Integrated NMR and MS analysis of plasma metabolome reveals major changes in inflammatory markers, one-carbon, lipid, and amino acid metabolism in severe and fatal COVID-19 subjects

Brazil has the second highest COVID-19 death rate while Rio de Janeiro is among the states with the highest rate in the country. Although effective vaccines have been developed, it is anticipated that the ongoing COVID-19 pandemic will transition into an endemic state. Under this scenario, it is worrisome that the underlying molecular mechanisms associated with the disease clinical evolution from mild to severe, as well as the mechanisms leading to long COVID are not yet fully understood. In this study, 1H Nuclear Magnetic Resonance spectroscopy and Liquid Chromatography-Mass spectrometry-based metabolomics were used to identify potential pathways and metabolites involved in COVID-19 pathophysiology and disease outcome. We prospectively enrolled 35 severe RT-PCR confirmed COVID-19 cases within 72 hours from intensive care unit admission, between April and July 2020 from two reference centers in Rio de Janeiro, and 12 samples from non-infected control subjects. Of the 35 samples from COVID-19 patients, 18 were from survivors and 17 from non-survivors. We observed that patients with severe COVID-19 had their plasma metabolome significantly changed if compared to control subjects. We observed lower levels of glycerophosphocholine and other choline-related metabolites, serine, glycine, and betaine, indicating a dysregulation in methyl donors and one-carbon metabolism. Importantly, non-survivors had higher levels of creatine/creatinine, 4-hydroxyproline, gluconic acid and N -acetylserine compared to survivors and controls, reflecting uncontrolled inflammation, liver and kidney dysfunction, and insulin resistance in these patients. Lipoprotein dynamics and amino acid metabolism were also altered in severe COVID-19 subjects. Several changes were greater in women, thus patient’s sex should be considered in pandemic surveillance to achieve better disease stratification and improve outcomes. The incidence of severe outcome after hospital discharge is very high in Brazil, thus these metabolic alterations may be used to monitor patients’ organs and tissues and to understand the pathophysiology of long-post COVID-19. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by grants from Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Marcos C. Gama-Almeida was recipient of CAPES scholarship. Claudio J. Struchiner was recipient of research grants from CNPq (PQ A1) and FAPERJ (Cientista do Nosso Estado). Tatiana El-Bacha was recipient of research grant from FAPERJ (process SEI-260003/002690/2020 and E-26/200.930/2022) and from CNPq (process 314971/2021-1, PQ2. Gilson C. dos Santos Jr was recipient of research grant from FAPERJ (process E-26/201.259/2021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The National Review Board of Brazil approved the study protocol (Comissão Nacional de Ética em Pesquisa [CONEP] 30650420.4.1001.0008), and written informed consent was obtained from all subjects or their caregivers. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All relevant data are within the manuscript and its Supporting Information files.