key: cord-0709553-c0jx5lvh
authors: Cartin-Ceba, Rodrigo; Khatua, Biswajit; El-Kurdi, Bara; Trivedi, Shubham; Kostenko, Sergiy; Imam, Zaid; Smith, Ryan; Snozek, Christine; Navina, Sarah; Sharma, Vijeta; McFayden, Bryce; Ionescu, Filip; Stolow, Eugene; Keiser, Sylvia; Tejani, Aziz; Harrington, Allison; Acosta, Phillip; Kuwelker, Saatchi; Echavarria, Juan; Nair, Girish B.; Bataineh, Adam; Singh, Vijay P.
title: EVIDENCE SHOWING LIPOTOXICITY WORSENS OUTCOMES IN COVID-19 PATIENTS AND INSIGHTS ABOUT THE UNDERLYING MECHANISMS
date: 2022-04-27
journal: iScience
DOI: 10.1016/j.isci.2022.104322
sha: 5553fe30fc2bb7b138c5964262dbfac741515b7e
doc_id: 709553
cord_uid: c0jx5lvh

We compared 3 hospitalized patient cohorts and did mechanistic studies to determine if lipotoxicity worsens COVID-19. Cohort-1 (n=30) compared COVID-19 patients dismissed home to those requiring intensive-care unit (ICU) transfer. Cohort-2 (n=116) compared critically ill ICU patients with and without COVID-19. Cohort-3 (n=3969) studied hypoalbuminemia and hypocalcemia’s impact on COVID-19 mortality. Patients requiring ICU transfer had higher serum albumin unbound linoleic acid (LA). Unbound fatty acids and LA were elevated in ICU transfers, COVID-19 ICU patients and ICU non-survivors. COVID-19 ICU patients (cohort-2) had greater serum lipase, damage-associated molecular patterns (DAMPs), cytokines, hypocalcemia, hypoalbuminemia, organ failure and thrombotic events. Hypocalcemia and hypoalbuminemia independently associated with COVID-19 mortality in cohort-3. Experimentally, LA reacted with albumin, calcium and induced hypocalcemia, hypoalbuminemia in mice. Endothelial cells took up unbound LA, which depolarized their mitochondria. In mice, unbound LA increased DAMPs, cytokines, causing endothelial injury, organ failure and thrombosis. Therefore, excessive unbound LA in the circulation may worsen COVID-19 outcomes.

The coronavirus disease pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents the greatest global public health crisis of our time. COVID-19 outcomes range from asymptomatic disease to death, with mortality being almost 50% for those requiring invasive mechanical ventilation (Domecq et al., 2021) . COVID-19 outcomes therefore may depend on disease modifiers and not just infection.

SARS-CoV-2 may infect adipocytes (Martínez-Colón et al., 2021) which store triglyceride and have the angiotensin-converting enzyme 2 (ACE2) receptor (Li et al., 2020a) . Rapid triglyceride breakdown by lipolysis can release large amounts of long chain (>12 carbon) non-esterified fatty acids (NEFAs) which can be toxic. Such lipotoxicity is described in severe pancreatitis, which like severe COVID-19 is worse in obesity (Lighter et al., 2020; Martinez et al., 2006) . In severe pancreatitis unsaturated fatty acids (UFAs) generated by visceral triglyceride lipolysis (Camhi et al., 2011; Navina et al., 2011) comprise 60-80% of NEFAs Noel et al., 2016) . UFAs worsen pancreatitis by causing lung, renal and circulatory failure Navina et al., 2011) . Intravenous UFAs infusion is a common model of acute lung injury (Kamuf et al., 2018; Moriuchi et al., 1998) . The above findings, and reports of pancreatitis or fat necrosis in 20-40% of COVID-19 full body autopsies (Hanley et al., 2020; Lax et al., 2020) suggest a potential role of lipotoxicity in worsening COVID-

Pancreatic lipase elevation without clinical pancreatitis correlates with worse outcomes in critical illnesses (Manjuck et al., 2005) , burns (Ryan et al., 1995) , trauma (Subramanian et al., 2016) , hemorrhagic shock (Malinoski et al., 2009 ), intracranial bleeding(Justice et al., 1994 , and neurosurgical intensive-care unit COVID-19 patients and mice with elevated LA cause MOF, thrombosis, and cytokine elevation noted in severe COVID-19.

