key: cord-0970885-85fml9ar
authors: Vazquez-Agra, N.; Marques-Afonso, A.-T.; Cruces-Sande, A.; Novo-Veleiro, I.; Pose-Reino, A.; Mendez-Alvarez, E.; Soto-Otero, R.; hermida-Ameijeiras, a.
title: Assessment of oxidative stress markers in elderly patients with SARS-CoV-2 infection and potential prognostic implications. An observational study
date: 2022-05-16
journal: nan
DOI: 10.1101/2022.05.11.22274952
sha: 98c9ca961b20ca8678f892c2eb7cd87581873718
doc_id: 970885
cord_uid: 85fml9ar

The aim of the study was to evaluate the correlation of plasma levels of thiobarbituric acid reactive substances (TBARS) and reduced thiols with morbidity, mortality and immune response in SARS-CoV-2 infection. This was an observational study that included inpatients with SARS-CoV-2 infection greater than 65 years old. Individuals were followed up until 12 months after hospital discharge. Demographic, clinical and laboratory variables were collected. Plasma levels of TBARS and reduced thiols were quantified as a measure of lipid and protein oxidation, respectively. Events of interest (fatal and non-fatal) were quantified at hospital discharge, third, sixth and twelfth-month post-discharge. The outcomes were differences in oxidative stress markers between groups of interest and time to a negative RT-qPCR and to significant anti-SARS-CoV-2 IgM titers. There were 61 patients (57% women) with a mean age of 83 years old. Patients with higher levels of TBARS and lower levels of reduced thiols had more risk of fatal and non-fatal events between admission and the first 12 months post-discharge. The presence of any event (fatal or non-fatal) at the end of the first 12 months post-discharge was correlated with TBARS levels, anti-SARS-CoV-2 IgM titers, lactate dehydrogenase, platelet count and neutrophil and lymphocyte count. We found a correlation between plasma reduced thiols and time to achieve significant anti-SARS-CoV-2 IgM titers. Assessment of some parameters related to oxidative stress could help to identify groups of patients with a higher risk of morbidity and mortality during and after SARS-CoV-2 infection.

6 147 SARS-CoV-2 IgM antibodies was performed by enzyme-linked immunosorbent assay (ELISA) and results were 148 provided quantitatively [23, 24] . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101/2022.05.11.22274952 doi: medRxiv preprint 176 177 Mortality was defined as a fatal event. We grouped it into four main categories as follows: I) In-hospital fatal events;

178 II) 3º-month post-discharge fatal events; III) 6º-month post-discharge fatal events and IV) 12º-month post-discharge 179 fatal events.

181 Non-fatal events were grouped into four main categories as follows: I) In-hospital non-fatal events (which referred 182 to the need for transfer during admission to a critical respiratory care unit); II) 3º-month post-discharge non-fatal 183 events; III) 6º-month post-discharge non-fatal events; IV) 12º-month post-discharge non-fatal events (which referred 184 to the need for readmission to hospital for respiratory or cardiovascular disease within 3, 6 and 12 months post-185 discharge).

187 Total events were defined as presence or absence of any fatal or non-fatal events for each of the groups listed 188 previously (In-hospital, 3º, 6º and 12º-month post-discharge). The other outcomes were time to significant anti-189 SARS-CoV-2 IgM titers (considering mean titers as the threshold) and time to a negative RT-qPCR. For an unknown population size, considering a variance of 0.5 and 0.05 with a threshold for the mean difference to 194 be detected of 0.5 and 0.05 units for TBARS and reduced thiols respectively, the sample size calculated to estimate 195 the mean difference between groups with a 95% confidence interval was a minimum of n = 30 patients [29] .

196 Variables were collected according to data provided by the regional digital health records (IANUS) belonging to the 197 Galician (Spain) Health Service (SERGAS). Most of the clinical variables were coded as qualitative ones and 198 laboratory parameters including TBARS and reduce thiols were collected as continuous quantitative variables.

199 Outcome variables were coded as dichotomous qualitative variables. 212 The Kolmogorov-Smirnov test was used to determine whether quantitative continuous variables were normally 213 distributed. Normally distributed variables were expressed as mean (m) and standard deviation (± SD) and non-214 normally ones were expressed as median and interquartile range (IQR). A missing value analysis was carried out on 215 those variables with more than 5% of missing values. 

