key: cord-0892528-f0xp9qd5
authors: Aparisi, Álvaro; Iglesias-Echeverría, Carolina; Ybarra-Falcón, Cristina; Cusácovich, Iván; Uribarri, Aitor; García-Gómez, Mario; Ladrón, Raquel; Fuertes, Raúl; Candela, Jordi; Tobar, Javier; Hinojosa, Williams; Dueñas, Carlos; González, Roberto; Nogales, Leonor; Calvo, Dolores; Carrasco-Moraleja, Manuel; San Román, J. Alberto; Amat-Santos, Ignacio J.; Andaluz-Ojeda, David
title: Low-density lipoprotein cholesterol levels are associated with poor clinical outcomes in COVID-19
date: 2021-07-06
journal: Nutr Metab Cardiovasc Dis
DOI: 10.1016/j.numecd.2021.06.016
sha: 04f9f26ef3ad472bfb7b2574fc81d5256e5bd8a5
doc_id: 892528
cord_uid: f0xp9qd5

Background and Aims Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the sole causative agent of coronavirus infectious disease-19 (COVID-19). Methods and Results We performed a retrospective single-center study of consecutively admitted patients between March 1st and May 15th, 2020, with a definitive diagnosis of SARS-CoV-2 infection. The primary end-point was to evaluate the association of lipid markers with 30-days all-cause mortality in COVID-19. A total of 654 patients were enrolled, with an estimated 30-day mortality of 22.8% (149 patients). Non-survivors had lower total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-c) levels during the entire course of the disease. Both showed a significant inverse correlation with inflammatory markers and a positive correlation with lymphocyte count. In a multivariate analysis, LDL-c ≤ 69 mg/dl (hazard ratio [HR] 1.94; 95% confidence interval [CI] 1.14-3.31), C-reactive protein > 88 mg/dl (HR 2.44; 95% CI, 1.41-4.23) and lymphopenia < 1,000 (HR 2.68; 95% CI, 1.91-3.78) at admission were independently associated with 30-day mortality. This association was maintained 7 days after admission. Survivors presented with complete normalization of their lipid profiles on short-term follow-up. Conclusion Hypolipidemia in SARS-CoV-2 infection may be secondary to an immune-inflammatory response, with complete recovery in survivors. Low LDL-c serum levels are independently associated with higher 30-day mortality in COVID-19 patients.

STRUCTURED ABSTRACT 32 33 Background and Aims: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the sole 34 causative agent of coronavirus infectious disease-19 . presented with complete normalization of their lipid profiles on short-term follow-up. 46

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel single-stranded RNA 76 virus [1] , and it is considered the sole causative agent of Coronavirus disease-2019 . According 77 to the World Health Organization, approximately 2,191,898 patients are currently dead after 101,406,059 78 confirmed cases worldwide [2] . Initial reports have suggested that SARS-CoV-2 binds to a human 79 angiotensin-converting enzyme-2 receptor to gain intracellular entry [1] after it causes some protein and 80 lipid conformational changes at the edges of cholesterol-rich lipid domains [3] . 81

Cholesterol is a precursor of steroid hormones and bile acids and is an essential constituent of cell 82 membranes that facilitates signal transduction. Besides, membrane cholesterol is a critical component to 83 facilitate viral entry into host cells, and evidence suggests that inflammation can alter circulating levels of 84 lipids [4] [5] [6] [7] . Interestingly, COVID19 is associated with a great inflammatory burden and inflammatory 85 markers have been associated with an increased mortality rate [1, 8] . Converseverly, studies in SARS-CoV 86 patients showed long-term metabolic dysregulations [9] . 87

This evidence led us to hypothesize that COVID-19 may disrupt cholesterol homeostasis and mirror 88 the lipid changes observed in other inflammatory conditions. Herein, we conducted a retrospective study 89 involving hospitalized SARS-CoV-2 infected patients to evaluate the relationship between COVID-19 and 90 lipid profiles. For this purpose, we also evaluated previous baseline and follow-up lipid profiles, as well as 91 inflammatory profiles. 92

lymphocyte, and monocyte counts were performed on Sysmex XN-1000® analyzer using the manufacture's 121 reagents (Sysmex Corporation, Japan). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), 122 lactate dehydrogenase (LDH), LDL-c, HDL-c, TC, and TG were tested on Roche Cobas 8100 sampling 123 system analyzer (Module Cobas® c 702, Roche Diagnostics, Switzerland) using manufacture's reagents. 124