Severe COVID-19 and unbound LA cause endothelial damage: We first noted that Unbound LA at clinically relevant concentration range of 2.5-10 M dose dependently depolarized mitochondria, (510/590 Emission ratio in Figure 4A ) in JC-1 loaded endothelial cells (HUV-EC). The phenomenon seemed to be triggered by unbound LA since LA (30 M) and its fluorescent tracer LA-coumarin were reversibly taken into HUV-ECs (middle row 60s-150S images, Figure 4B ). While LA uptake depolarized HUV-EC mitochondria as evidenced by reduced fluorescence of the mitochondrial membrane potential sensitive dye MitoTracker Red CMXRos (MT-Red, top row), addition of albumin along with a fluorophore (Albumin-647, at 180s, bottom row), reversed the uptake and depolarization. Thus, unbound LA may depolarize endothelial mitochondria and injure them. This hypothesis is supported by the higher endothelial injury marker; soluble E-selectin in ICU COVID-19 patients (vs.

non-septic COVID-19 controls), and LA treated mice . In later sections (Figure5J, black arrows) we also noted morphological evidence of LA induced endothelial injury in vivo. Similar to COVID-19 patients, LA treated mice had higher soluble ICAM-1, and DAMPs (i.e.ds-DNA, HMGB-1, and histone-DNA complex levels) (Figure4E-J) which as we shall discuss later can be pro-thrombotic and pro-inflammatory.

Severe COVID-19 induced cytokine elevation, organ failure and thrombosis in ICU patients are replicated by unbound LA in-vivo: COVID-19 ICU patients, had significantly higher levels of the proinflammatory cytokines CXCL1, IL-1, IL-6, MCP-1, and TNF- compared to the non-COVID group (Table 6) . LA treated mice also had a similar pattern of cytokine elevation vs. controls (right side Table 6 , S21). However, in PA administered mice, while IL-6 elevation was noted, it was lower than in mice given LA (right side Table 6 ).

COVID-19 patients were in a prothrombotic state evidenced by a higher rate of deep venous thrombosis (DVT) and pulmonary embolism (PE), irrespective of septic shock (Bottom rows of Table 7 , S22), while MOF was higher after excluding septic shock (Top rows of Table 7 , S22). ECMO utilization were also seen more frequently in the COVID-19 group (Table 7, S22) . Similar to COVID-19 patients, LA induced MOF in mice ( Figure 5) necessitating euthanasia by 72 hours. Shock was evidenced by a drop in carotid artery pulse distention (Pulse J o u r n a l P r e -p r o o f dist., Figure 5A ). LA also caused hypothermia ( Figure 5B ) suggesting a severe systemic inflammatory response.

Apart from shock, LA also induced other parts of MOF. LA induced renal failure was noted as a large increase in serum BUN, and creatinine ( Figure 5C , D). LA also increased TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) positivity in the renal tubules ( Figure 5E , H yellow arrows). LA induced lung injury was noted as TUNEL positivity in the lung alveoli ( Figure 5F , J red arrows), and vessels (J, black arrows). Interestingly, this was associated with pulmonary thrombi ( Figure 5J , 6J). LA induced lung injury was also noted as increased lactate dehydrogenase (LDH) and higher annexin V positive cells in the Bronchoalveolar lavage (BAL; Figure 5K -L). Specifically, type-II pneumocyte injury was noted as increased dual Thyroid transcription factor-1 (TTF-1), Annexin V positive cells, or CD208+ Annexin V+ cells ( Figure 5M -O) in the BAL of LA treated mice. Therefore, LA induced shock, renal and lung injury in mice.

