key: cord-0987581-1qs6sj3g
authors: Arrieta, Francisco; Martinez-Vaello, Victoria; Bengoa, Nuria; Jiménez-Mendiguchia, Lucía; Rosillo, Marta; de Pablo, Angélica; Voguel, Cristina; Martinez-Barros, Hilario; Pintor, Rosario; Belanger-Quintana, Amaya; Mateo, Raquel; Candela, Angel; Botella-Carretero, José I.
title: Serum zinc and copper in patients with COVID-19 and zinc supplementation in parenteral nutrition
date: 2021-08-31
journal: Nutrition
DOI: 10.1016/j.nut.2021.111467
sha: 2a56a4d1e49de9ad7a2cf73a8ba279aedec31254
doc_id: 987581
cord_uid: 1qs6sj3g

Objective : Zinc and copper are important to protect cells from oxidative stress and to enhance immunity. An association between low zinc levels and the severity of acute respiratory distress syndrome has been shown for patients with COVID-19. We aimed to study serum zinc and copper concentrations in severe COVID-19 patients and its supplementation in parenteral nutrition (PN). Methods : Thirty-five COVID-19 patients in need for PN were studied in a retrospective design. Serum samples were collected at three time points: at the start of PN, between three and seven days after, and at the end of PN. Results : Patients were on PN for a mean of 14 days, with a mean daily supplemental zinc of 14.8±3.7 mg/day. Serum zinc increased during PN administration from 98.8±22.8 to 114.1±23.3 µg/dL (Wilk´s λ=0.751, F=5.459, P=0.009). Conversely, serum copper did not vary from baseline (107.9±34.2 µg/dL) to the end of the study (104.5±37.4 µg/dL, Wilk´s λ=0.919, F=1.453, P=0.248). Serum zinc within the first week after starting PN and at the end of PN inversely correlated with total hospital stay (r=-0.413, P=0.014 and r=-0.386, P=0.022, respectively). Patients in critical condition presented lower serum copper (z=2.615, P=0.007). Mortality was not associated with supplemental zinc or with serum zinc or copper concentrations at any time of the study (P>0.1 for all analyses). Conclusions : Serum zinc concentrations during PN support were inversely associated with length of hospital stay but not with mortality. Serum copper concentrations were lower in patients in critical condition but not associated with prognosis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the coronavirus disease 2019 (COVID- 19) pandemic, which has affected more than one hundred seventy million individuals and is the cause of almost four million deaths worldwide as of this writing [1] . An important proportion of COVID-19 patients may present severe pneumonia requiring hospital admission and some to an intensive care unit (ICU). In these patients an excessive release of inflammatory cytokines, activation of pro-coagulating factors, and increased oxidative stress may occur [2] . The disease can then progress to acute respiratory distress syndrome and other multiple organ failures such as acute cardiac, liver or kidney injury [3, 4] . In this situation, artificial nutrition may be needed to avoid further malnutrition either as parenteral nutrition (PN) or as enteral nutrition (EN) [5, 6] .

Apart from macronutrients, several micronutrients are usual components of artificial nutrition based on average requirements. Among these, some trace elements such as zinc and copper are important to protect cells from oxidative stress especially in critical care patients [7] . Critically ill patients develop severe stress and a state that may raise the utilization and metabolic replacement of some of these trace elements, especially zinc, depleting their body reserves [8] . In addition, trace element-dependent enzymes are of great importance in the antioxidant defense mechanisms to protect cells from reactive oxygen species [9] . Accordingly, low serum levels of zinc and copper were observed at ICU admission and those with the higher serum concentrations had significantly lower mortality [10, 11] .

Recently, an association between low zinc levels and the severity of acute respiratory distress syndrome has been shown for patients with severe COVID-19 [12] . Therefore, given this previous knowledge, we aimed to study serum zinc and copper concentrations in severe COVID-19 patients and zinc supplementation in PN, as well as their associations with inflammatory markers and prognosis.

Thirty-five COVID-19 patients in need for PN were studied in a retrospective design.

Inclusion criteria were the following: confirmation of COVID-19 by the presence of SARS-CoV-2 in respiratory specimens by real-time polymerase chain reaction (RT-PCR) in pharyngeal swabs, severe pneumonia requiring high flow oxygen, the need for artificial nutrition in the form of PN, and available serum samples for zinc and copper measurements. The latter were collected at three time points: at the start of PN, between three and seven days after, and at the end of PN. All patients received our standard treatment protocol for COVID-19 including glucocorticoids, low-molecular-weight heparin, and tocilizumab if indicated.

The study protocol was approved by the Institutional Ethics Committee of our center (study code 147/20) and performed according to the Declaration of Helsinki.

Written or verbal informed consent was obtained from all patients.