228 Quantitative variables were normally distributed and we only registered more than 5% of missing values in alcohol 229 intake, smoking habit (current/former smokers) and level of physical dependence (Barthel index), which were found 230 to be missing values at random. There were 61 patients (57% women) with a mean age of 83 years old who were 231 included in the study, and among them, 42 (69%), 31(51%) and 15(25%) suffered from AHT, HLP and DM 232 respectively. A total of 27(44%) and 21(34%) individuals were affected of HF and AF. Approximately one out of 233 four individuals had tobacco or alcohol abuse and most of them were former users (data not shown). The mean . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101/2022.05.11.22274952 doi: medRxiv preprint 234 levels of TBARS and reduced thiols were 3.11 ± 0.09 μmol/L and 0.46 ± 0.07 mmol/L respectively. The mean 235 levels of anti-SARS-CoV-2 IgM titers were 40 ± 38 U/mL. General results are shown in Table 1 and complete 236 clinical features are provided in S1 Table. 237 257 (E-F) In-hospital and post-discharge total events. Results are shown in mean ± standard deviation.

258 TBARS-Tiobarbituric acid reactive substances. **Refers to P-value <0.05.

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101/2022.05.11.22274952 doi: medRxiv preprint 260 Non-fatal events 261 262 The results of the comparison groups are shown in Table 2 and extended in S3-4 Tables. In general terms current or 263 former smokers presented a higher number of non-fatal events with statistically significant differences in the 6º-264 month and 12º-month post-discharge non-fatal events groups. Regarding laboratory variables, we only observed 265 relevant differences in LDH and PTC for 6º-month and 12º-month post-discharge non-fatal events respectively. 279 ± SD. These differences reached statistical significance in the following groups: In-hospital (no: 3.01 ± 1.02, yes:

280 4.02 ± 1.33; P= 0.030), 3º-month (no: 2.84 ± 0.91, yes: 3. 63 ± 1.23; P= 0.006), 6º-month (no: 2.7 ± 0.80, yes: 3.51 281 ± 1.25; P= 0.007) and 12º-month (no: 2.75 ± 0.78, yes: 3.33 ± 1.19; P= 0.043) total events. The levels of reduced 282 thiol groups measured in mmol/L and expressed as m ± SD were lower in patients who suffered a non-fatal event.

283 Such differences reached statistical significance in the following groups: In-hospital total events (no: 0.47 ± 0.07, . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101/2022.05.11.22274952 doi: medRxiv preprint 284 yes: 0.37 ± 0.05; P= 0.035). The results are extended in Fig 1E and 1F . . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101/2022.05.11.22274952 doi: medRxiv preprint 303 Figure 3a -b shows the survival analysis using the mean levels of TBARS and reduced thiols as the cut-off point. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101/2022.05.11.22274952 doi: medRxiv preprint 313 Considering a cutoff for significant anti-SARS-CoV-2 IgM titers above the mean (40 U/mL), patients with levels of 314 reduced thiols below and above the mean (0.46 mmol/L) showed relevant differences in time to reach significant anti-315 SARS-CoV-2 IgM titers. No cut-off point for TBARS levels showed relevant results. Figure 4a -b shows the survival 316 analysis using the mean levels of TBARS and reduced thiols as the cut-off point. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 16, 2022. ; https://doi.org/10.1101/2022.05.11.22274952 doi: medRxiv preprint

and some inflammatory mediators due to redox up regulation of some transcription factors such as Nrf2, NF-KB and the 344 NLRP3 inflammasome, as well as their downstream targets

However, non-inflammatory cellular pathways related to redox imbalance and their role in SARS-CoV-2 infection 347 severity and prognostic are not yet fully understood. Indeed, if the role that redox status may play in the prognosis of

SARS-CoV-2 infection is poorly understood, the consequences of redox imbalance in the medium and long term are 349 still incalculable although it is possible that mortality and admissions one year ahead could be influenced by an

Literature widely supports the role of lipid peroxidation products in the prognosis of some inflammatory 354 diseases and there are some investigation lines trying to discover metabolic pathways related to lipid peroxidation and 355 its role in the severity and evolution of patients with SARS-CoV-2 infection

According to literature, the results related to 12º-month post-discharge total events could suggest at least three 361 concomitant and interrelated processes in SARS-CoV-2 infection with a prognostic role in the medium and long term as 362 follows: I) Impaired oxidative metabolism, tissue hypoxemia and anaerobic metabolism represented by between-group 363 differences in some oxidative stress markers (TBARS) and LDH

II) endothelial dysfunction, platelet activation and a 364 procoagulant state represented by between-group differences in PTC; and III) inflammation and immune activation 365 represented by between-groups differences in neutrophyl and lymphocyte counts, as well as anti

In the end, multiple studies have shown the existence of mechanisms related to redox imbalance and a proinflammatory 369 status in SARS-CoV-2 infection. Some of these studies have also investigated the existence of prognostic differences 370 based on certain inflammatory and oxidative stress markers