On focussing on prothrombotic mechanisms, we noted COVID-19 patients in the ICU had higher coagulation factor III (CFIII; Figure 6A ) and tissue factor pathway inhibitor (TFPI; Figure 6B ) supporting a prothrombotic state compared to controls. Similarly, 8 of 12 mice given LA had portal venous thrombi ( Figure   6C -D) unlike control mice. A prothrombotic state induced by LA was noted as increased fibrinogen, plasminogen activation inhibitor-1 (PAI-1), TFPI levels. Elevated Soluble Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) or CD31 ( Figure 6E -H) levels suggested LA induced endothelial injury in vivo. On immunohistochemistry of control mouse lungs, PECAM-1/CD31 normally localized to pulmonary vessel endothelium (yellow Figure 6I ), and immune cells as described previously (Lertkiatmongkol et al., 2016) . LA however, dramatically increased PECAM-1/CD31 expression in the alveolar capillaries and pulmonary vascular thrombi (red squares, oval, Figure 6J ), with the mice developing thrombocytopenia and an elevated activated clotting time ( Figure 6K -L). Electron microscopy of pulmonary vessels showed LA causes loss of or lifting of endothelial cells (* in Figure 6O ) from the basement membrane, wherein fibrin strands attached (green arrows Figure 6O ) and extended inwards towards large platelet aggregates ( Figure 6P ).

Administration of LA along with calcium and albumin (fatty acid free) inhibited the increase in unbound FA, without affecting the increase in serum LA or total NEFA ( Figure at 72 hours (0/7 with LA vs. 6/6 with LA +Ca, Alb). Thus, in mice unbound LA may mediate most of the severe COVID-19 like outcomes noted in humans.

Finally, it has been reported that patients with diabetes mellitus could be linked to underlying elevated FAs as compared to non-diabetics (Bergman and Ader, 2000) . Furthermore, LA plasma levels have been reported to be significantly higher in women (Lohner et al., 2013) . In order to evaluate if the presence of diabetes or the female gender could confound the results, we analyzed the ICU cohort (Cohort 2) firstly by removing all diabetic patients (Table S23 ) and secondly by removing women from the cohort (Table S24) . No major differences were noted as compared to the initial results. Therefore, the prothrombotic state, MOF and reduced survival noted in severe COVID-19 patients is likely due to elevated unbound UFAs like LA.

Here we present evidence in humans, along with mechanisms in animal, and in-vitro studies explaining how unbound LA may worsen COVID-19 outcomes. Prospectively, in hospitalized patients we note that in comparison to mild COVID-19 patients, those progressing to severe COVID-19 requiring ICU admission have higher UFAs, including LA and OA, which are also increased in the unbound form. Severe COVID-19 ICU patients had higher lipase, LA levels, UFAs, inflammatory cytokines, thrombotic events, hypoalbuminemia, and hypocalcemia compared to non-COVID patients. Unbound FAs were elevated in both COVID-19 and septic shock patients who also had a similar rate of MOF. Cumulatively, COVID-19 patients required more organ support therapies including ECMO and mechanical ventilation. The retrospective hospitalized COVID-19 cohort had hypocalcemia and hypoalbuminemia independently associated with hospital mortality and ventilator requirements after adjusting for age, gender, BMI, race, and medical comorbidities.

Experimentally in mice, unbound LA induced the widely reported hypoalbuminemia, hypocalcemia, DAMP release, cytokine storm, thrombosis, and MOF phenotype which we note, and others have reported in J o u r n a l P r e -p r o o f severe COVID-19. These are consistent with well-known models of acute lung injury indued by intravenous UFAs (Kamuf et al., 2018; Moriuchi et al., 1998) . PA, the most abundant saturated NEFA, despite being administered at higher doses than LA, did not enter the circulation, perhaps due to its extreme hydrophobicity (Khatua et al., 2021) . Administering intraperitoneal PA with a solvent (dimethyl sulfoxide) or directly through an incision were equally harmless (data not shown).

While albumin binds LA more strongly than calcium (H= -154± 54 KJ/mol vs. -17.1±1.5 KJ/mol; Figure   3A , B), albumin's molar amounts in normal serum (600-800 M) are lower than calcium (2-2.5mM). However, albumin's stronger binding to LA put it upstream of calcium in preventing lipotoxicity ( Figure 8 ). Therefore, despite the increase in total LA concentrations induced by giving prebound LA ( Figure 7B ); the prebinding kept unbound FAs low, at control mouse levels ( Figure 7C ). This is mechanistically consistent with the energetically favorable pre-binding of LA by albumin preventing the increase in unbound FAs despite an increase in total LA.