The type of composition of artificial nutrition administered to the included patients were recorded. PN was delivered through a central line as soon as the patient was hemodynamically stable. Individualized formulae were prepared by the hospital pharmacy and whenever possible, commercial "Ready To Use" (RTU) bags were initially employed (Olimel N9, Baxter Ltd, Illinois, US). Some patients were started on peripheral PN (Periolimel N9, Baxter Ltd, Illinois, US, or Isoplasmal, Braun Medical Inc, Melsungen, Germany).

We aimed at 20-25 kcal/Kg/day, with a proportion of 3-6 g/Kg/day for glucose, 1.0 g/Kg/day for amino-acids and less than 1 g/Kg/day for lipids, with 7-10 g/day of essential fatty acids. Vitamins (Cernevit, Baxter Ltd, Illinois, US) and trace elements (Supliven, Fresenius Kabi, Bad Homburg, Germany) were added by the hospital pharmacy. The latter contains 1.05 mg of zinc chloride per ml. Supplemental zinc was also added to PN bags when needed in the form of zinc sulphate (Oligozinc, 10 mg in 10 ml vials, Fresenius Kabi, Bad Homburg, Germany) to maintain serum zinc in the normal range. Mean daily zinc doses were calculated during PN from all parenteral sources. For those patients with stays above 7 days and whenever possible, EN was started with a standard fiber-free formulation, and PN gradually tapered as EN was tolerated and increased. After PN withdrawal, parenteral zinc supplementation stopped. Therefore, the analysis was restricted to the period of PN administration.

Serum biochemical variables were measured with an Architect c16000/i2000-analyzer (Abbot Diagnostics, UK). Serum copper was analyzed by atomic absorption spectrophotometry (AAnalyst 800, Perkin Elmer, CA, USA). Serum zinc was analyzed by a colorimetric method (Sentinel Diagnostics, Milano, MI, Italy). The normal ranges were 60-150 µg/dL for zinc and 75-150 µg/dL for copper. Immunoanalysis was employed for the measurement of C-reactive protein (CRP), procalcitonin (Abbott, US) and D-dimer (Siemens, Germany), and interleukins 6 (IL-6) and 12 (IL-12) by enzymelinked immunosorbent assay (Invitrogen, US). The intra-and interassay coefficients of variation were below 10%.

Sample size was calculated with the online software package GRANMO 7.12 (https://www.imim.es/ofertadeserveis/software-public/granmo/index.html). In order to find a mean difference of 15 µg/dL in circulating zinc and copper between baseline levels and at the end of follow-up, with a standard deviation (SD) of 25 µg/dL, a minimum sample size of 22 subjects was needed for a bilateral contrast with an α of 0.05 and 1-β of 0.20 and no expected losses.

Results are expressed as means ± SD unless otherwise stated. The Kolmogorov-Smirnov statistic was applied to continuous variables. Logarithmic or square root transformations were used as needed to ensure normal distribution of the variables. To compare discontinuous variables, we used the χ2 test and Fisher's exact test as appropriate. Unpaired t tests or Mann-Whitney U tests were used to compare the central tendencies of the different groups, as appropriate. General linear modelrepeated measures test was used to analyze continues variables measured several times during the study period. Bivariate correlation was employed to study the association between two continuous variables using Pearson or Spearman's tests as appropriate. Analyses were performed using SPSS 18 (SPSS Inc, Chicago, Illinois). P < 0.05 was considered statistically significant.

The baseline characteristics of the 35 included patients are shown in Table 1 . Most of the patients were males, less than half with hypertension and less than a third with obesity or previous type 2 diabetes mellitus. Twenty-seven patients were critical ones admitted to the ICU whereas the other eight patients were in respiratory intermediate care. Patients were on total PN for a mean of fourteen days, with a mean daily supplemental zinc of 14.8 ± 3.7 mg/day (Table 1) . Only one patient had zinc deficiency at baseline according to our hospital reference range.

At follow-up there was an increase in alanine-amino transpherase (ALT), gamma glutamyl transpeptidase (GGT) and D-dimer, whereas serum IL-6, IL-12, total proteins and albumin remained stable (Table 2) 

We have shown in the present study that supplementation of zinc in PN produced an increase in serum zinc concentrations in patients with severe COVID-19 infection.

Serum zinc concentrations during PN support were inversely associated with length of hospital stay but not with mortality. This association was observed in patients both at ICU and at intermediate care. In addition, serum copper concentrations were lower in those patients at ICU but not associated with prognosis.

Zinc is a highly abundant element on earth and it is an essential micronutrient.