RT-qPCR results and serological titers during SARS-CoV2 infection in terms of mechanisms, duration and prognosis of 372 infection. However, to date there is an evidence gap on the implications that redox status during SARS-CoV-2 infection 373 could have in the medium to long term and we do not know how redox status might affect the time

Clinical manifestation, diagnosis, 442 prevention and control of SARS-CoV-2 (COVID-19) during the outbreak period

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 444 and coronavirus disease-2019 (COVID-19): The epidemic and the challenges

Vascular Inflammation and Oxidative 447 Stress: Major Triggers for Cardiovascular Disease

Redox regulation of cardiovascular inflammation -Immunomodulatory 450 function of mitochondrial and Nox-derived reactive oxygen and nitrogen species

Coronavirus disease 2019 in elderly patients: Characteristics and 453 prognostic factors based on 4-week follow-up

The trinity of COVID-19: immunity, inflammation and 455 intervention

Oxidative Stress as Key Player in Severe Acute Respiratory Syndrome Coronavirus 457 (SARS-CoV) Infection

Role of Oxidative Stress on SARS-CoV 459 (SARS) and SARS-CoV-2 (COVID-19) Infection: A Review

Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological 462 samples: Analytical and biological challenges

The thiol pool in human plasma: the central contribution of albumin to redox 464 processes

Oxidized lipid-466 associated protein damage in children and adolescents with type 1 diabetes mellitus: New diagnostic/prognostic clinical 467 markers

FUNCTIONAL EVALUATION: THE BARTHEL INDEX

ESC/ESH Guidelines for the 471 management of arterial hypertension: The Task Force for the management of arterial hypertension of the European 472 Society of Cardiology and the European Society of Hypertension: The Task Force for the management of arterial 473 hypertension of the European Society of Cardiology and the European Society of Hypertension

ESC/EAS Guidelines for the 476 management of dyslipidaemias: lipid modification to reduce cardiovascular risk

Pharmacologic Approaches to Glycemic Treatment: Standards of Medical Care 479 in Diabetes-2020

ESC Guidelines for the 481 diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and 482 chronic heart failure of the European Society of Cardiology (ESC)

Chronic Kidney Disease: Synopsis of the 2020 KDIGO Clinical Practice Guideline

Rapid in-person cognitive screening in 491 the preoperative setting: Test considerations and recommendations from the Society for Perioperative Assessment and 492 Quality Improvement (SPAQI)

Association of Inflammatory Markers with

Mortality in COVID-19 Infection

Lactate Dehydrogenase Predict Early Invasive Ventilation in Patients With COVID-497 19

Interleukin-6 receptor blockade 499 with subcutaneous tocilizumab improves coagulation activity in patients with COVID-19

Real-time RT-PCR for COVID-19 diagnosis: challenges and prospects

How to interpret and 504 use COVID-19 serology and immunology tests

Optimized steps in fluorometric determination of thiobarbituric acid-reactive 507 substances in serum: importance of extraction pH and influence of sample preservation and storage

Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction

Simple assay for the level of total lipid peroxides in serum or plasma

Tissue sulfhydryl groups

Fundamentals of estimating sample size

Shedding Light on the Inhibitory Mechanisms of SARS-CoV

CoV-2 Spike Proteins by ACE2-Designed Peptides

Rationale for the use of N-acetylcysteine in both prevention and adjuvant 520 therapy of COVID-19

Anti-influenza A virus activity of rhein through 522 regulating oxidative stress, TLR4, Akt, MAPK, and NF-κB signal pathways

Identification of oxidative stress 525 and Toll-like receptor 4 signaling as a key pathway of acute lung injury

Levels of serum sialic acid and thiobarbituric acid reactive substances in 528 subjects with impaired glucose tolerance and type 2 diabetes mellitus

Fatal lymphocytic cardiac damage 531 in coronavirus disease 2019 (COVID-19): autopsy reveals a ferroptosis signature

Profile of oxidative stress 534 biomarkers in COVID-19: correlation with clinical inflammatory and biochemical parameters. Free Radical Biology 535 and Medicine

A sensitive indicator for the severity 537 of COVID-19: thiol

Impact of Thiol-Disulfide Balance on the Binding of Covid-19 Spike Protein with

Angiotensin-Converting Enzyme 2 Receptor

Lactate dehydrogenase levels predict 541 coronavirus disease 2019 (COVID-19) severity and mortality: A pooled analysis

Platelet activation and 544 platelet-monocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19

Combination of cycle 547 threshold time, absolute lymphocyte count and neutrophil:lymphocyte ratio is predictive of hypoxia in patients with 548 SARS-CoV-2 infection

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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