While we cannot comment on the exact proportion of unbound LA neutralized by calcium, the therapeutic role of calcium is supported by it preventing LA induced hypocalcemia ( Figure 7D , F) and its energetically favorable binding to LA ( Figure 3B ). This is clinically relevant since we note hypocalcemia with severe COVID-19 (Figure 2 ), and calcific fat necrosis is noted in autopsies of COVID-19 patients (Lax et al., 2020) . The protective role of calcium against lipotoxicity is also supported by previous studies showing extracellular calcium deposits in fat necrosis, and extracellular calcium supplementation to reduce lipotoxic cell injury and delay organ failure . Mechanistically, unbound LA relevant to COVID-19 concentrations was reversibly taken into cells, and depolarized mitochondria ( Figure 4A -B). LA which reduces transendothelial resistance, causing macromolecular capillary leakage (El-Kurdi et al., 2020; Khatua et al., 2021) , reacted with albumin and calcium, explaining the rapid hypoalbuminemia and hypocalcemia we note in humans and mice (Figure 2-3) . Such hypoalbuminemia cannot be explained by reduced albumin synthesis, since albumin has a 25 day half-life(Levitt and Levitt, 2016). These findings and concepts are summarized in Figure 8 .

While most COVID-19 infections are mild or self-limited, in this study a severe phenotype-like picture could be induced by an UFA increased in the blood of patients with severe COVID-19, i.e., LA. This along with J o u r n a l P r e -p r o o f the use of intravenous UFAs to induce lung injury (Kamuf et al., 2018; Moriuchi et al., 1998) supports LA's role in worsening COVID-19. Our ICU COVID-19 patients, as in previous studies (Domecq et al., 2021; Richardson et al., 2020) developed respiratory failure (Domecq et al., 2021; Li et al., 2021) , MOF, and VTE events. Similarly, endothelial injury(Ackermann et al., 2020b), lung injury, and vascular occlusions were also induced by unbound LA in mice ( Figure 5, 6 ). LA induced injury in the BAL without coexisting pneumonia or pancreatitis suggest unbound LA may worsen COVID-19. Interestingly, the elevation of unbound FAs, UFAs, and LA in ICU nonsurvivors (Table S6) irrespective of etiology suggests a broader relevance of such elevations. Please note that since our COVID-19 patients had an even fluid balance, we gave our mice water ad libitum, with subcutaneous saline supplementation at 10 % bodyweight/ day. Double bonds in a FA like LA increase its lipolytic generation, and the aqueous stability of its monomers even without a carrier like albumin (Khatua et al., 2021) . Saturation in contrast makes long chain fatty acids too hydrophobic to exist as unbound monomers. This likely explains the lack of PA elevation, cytokine response in PA administered mice (Tables 3,6) and the lack of MOF as previously reported (Khatua et al., 2021) . Double bonds explain the elevated UFAs and unbound FA levels in COVID-19 non-survivors (Table S6) , those with septicshock [who had increased palmitoleic acid (C16:1), the shorter chain of which increases aqueous stability] and mice given LA alone (Table 3) . Previously, in the presence of albumin 300-600 M LA concentrations were shown to depolarize mitochondria Patel et al., 2016) . In contrast, in the absence of albumin we note 2.5-30 M unbound LA is sufficient to depolarize endothelial (HUV-EC) cell mitochondria ( Figure 4A Figure 5A ) and hypoalbuminemia in severe COVID-19 and LA treated mice (Figures 2,3) .

Overall, the shock, renal and lung injury we note in LA treated mice may explain the MOF noted in COVID-19 patients who also had elevated LA. The pro-inflammatory state triggered by LA is supported by the higher cytokines (Table 6) LA itself is a precursor of arachidonic acid (Gao et al., 2010) , which is elevated in our COVID-19 ICU patients (Table 3) There is a potential role for early albumin and calcium supplementation to prevent lipotoxicity in COVID-19. We have described experimentally what occurred when albumin was administered with LA along with calcium: it inhibited the increase in unbound FA and prevented DAMP increase, coagulation abnormalities, and MOF development; resulting in improved survival in mice. The potential utility of albumin therapy in COVID-19 J o u r n a l P r e -p r o o f patients was recently described by an Italian group (Violi et al., 2021) . In an observational prospective study performed in 29 SARS-CoV-2 patients treated with anticoagulant alone or anticoagulant plus albumin supplementation for 7 days, the investigators demonstrated a significant decrease of D-dimer only in the albumin-treated patients, who also had significantly reduced mortality (0/10 vs. 8/19 without albumin supplementation; p=0.02).

