of NC activity. Effects of NSM, NC, ceramides and sphingomyelins remained significant after Bonferroni correction. SARS-CoV-2 antibody levels in convalescent patients were associated with age but none of the sphingolipid parameters. Based on our data, COVID-19 is associated with a dysregulation of sphingolipid homeostasis in a severity-dependent manner, particularly focused around a reduction of sphingomyelins and an accumulation of ceramides by increased enzyme activities leading to ceramide elevation (ASM, NSM) combined with a decreased activity of enzymes (NC) reducing ceramide levels. The potential of a combined sphingolipid/enzyme pattern as a diagnostic and prognostic marker and therapeutic target deserves further exploration.

Article activity feed

  1. Rapid Reviews Infectious Diseases

    Undurti Das

    Review 3: "COVID-19 and its clinical severity are associated with alterations of plasma sphingolipids and enzyme activities of sphingomyelinase and ceramidase"

    This preprint examines the modulated sphingolipid metabolic pathway in COVID-19 patients and finds case severity correlates with increased metabolic flux toward systemic ceramide production. Reviewers found the study potentially informative, but needs more mechanistic studies.

  2. Rapid Reviews Infectious Diseases

    Johnny Stiban

    Review 1: "COVID-19 and its clinical severity are associated with alterations of plasma sphingolipids and enzyme activities of sphingomyelinase and ceramidase"

    This preprint examines the modulated sphingolipid metabolic pathway in COVID-19 patients and finds case severity correlates with increased metabolic flux toward systemic ceramide production. Reviewers found the study potentially informative, but needs more mechanistic studies.

  3. Rapid Reviews Infectious Diseases

    Ralf Alexander Claus, Daniel Thomas-Rüddel

    Review 2: "COVID-19 and its clinical severity are associated with alterations of plasma sphingolipids and enzyme activities of sphingomyelinase and ceramidase"

    This preprint examines the modulated sphingolipid metabolic pathway in COVID-19 patients and finds case severity correlates with increased metabolic flux toward systemic ceramide production. Reviewers found the study potentially informative, but needs more mechanistic studies.

  4. Rapid Reviews Infectious Diseases

    Strength of evidence

    Reviewers: Johnny Stiban (Birzeit University) | 📒📒📒◻️◻️
    Ralf A. Claus, Daniel Thomas-Rüddel (Jena University Hospital) | 📗📗📗📗◻️
    Undurti Das (UND Life Sciences)📒📒📒◻️◻️

  5. ScreenIT

    SciScore for 10.1101/2022.01.19.22269391: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: The study was approved by the Ethics Committee of the Medical Faculty of the Friedrich-Alexander University Erlangen-Nürnberg (174_20B, April 30, 2020 for patients and 357_19B, October 18th, 2019 for convalescent plasma donors).
    Consent: All participants provided written informed consent.
    Sex as a biological variableBecause of the importance of sex differences in science generally (Tannenbaum et al. 2019) and sex differences for some parameters as well as known sex-dependent effects of sphingolipid enzymes (Mühle et al. 2014; Muhle et al. 2019b; Mühle et al. 2018), we also analyzed men and women separately.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Determination of SARS-CoV-2 antibody levels: Levels of antibodies against SARS-CoV-2 in plasma donors (patients after convalescence) were determined by two enzyme immunoassays: LIAISON® SARS-CoV-2 S1/S2 IgG (DiaSorin Deutschland GmbH, Dietzenbach, Germany, n=27), a semiquantitative assay, and SARS-CoV-2-EIA (EUROIMMUN AG, Lübeck, Germany, n=23)
    SARS-CoV-2-EIA
    suggested: None
    Software and Algorithms
    SentencesResources
    Spot intensities were detected on a Typhoon Trio scanner and quantified using the ImageQuant software (GE Healthcare Life Sciences, Buckinghamshire, UK).
    ImageQuant
    suggested: (ImageQuant, RRID:SCR_014246)
    Statistical analyses: For statistics, SPSS for Windows 28.0 (SPSS Inc., Chicago, IL) was used and means with standard deviation (SD) are reported (SPSS custom tables function) where the t-test was applied to analyze differences.
    SPSS
    suggested: (SPSS, RRID:SCR_002865)
    Graphs were prepared using GraphPad Prism 8.4.3 (Graph Pad Soft-ware Inc., San Diego, CA, USA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: We detected the following sentences addressing limitations in the study:
    However, it is also subject to several limitations. We have not taken into account the SARS-CoV2-variant causing infection and our data might not be generalizable to all new variants including omicron. Our samples are restricted to one university hospital and to not include healthy or asymptomatic individuals who could be expected to show comparable parameters to convalescent patients. It would be valuable to analyze the sphingolipid pattern during the course of the disease to study the dynamics and to correlate early patterns with progression and outcome to develop predictive markers. Typical risk factors for a lethal COVID-19 course including age, obesity or hypertension are also associated with the ASM/Cer system (Kornhuber et al. 2021). We were therefore cautious to include age as a cofactor in our statistical models because our groups taken from available hospitalized patients during early pandemic differed significantly in age. Due to a lack of availability, the body mass index as marker of obesity could not be included in our analyses. Future studies should pay attention to collect and integrate full medical data including biochemical laboratory parameters and clinical parameters of disease progression as well as medications which could have additional confounding effects such as FIASMAs. Due to lacking material, we did not analyze relevant cellular enzymes in peripheral blood mononuclear cells of patients such as lysosomal ASM as well as sphingomyelin synthase (Muhle ...

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    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.

  6. Version published to 10.1101/2022.01.19.22269391v1 on medRxiv