DOI: 10.25338/b8m92x
title: Lipid mediators detected in COVID-19 patients and healthy controls

description: Polyunsaturated fatty acids are metabolized into regulatory lipids
 important for initiating inflammatory responses in the event of disease or
 injury and for signaling the resolution of inflammation and return to
 homeostasis. The epoxides of linoleic acid (leukotoxins) regulate skin
 barrier function, perivascular and alveolar permeability and have been
 associated with poor outcomes in burn patients and in sepsis. It was later
 reported that blocking metabolism of leukotoxins into the vicinal diols
 ameliorated the deleterious effects of leukotoxins, suggesting that the
 leukotoxin diols are contributing to the toxicity. During quantitative
 profiling of fatty acid chemical mediators (eicosanoids) in COVID-19
 patients, we found increases in the regioisomeric leukotoxin diols in
 plasma samples of hospitalized patients suffering from severe pulmonary
 involvement. In rodents these leukotoxin diols cause dramatic vascular
 permeability and are associated with acute adult respiratory like
 symptoms. Thus, pathways involved in the biosynthesis and degradation of
 these regulatory lipids should be investigated in larger biomarker studies
 to determine their significance in COVID-19 disease. In addition,
 incorporating diols in plasma multi-omics of patients could
 illuminate the COVID-19 pathological signature along with other
 lipid mediators and blood chemistry.
description: Detailed
 Methods. 
 This is a retrospective study using prospectively
 collected plasma samples and clinical data. For oxylipin analysis,
 heparinized plasma was collected from six patients with
 laboratory-confirmed SARS-CoV-2 infection and admitted to the University
 of California Davis Medical Center in Sacramento, CA and 44 samples from
 healthy controls carefully chosen from a recently completed clinical
 study. For comparison of cytokines, plasma from healthy volunteers was
 collected from the California Central Valley Delta Blood Bank (Stockton,
 CA) prior to the COVID-19 pandemic. The methods used for blood collection,
 plasma processing, use of anti-coagulants/antioxidant/preservatives, and
 flash-freeze protocol were well-matched between case and control groups.
 The UC Davis and UC San Diego Institutional Review Boards approved the use
 of anonymized biospecimens for this study.
 <b>Lipid mediator Profiling          </b>
 Plasma (200
 μL) samples were aliquoted to a cocktail solution including 600 μL of
 methanol with 10 μL of 500 nM of surrogate solution including 9
 isotope-labeled oxylipins (d4 PGF1a, d4 PGE2, d4 TXB2, d4 LTB4, d6 20
 HETE, d11 14,15 DiHETrE, d8 9 HODE, d8 5 HETE, d11 11,12 EpETrE). Before
 the extraction, the samples were vortexed and centrifuged at 3000 rpm in a
 biosafety hood. The supernatants were then loaded on prewashed SPE
 cartridges and washed with two column volumes of 5% MeOH solution before
 elution by 0.5 mL of MeOH and 1.5 mL of ethyl acetate. The eluents were
 dried under vacuum using the Nutec MaxiVac vacuum concentrator
 (Farmingdale, NY USA) before reconstitution with 50 μL of 100 nM CUDA
 solution in methanol. Then, the extracted samples were analyzed using the
 UPLC/MS/MS system (Waters Acquity UPLC (Milford, MA, USA) hyphenated to AB
 Sciex 6500+ QTrap system (Redwood City, CA USA). The detailed parameters
 for the UPLC/MS/MS method were described previously (13, 14).
 Statistical analysis To test for
 differences between the COVID-19 and the control group cytokine levels,
 cytokine levels were log<sub>10</sub> transformed to fit a
 normal distribution and analyzed in Graphpad Prism (version 8.4.3) using
 the Wilcoxon rank-sum test with COVID positive and negative status as the
 main effect. Lipid mediator
 results were analyzed using MetaboAnalyst (https://www.metaboanalyst.ca/) and scaled using autoscaling before analysis. Multiple data sets described below were integrated to prioritize the oxylipins as possible biomarkers contributing to the severity of COVID. Oxylipins were analyzed by multiple independent t-tests using patient vs. control as the variable and the two-stage step-up method of Benjamini, Krieger and Yekutieli to determine a false discovery rate (15) to generate the volcano plot. The lipid mediators were then ranked by their effect sizes (i.e., the fold-difference between mean analyte concentration in each group). The analytes with the largest effect sizes were further evaluated by random effect ANOVA models. We minimized type 1 errors by testing for between-group differences among the analytes with the largest effect sizes and to improve the likelihood of identifying analytes that showed best potential to seve as biomarkers of disease severity. Each analyte with an effect size above 8 (i.e., analyte concentrations &gt;8-fold different) was used as a response variable. Random effect ANOVAs were run with ‘patient’ as a random effect to account for the multiple measurements from the same patient, and the fixed effect was ‘group’ (i.e., COVID positive or control). The log<sub>10</sub>-transformation of the analyte concentrations was applied. The analysis was done in JMP Pro Version15.