In summary, we note that during severe COVID-19 infection, the lipolytic release of UFAs like LA, perpetuated by the hypoalbuminemia and hypocalcemia induced by LA, may result in cellular uptake of the unbound FAs like LA, resulting in mitochondrial injury. This injury to endothelial and other cells in vivo may result in shock, renal failure, DAMP release, and the consequent cytokine storm, MOF, and thrombosis that we note during severe COVID-19 infection.

We acknowledge several limitations. First, our prospective cohorts are small, and the cohort-2 non-COVID-19 ICU patients are not an ideal control group since these included septic patients with whom they could share some pathophysiologic mechanisms. However, on excluding septic patients, the most important lipotoxic, inflammatory, and thrombotic abnormalities persisted. Additionally, while organ failure was increased in the COVID-19 ICU group, mortality did not achieve statistical significance compared to the non-COVID group. To address this, we compare the NEFA profile between ICU survivors and non-survivors and note that NEFA, UFAs and specifically LA and unbound fatty acids were higher in non-survivors (Table S6) . We also do not have an obese animal model of COVID-19 to test the hypothesis that unbound fatty acids worsen COVID-19 in these models, nor do we provide proof of albumin having a therapeutic role in these. This issue is partly addressed by the human study showing that COVID-19 patients receiving albumin along with anticoagulation had improved survival than those who received anticoagulation only (Violi et al., 2021) . Another limitation included the lack of lipase levels in the large retrospective cohort; however, large meta-analyses have shown lipase elevation to be associated with worse COVID-19 outcomes in the absence of clinical pancreatitis (Yang et al., 2022) . Therefore, while this study provides preliminary evidence of, and mechanisms supporting lipotoxic exacerbation of COVID-19, its findings need to be confirmed in larger studies. 

Each circle represents an individual patient. Values in all groups from day 1 to day 4 were lower than those on day 0 on ANOVA. The P value shown (P<0.0001) was calculated on Mann-Whitney U test between survivors and nonsurvivors for each day separately. Patients requiring ventilator support and no ventilator are compared similarly. Survivors and patients not requiring mechanical ventilation are shown in green. The lines across connect the median values for survivors (black) and non-survivors (red). The error bars represent the interquartile range. (D, F, H, J) . Each dot represents an individual patient or mouse. The P-values shown above were calculated using a Mann-Whitney test. P values comparing the two groups of humans, or two groups of mice J o u r n a l P r e -p r o o f were calculated using a Mann-Whitney U test. The red L shaped structures denote unsaturated fatty acids like linoleic acid, which are present in excess while the blue straight lines denote saturated fatty acids. When the amount of unsaturated fatty acids like LA exceeds the ability of albumin to bind them, the unbound LA reacts with calcium causing hypocalcemia. Excess unbound LA is taken up by cells, causing mitochondrial depolarization, and consequent epithelial and endothelial injury along with DAMP release, the latter among which causes the cytokine storm. The endothelial injury causes vascular leak, and hypoalbuminemia, along with release of procoagulant coagulation factor III (CFIII) from basement membranes, which with DAMPs promote thrombosis. These along with plasminogen activation inhibitor-1 (PAI-1) result in a prothrombotic state, worsening organ failure, which can result in death. 

All deidentified data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

The age, sex of the subjects in the various cohorts in the study are mentioned in the respective Tables. The cohorts are detailed below.

Hospitalized pre-ICU COVID-19 Cohort (Cohort 1): The study was from 5/12/2020, till 12/03/2020 on consented inpatients at Mayo Clinic Hospital (MCH; Phoenix, AZ), approved by the Mayo Foundation Institutional Review Board (IRB) and conformed to the regulatory standards of the institution. Inclusion criteria:

≥18 years old with positive SARS-CoV-2 PCR within 14 days, and sera available within 1 day of admission and dismissal to home (mild COVID) or ICU transfer (severe COVID).