The methodology for direct LDL-c, HDL-c, and TC methods are based on a standard homogeneous 125 enzymatic colorimetric assay. We tested interleukin 6 (IL6) on IMMULITE® 2000 immunoassay system 126 using the manufacture´s luminescent immunoassay reagents (IMMULITE® 2000 IL6, Siemens Healthcare 127 Diagnostic, Germany). Procalcitonin (PCT) measurement in plasma was performed by 128 electrochemiluminescence immunoassay on a chemistry analyzer (Cobas 6000, Roche Diagnostics) limit 129 of detection, as below 0.02 ng/ml. We measured serum C-reactive protein (CRP) by particle enhanced 130 immunoturbidimetric (e501 Module Analyser, Roche Diagnostics). The limit of detection was set below 131 0.3 mg/L. 132

We reported categorical variables as absolute values and percentages. Continuous variables were 134 reported as median (interquartile range [IQR] ). The normal distribution of continuous variables was verified 135 with the Kolmogorov-Smirnov test and q-q plot. Categorical variables were compared with the chi-square 136 test and the Fisher exact test when necessary. We compared continuous variables with the Mann-Whitney 137 U test. A Spearman test was performed to analyze the correlation between lipid variables with the rest of 138 the serum markers. We assessed the accuracy of analyzed variables to identify non-survivors patients by 139 using the area under the receiver operating characteristic curve analysis (AUROC). We determined the 140 optimal operating point (OOP) in the AUROC as the one that equaled sensitivity and specificity regarding 141 mortality, and we used it as the cut-off point in the lipid profiles. We analyzed time to 30-day mortality by day mortality in the global study population. Proportional hazard assumptions were verified by Shoenfeld 145 residual test and check using the log(-log(survival)) plots. Sensitivity subgroup analyses were performed to 146 determine possible differences in LDL-c levels as markers of poor prognosis by age, sex, and plasma lipids. 147

We performed the statistical analyses with the use of R software, version 3.6.1 (R Project for Statistical 148

Computing) and IBM SPSS Statistics, Version 25.0. Armonk, NY: IBM Corp. Differences were statistically 149 significant when the p-value was < 0.05. 150

The main baseline and clinical characteristics at admission are listed in Table 1 The results of changes in the concentration of lipid and inflammatory markers over time are 182 summarized in Figure 1 and Table 2 

A Spearman correlation analysis assessed the relationship of lipid parameters with all the gathered 187 analytical parameters. Interestingly, LDL-c and TC levels at admission were inversely correlated with the 188 levels of CRP (r= -0.217 and -0.209, respectively; p<0.001), PCT (r= -0.313 and -0.229; p <0.001) and IL6 and LDL-c (R=-0.273; p<0.001) with age at the time of admission. 194

We analyzed the diagnostic performance accuracy of lipid profiles to predict 30-day mortality using 196 the area under the receiver operating characteristic curve analysis (AUROC). The best estimated threshold 197 values for LDL-c and TC were those calculated by the optimal operating point (OOP) in the AUROC as 198 the one that equaled sensitivity and specificity regarding mortality. These cut offs were 69 mg/dl on 199 admission and 75 mg/dl in the 7 th day of hospitalization for LDL-c. For TC estimated cut-off were 132 200 mg/dL and 147 mg/dL (see Figure S1 ). Threshold values for LDL-c and TC were also calculated from the 201 quartiles, but had a worse balance between sensitivity and specificity, so they were not selected for 202 multivariate analysis. 203

Independent predictors of mortality were estimated through a Cox uni-and multivariate regression 205 analysis. The variables included in the multivariate model were those with a p-value <0.05 on the univariate 206 analysis (age, hypertension, diabetes, dyslipidemia, ischemic heart disease, chronic renal disease, 207 angiotensin receptor antagonist, angiotensin-converting enzyme inhibitors, statins, lymphopenia, CRP, 208 antiviral treatment, anticoagulation, total cholesterol, and LDL-c However, statins were not independently associated with mortality. A sensitivity subgroup analysis was 214 performed to identify potential differences at the time of admission for LDL-c (See Figure S2) . Overall, <75mg / dL determined on the 7th day of admission, were the only variables associated with 30-day 218

The unadjusted survival Kaplan-Meier curves for 30-day global mortality were performed and 220 shown in Figure 2 . Those patients with LDL-c levels <69 mg/dl at the time of admission and <75 mg/dl on 221 the 7th day showed a 20% higher 30-day mortality rate than the rest of the patients. On the other hand, it 222 was observed that the lower the LDL-c levels, the higher the mortality on day 30, represented by Kaplan-223

Meier curves (Suppl. Figure 3) . The mechanism underlying the observed altered cholesterol homeostasis is likely multifactorial and 233 complex. Serum ALT, AST, and LDH levels were moderately increased in non-surviving patients, 234

indicating mild liver-function impairment, which could be a contributing factor by disrupting uptake and 235 biosynthesis of lipoproteins [12] . Nonetheless, a specific type of viral infections can lead to alteration of 236 lipid metabolism in the acute and chronic phases as a response to an ongoing inflammatory state [6, 7] . 237

