Low Zinc Levels at Admission Associates with Poor Clinical Outcomes in SARS-CoV-2 Infection

Abstract

Background: Zinc is an essential micronutrient that impacts host-pathogen interplay at infection. Zinc balances immune responses, and also has a proven direct antiviral action against some viruses. Importantly, zinc deficiency (ZD) is a common condition in elderly and individuals with chronic diseases, two groups with an increased risk for severe severe coronavirus disease 2019 (COVID-19) outcomes. We hypothesize that serum zinc content (SZC) influences COVID-19 disease progression, and thus might represent a useful biomarker.

Methods: We ran an observational cohort study with 249 COVID-19 patients admitted in Hospital del Mar. We have studied COVID-19 severity and progression attending to SZC at admission. In parallel, we have studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) replication in the Vero E6 cell line modifying zinc concentrations.

Findings: Our study demonstrates a correlation between serum zinc levels and COVID-19 outcome. Serum zinc levels lower than 50 µg/dL at admission correlated with worse clinical presentation, longer time to reach stability, and higher mortality. Our in vitro results indicate that low zinc levels favor viral expansion in SARS-CoV-2 infected cells.

Interpretation: Low SZC is a risk factor that determines COVID-19 outcome. We encourage performing randomized clinical trials to study zinc supplementation as potential prophylaxis and treatment with people at risk of zinc deficiency.

Keywords: SARS-CoV-2; clinical outcomes; zinc.

Figure 1

Correlation between serum zinc levels at admission, inflammatory markers, and clinical outcome. (A) Zinc and IL-6 at admission. (B) Zinc at admission and highest value of IL-6 during the episode. (C) Zinc and C-reactive protein at admission. (D) Serum zinc content at admission and time to clinical stability.

Figure 2

Evaluation of zinc homeostasis in SARS-CoV-2 infection. (A,B). Intracellular zinc content measurement using FluoZin-3AM and Zinquin probes in Vero E6 cells. (A). Flow cytometry in cells incubated for 30 min with 0, 10, and 50 µM extracellular Zn²⁺ content. Intensity expressed in arbitrary units (AU) (n = 3; * p < 0.05, *** p < 0.001; Bonferroni-corrected one-way ANOVA compared to 0 Zn²⁺). (B). Confocal images of living cells incubated with FluoZin-3AM (up) or Zinquin (bottom) and Lysotyracker in 50 µM Zn²⁺ extracellular medium. Scale bar = 10 µm. (C). Viability MTT assay in cells incubated with 0, 10, and 50 µM extracellular Zn²⁺ content for 48 h. Data expressed in percentage compared to control condition (10 µM Zn²⁺) (n = 12; Bonferroni-corrected one-way ANOVA compared to 10 µM Zn²⁺). (D). Quantification in supernatant of viral RNA copies by qPCR in cells infected with SARS-CoV-2 and collected at 48 h. Data expressed in percentage compared to control condition (10 µM Zn²⁺) (n = 3; ** p < 0.01; Bonferroni ANOVA compared to 10 µM Zn²⁺).

Figure 3

Evaluation of zinc potentiation of chloroquine antiviral action. (A) Viability MTT assay in cells incubated with 10 µM chloroquine (CQ) in 0, 10, and 50 µM Zn²⁺ content for 48 h (n = 15; Bonferroni-corrected one-way ANOVA compared to 10 µM Zn²⁺). (B) Quantification in the supernatant of viral RNA copies by qPCR in cells infected with SARS-CoV-2 treated with 10 µM CQ in 0, 10, and 50 µM Zn²⁺, and collected at 48 h. Data expressed in percentage compared to control condition (10 µM Zn²⁺ without CQ, Figure 2D) (n = 3; Bonferroni-corrected one-way ANOVA compared to 10 µM CQ in 10 µM Zn²⁺). (C) Flow cytometry in cells incubated for 30 min with 0, 10, and 50 µM extracellular Zn²⁺ content using FluoZin-3AM and Zinquin probes. Intensity expressed in arbitrary units (AU) (n ≥ 4; *** p < 0.001; Bonferroni-corrected one-way ANOVA compared to 0 µM Zn²⁺ from each probe). (D–F) Western blot analysis from cells incubated for 24 h in 0, 10, and 50 µM Zn²⁺ with or without 10 µM CQ. Antibodies against microtubule-associated proteins 1A/1B light chain 3B (LC3), p62, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (D) Representative blot. (E,F) Quantification of the bands for LC3 ((E) n ≥ 10) and p62 ((F) n ≥ 7) (Bonferroni-corrected one-way ANOVA compared to 10 µM Zn²⁺ or 10 µM CQ in 10 µM Zn²⁺; ** p < 0.01, *** p < 0.001 unpaired t-test comparing conditions with and without CQ).