Prospective ICU cohort (Cohort 2): Study was from May 1 st , 2019 to October 31 st , 2020 in a 30-bed multidisciplinary ICU at MCH, with IRB approval as above. Inclusion criteria: Consecutive critically ill patients ≥18 years of age admitted to the ICU; for COVID-19 patients, a positive SARS-CoV-2 PCR test within 14 days was required for inclusion. Exclusion criteria included DNR/DNI and comfort care patients, ICU readmissions, and patients who had not agreed to the use of their medical records for research. Data collection included patient characteristics, hospital course, laboratory values, and interventions on all enrolled patients during their hospitalization. The APS, APACHE IV score, and predicted hospital mortality rates based on these scores were calculated using an online APACHE IV calculator (Zimmerman et al., 2006) . SOFA score (Moreno et al., 1999; Vincent et al., 1996) was documented daily from day 1 to day 7. Organ dysfunction was defined as a SOFA score of 1 or 2 points and organ failure as a SOFA score ≥ 3. MOF was defined as two or more organ failures (Moreno et al., 1999; Vincent et al., 1996) J o u r n a l P r e -p r o o f . There were 4 groups: Controls, LA, PA treated and those administered LA with calcium and albumin (LA+ Ca, Alb.) . There were at least 7 mice per group. LA was given intraperitoneally at 0.2% body weight (El-Kurdi et al., 2020; Khatua et al., 2021; . These mice received 1.0 ml saline subcutaneously 3 times per day. Co-administration of a similar does of LA with calcium and albumin (LA+ Ca, Alb.) was done after gently mixing 500l LA (at 37 0 C for 2 hours) with 10 ml of 25% albumin (fatty acid free in saline) containing 20mM calcium. This was delivered intraperitoneally at 1.2ml/ 30 gm body weight. PA (333 mg/Kg) was administered intraperitoneally either alone via a sterile peritoneal incision, or via intraperitoneal injection having PA dissolved in 50% dimethyl sulfoxide. While some mice overlapped with previous cohorts (El-Kurdi et al., 2020; Khatua et al., 2021; , the current studies were separate from previous ones reported. Mice were monitored daily thereafter for general appearance, grooming, posture, activity, food intake, and vitals for the next 3 days. Rectal temperature was measured with a clinical thermometer; carotid artery pulse distention was measured using a neck collar of a MouseOx pulse oximeter (Starr Life sciences, Oakmont, PA). Mice were euthanized on day 3 or when unable to ambulate, moribund or if noted to be in distress (El-Kurdi et al., 2020) . Vitals prior to this euthanasia are the ones reported. Blood parameters measured were the ones at the time of euthanasia. Creatinine, ionized calcium, and BUN were measured using the CHEM8+ cartridge in the i-STAT 1 blood analyzer (Abbott Point of Care, Orlando, Florida, USA). All protocols were approved by the institutional animal care and use committee of the Mayo Clinic Foundation.

Bronchoalveolar lavage (BAL) analysis: BAL was performed via tracheal incision at the time of euthanasia. To collect mice lungs BAL fluid, an incision was made on the disinfected neck skin. The trachea was exposed by blunt dissection. After tracheal incision with a scalpel, a catheter was placed in the exposed trachea. which was connected to a syringe filled with 1 mL of sterile saline solution. A cotton ligature was placed around the trachea and catheter to avoid flowing back of the fluid to the upper airways. The lungs were flushed with saline gently, preventing the collapse of the lung airway. The aspirated fluid was collected and centrifuged (400g, 10 min., 4 0 C) to pellet the cells, which were immediately as below. The supernatant was analyzed for lactate dehydrogenase (LDH) leakage and total proteins. A colorimetric LDH leakage assay was performed with Roche 1. Fatty acid extraction: Kangani and Delany Plasma Free Fatty Acid Extraction i. Add 5L of internal standard (in standards section) to the 160L of de-albuminated dried Folch extract resuspended in PBS (equivalent to 44L of serum).

ii. Add 2mL of a 40:10:1, Isopropanol: Hexane: Hydrochloric acid (1M) mixture for each sample and transfer to a 13mL glass tube with PTFE lined screw caps using a pasteur pipette. Mix 50mg of BHT per liter of the 40:10:1 solvent mixture.

iii. Thoroughly vortex mixture for 30 minutes followed by a 10-minute incubation at room temperature.

iv. Add 1.89mL of LCMS grade water then 4mL of hexane to each tube and vortex for 5 minutes.