Although COVID-19 pathophysiology is not fully understood, COVID-19 severity and death are 238 associated with a hyperinflammatory state due to a dysregulated immune system [8, 13] . The clinical profile 239 of the patients included in this study shows a similar trend, with systemic inflammation being a major 240 contributor to mortality, but also SARS-CoV-2-mediated dyslipidemia. We observed a paradoxical lipid 241 metabolism with a U-shaped curve of lipid levels among survivors with similar findings previously described in inflammatory diseases [14] . There are a number of possible explanations from an 243 immunological point of view. 244 HDL-c might become pro-inflammatory under specific conditions that increase reactive oxygen 245 species and myeloperoxidase activity. It can also modify their levels and Apoprotein-AI concentration; 246 hence, altered reverse cholesterol transport [4, 15] . LDL-c can be oxidized when its HDL-c counterpart loses 247 its antioxidative properties, or if oxidized phospholipids accumulate. They are identified as damaged-248 associated molecular patterns (DAMPs) by scavenger receptors, activate the inflammasome [4] and the 249 immune system [13] . Low LDL-c levels may also be the consequence of an increased vascular leakage in 250 the lung parenchyma as a result of endothelial damage [12, 13] . 251

Finally, an increased concentration of pro-inflammatory cytokines may be responsible for a drastic 252 decrease in plasma LDL-c levels during the acute-phase response. Direct effects of cytokines might explain 253 the altered lipid concentrations [14] by up-regulating ox-LDL uptake or overriding suppression of LDL-c 254 receptor through the expression of scavenger receptors. These changes observed with inflammation can 255 increase the odds of cardiovascular disease through the formation of foam cells and endothelial damage 256 [15, 16] . 257

Sepsis is defined as the presence of infection with a detrimental host response with organ damage 258 [17] . Low HDL-c levels have been associated before with an increased risk of sepsis [18, 19] and adverse 259 outcomes [20] [21] [22] [23] . In fact, Maile et al. [24] or Guirgis et al. [25] also suggested that low baseline LDL-c 260 levels are associated with an increased risk of mortality and sepsis, respectively. By analogy, similar 261 findings should be identified in SARS-CoV-2 patients. 262

In particular, low HDL-c levels in SARS-CoV-2 patients have been associated with disease severity 263 [26, 27] , but our results are in agreement with those recently published in which low LDL-c levels were 264 associated with COVID-19 severity [12, 28] . However, we also observed an association with an increased patients, without a contributing role to mortality [29] . 268

The observed associated mortality in this cohort of COVID-19 patients may be explained by other 269 mechanism. In this sense, LDL-c transports a large percentage of plasma Coenzyme Q10 (CoQ10), which 270 has a significant antioxidant capacity, avoiding peroxidative damage to the cellular membranes [30, 31] . 271

Low LDL-c levels can cause a decrease in plasma CoQ10 levels, which can lead to endothelial dysfunction, 272 organ damage and death, as observed in COVID-19 patients [32] . Furthermore, the incidence of severe 273 COVID-19 among elderly has been the greatest. Aging is associated with increased circulating levels of 274 ox-LDL; thus, it could trigger a vicious cycle due to higher basal levels [33] . All these mechanisms justify 275 that patients with low LDL-c levels have a reduced defensive, energetic and metabolic reaction capacities 276 to be able to properly manage a situation of aggression and organ stress such as COVID- 19. 277 Overall, low LDL-c levels may reflect a pro-inflammatory phenotype of severe SARS-CoV-2 278

infection, but they may also induce multiple systemic reactions through a complex interplay. Therefore, in 279 the appropriate scenario, we might hypothetically consider low LDL-c levels as a plausible candidate as a 280 routine risk marker during admission and disease progression. Nevertheless, we did not explore role of 281 statins given the lack of association with mortality despite their pleiotropic properties [34] . Additionally, 282

we cannot rule out a catabolic state or high immune cell turnover as a cause of low LDL-c levels in COVID-283 19 patients. The presence in our study of a statistically significant positive correlation between LDL-c levels 284

and blood lymphocyte count, the latter being an independent variable associated with 30-day mortality 285 together with LDL-c in multivariate regression analysis, can support this theory. 286

Our work presents certain limitations. These observations should be considered hypothesis-287 generating only due to the intrinsic retrospective nature of the present work. Moreover, the data were subject 288 to selection bias, and the generalizability of the results may be reduced by the fact that we did not evaluate 289 outpatients. We could not measure apoproteins or oxidized forms of main lipoproteins, which may play a J o u r n a l P r e -p r o o f

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