v. Centrifuge at 1000g for 10 minutes at 4°C vi. Transfer the hexane upper phase to a new glass tube using a pasteur pipette, then evaporate using nitrogen and no heat.

vii. On ice, add 200L of dichloromethane to all the vials, followed by 4L of diisopropylethylamine, then 8L of dimethylamine.

viii. Add 2L of Deoxo-Fluor to the wall of the vial, immediately cap and vortex for 5 seconds to mix.

ix. Incubate the samples at -20°C for 5 minutes, then at room temperature for 10 minutes.

x. Transfer the samples to screw top glass tubes with PTFE lined screw caps containing 2mL of LCMS grade water, vortexing briefly to stop the reaction.

xi. Add 4mL of hexane and vortex the mixture for 15 minutes.

xii. Centrifuge the mixture at 3000 RPM for 10 minutes.

xiii. Transfer the organic upper phase to a new test tube using a pasteur pipette and evaporate using nitrogen and heat at 40°C. xiv. Resuspend in 200L of hexane with caffeine and transfer to an auto-sampler vial.

The GC is an Agilent GC 7890B system with an Agilent 5977A MSD (single quadrupole) attached. The MS source is set at 275°C whereas the quad is set at 150°C. The carrier gas of Helium has a flow set at 1mL/min. The injection volume is set at 1L with the injector port temperature set at 260°C. The oven is programmed to begin at 140°C for 2 minutes, with a ramp up at 20°C/min to 200°C which is held for 4 minutes. A second ramp is started at 5°C/min up to 260°C with no holding time, where the final ramp beings at 10°C/min up to a maximum of 300°C that is held for 15 minutes. The GC is equipped with a capillary column from Agilent (HP-5MS UI, 30m x .25mm I.D with a .25um film thickness).

4. Use of standards xv. A standard curve is taken through the entire extraction and derivatization process that is made up of a combination of C12:0, C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, C18:3 and C20:4 in heptane. The curve was made to 20M and was diluted in a serial dilution to 10M, 5M, 3M, 2M, 1m and 0.5M the curve also includes the internal standards. xvi. A mixture of Internal standards of C16:2, C17:0, and C19:2 is added to every sample at 4.545M, 9.09M and 9.09M respectively and used to determine extraction efficacy.

xvii. Caffeine is used at 5ppm as a GC standard 5. Calculation of results xviii. Extraction blanks of hexane are subtracted out of the GC response and then each individual fatty acid is calculated for every sample from solving a linear equation generated from the standard curve. The values are then adjusted accounting for the extraction efficacy of each internal standard value targeting the theoretical values of 4.545M and 9.09M.

DAMPs: ds-DNA was measured using the Quant-iT PicoGreen dsDNA reagent (Life Technologies, Carlsbad, CA).

Histone complex DNA fragments were measured using a kit from Sigma-Aldrich (Saint-Louis, MO), or HMGB-1 from FineTest (Wuhan Fine Biotech Co., Ltd, Wuhan, China) were measured using ELISA.

J o u r n a l P r e -p r o o f 8.6 (5.7-11.2) 6.7 (5.3-10) 0.11 5.9 (4.9-7.8) 6.7 (6.1-8.3) 8.7 (7.0-13.9.5) >0.1

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), and sE-selectin, Soluble intercellular adhesion molecule-1 (sICAM-1), coagulation factor III (or tissue factor), and tissue factor pathway inhibitor (or TFPI) were measured and analyzed using Luminex Assay

MCP-1, TNF-α, sE-selectin, s-ICAM1, PECAM-1 and PAI-1 were measured using MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel (Millipore

Calcium, albumin; Pointe Scientific, Canton, MI, USA) were done as per the manufacturer's protocol

ACT were measured from freshly drown cardiac blood by using i-STAT Celite ACT Cartridge on i-STAT1 analyzer

Freshly isolated mouse blood was diluted with a 1% ammonium oxalate solution in 1:20 ratio and allowed 15-20 minutes to lyse all erythrocytes while the leukocytes, platelets, and reticulocytes remain intact. The solution was placed on Hemocytometer and put cover glass on it

) ELISA were done from mouse serum as per manufacturer protocol

Histologic analysis: Lung and liver tissue of mice were procured at the time of necropsy (CO2 euthanasia) and were fixed with 10% neutral buffered formalin (Fisher Scientific) and embedded in paraffin and sectioned. Liver paraffin section (5 microns) slides stained by hematoxylin and eosin (H&E) were used to identify portal venous thrombi by a pathologist (S. N.) blinded to the treatment received. TUNEL staining of the lungs and kidneys were done using the ApopTag Peroxidase In Situ Apoptosis Detection Kit" from EMD Millipore (Catalog number #S7100) as per validated manufacturer's protocol

PECAM-1 IHC: PECAM-1: Lungs paraffin section (5 microns) slides were stained with Anti-CD31/PECAM-1

the Pathology Research Core at Mayo Clinic

IN VITRO STUDIES: All data shown are representative of 5 independent experiments. For mitochondrial depolarization studies and mitochondrial uptake studies we show representative curves and images respectively

2019) to ensure a sTable emulsion for the duration of in vitro studies. LA-coumarin was custom synthesized by reacting Linoleic acid alkyne with 7-hydroxycoumarin azide at Nanosyn Inc

Isothermal titration calorimetry: The interaction of calcium or albumin with LA was carried out as described elsewhere

Similarly, albumin (stock 0.85mM) was injected into LA (1 mM), or LA (1mM stock) into albumin (0.16 mM) at 37 °C. The thermodynamic parameters (Kd, n and ΔH) for calcium-LA and albumin-LA was calculated from NanoAnalyze Software

Cells were loaded with MitoTracker-Red CMXRos (Excitation 580nm, Emission 600nm; ThermoFisher) in a complete F-12K Medium at 37°C for 30 min and then washed twice with HEPES

Fluor oil immersion objective. For localization of LA, cells were exposed to LA (30M) along with tracer amounts

Singh and McNiven, 2008) and stored on ice till the assay was conducted. LA was added after 100 seconds at the indicated concentrations to the stirred cell suspensions in a quartz cuvette at 37°C. Mitochondrial inner membrane potential (m) was determined by excitation at 490nm, and alternate measuring emission at 510 and 590 nm using the F2100 Hitachi Fluorescence Spectrophotometer

Heart rate, median (IQR) 98

Respiratory rate, median (IQR)

Fluids in first 24 hours, median (IQR)

Urine output, first 24 h, median (IQR)

Fluid balance 24 hours (mL), median (IQR)

Acute physiologic score, median (IQR)

Sodium (mmol/L), median (IQR)

Creatinine (mg/dL), median (IQR)

Blood urea nitrogen (mg/dL), median (IQR)

Glucose (mg/dL), median (IQR)

Lactate (mmol/L), median (IQR)

Bilirubin (mg/dL), median (IQR)

/L), median (IQR) 19

Albumin (g/dL), median (IQR)

ICU: intensive care unit, APACHE: Acute Physiology and Chronic Health Evaluation, SOFA: Sequential Organ Failure Assessment

Significantly different from control mice. ** Significantly different from control and Palmitic acid (PA) treated mice

• 3 cohorts of hospitalized COVID-19 patients with different severities were studied • Severe COVID-19 increased serum linoleic acid (LA) and unbound fatty acid levels • Endothelial cell uptake of unbound LA dose-dependently depolarized mitochondria • Unbound LA increased cytokines, endothelial injury

The data were collected every 10 seconds. The net increase over time point before addition was plotted vs.time. The increase in 510/590 emission ratio was used as a measure of mitochondrial depolarization.

Continuous parametric variables were reported as means and standard deviations while non-parametric continuous variables were reported as medians and interquartile ranges. Unpaired Student's t tests were used to compare continuous variables with normal distribution and the Wilcoxon rank test for skewed distribution. ANOVA with multiple comparisons was used for comparisons of multiple groups. For comparison of categorical variables, chi-square tests were used if the number of elements in each cell was ⩾ 5; Fisher's exact test were used otherwise. A p value ⩽ 0.05 was considered statistically significant. Univariate and multivariate regression analyses for the retrospective cohort study were conducted for the baseline variables abstracted identifying associations between variables and in-hospital mortality. All 

White blood count (x10 9 /L), median (IQR) 6.4 (5.1-9.2) 5.6 (4. J o u r n a l P r e -p r o o f