After its ingestion and absorption through the small intestine, it is bound predominantly to albumin as well as to other proteins, including prealbumin, α2-macroglobulin, transferrin, ceruloplasmin, haptoglobin, immunoglobulins and complement. Serum circulating zinc accounts for only 0.1% of total body stores [13] . There are two families of proteins that are responsible for the movement of zinc across membranes, which include zinc-importer proteins that transport zinc into the cytosol and the zinc transporter (ZnT) family proteins transporting zinc outside the cytosol [14] . Most of the zinc body content is found in muscle and bone, but many other organs such as the liver, intestine, kidney, skin, lung, brain, heart, and pancreas also contain significant concentrations. Its role within the human body is extensive in reproduction, immune function, wound repair, and, on the microcellular level, macrophage, neutrophil, natural killer cell, and complement activity [15] .

Notably, major risk groups for COVID-19, the elderly, men more than women, obese individuals and patients with diabetes are all at risk of zinc deficiency [16] .

Further, critically ill patients may raise the utilization of zinc, depleting their body reserves [8] and compromising the antioxidant defense mechanisms to protect cells from reactive oxygen species [9] . The alteration of zinc metabolism was more pronounced in septic patients than in non-infected critically ill patients. Specifically, sepsis was associated with lower plasma zinc concentrations [17] . A previous study reported that low serum zinc was very common at the onset of acute respiratory failure in the ICU but had no predictive value for 30-day mortality, ICU length of stay, and time of invasive mechanical ventilation [18] . In another study with pediatric population, plasma zinc correlated with measures of inflammation on day 3 and was associated with the degree of multi-organ failure in the ICU [19] . Therefore, zinc supplementation has been suggested as a novel strategy to influence severe COVID-19 as it may enhance the antiviral immune response [20, 21] . Indeed, much evidence has accumulated over the past 50 years to demonstrate the antiviral activity of zinc against a variety of viruses, and via numerous mechanisms [22] . Although the exact underlying mechanism of zincinduced antiviral response is not well understood, it has been demonstrated that zinc has the potential to inhibit viral binding to the mucosal cells, and eventual replication, possibly by generating antiviral interferon. In addition, it has been proposed that zinc may reduce Sirtuin-1 mediated cell surface angiotensin converting enzyme 2 expression, the binding site for SARS-CoV-2 spike protein [23] .

Our results, although limited by the nature of a small retrospective study, show some interesting data on zinc supplementation in PN and the prognosis of patients with severe COVID-19. Further, some of them are in agreement with recent reported data showing an association between low zinc levels and the severity of acute respiratory distress syndrome in patients with severe COVID-19 [12] . The latter study included 269 patients with severe COVID-19 admitted to an ICU with a mean age of 70y, mostly males and with a median body mass index of 30. They measured serum zinc at admission and found an association of of low zinc levels and severe acute respiratory distress syndrome even after adjusting for baseline variables [12] . Although we did not find an association between prognosis and baseline zinc concentrations, we only identified one patient with zinc deficiency in contrast to the data reported in that previous study that showed a prevalence of low zinc levels of 79.6% [12] . Our study has the advantage of serum zinc measurements at follow-up, and a direct quantification of the parenteral zinc administered with PN. We were able to show a dynamic picture of serum zinc changes and the association of serum zinc levels after PN support with the length of hospital stay.

Copper is a component of the Cu/Zn superoxide dismutase and serves as a free radical scavenger [7] . In relatively small amounts, it is an essential cofactor of a broad array of molecules [24] . Common dietary sources of copper are meat, shellfish, seeds, legumes, nuts, whole grains, potatoes, and chocolate. After intestinal absorption, it circulates mainly bound to serum albumin and is carried to the liver. A small amount of copper is excreted in bile whereas the rest binds to ceruloplasmin -a copper dependent ferroxidase -and is released back into the bloodstream [25] . Increased serum copper concentrations during ICU care were associated with a significantly lower mortality in a recent study [10] . Another previous one showed that copper supplementation produced significantly fewer 30-day infectious episodes and a significantly shorter length of stay in patients with major burns, but a deep analysis showed that the results were not associated with copper levels but with the increased amount of supplemented selenium [26, 27] . To our knowledge, there has been no reports of studies dealing with copper supplementation in COVID-19 patients, although it has been recently proposed as an adjunct therapy in these patients from a theoretical point of view [28] . Ours are then novel data that show an association of lower serum copper concentrations with critically ill COVID-19 patients.

It has been suggested that the altered plasma concentrations of zinc, selenium, and copper in patients with critical illness may be primarily due to the effects of the systemic inflammatory response and may not reliably indicate their status [29] . We did not measure zinc and copper in red blood cells, so this could be a limitation of our study. However, erythrocyte concentrations of trace elements may reflect the sufficiency or deficiency status at baseline but may not be sensitive enough to detect rapid changes as it occurs in critically ill patients [10, 11] .

In conclusion, serum zinc concentrations during PN support were inversely associated with length of hospital stay but not with mortality in severe COVID-19

patients. In addition, serum copper concentrations were lower in those patients at ICU but not associated with prognosis. Therefore, from the clinical experience, prophylactic supplementation of PN with zinc could be considered in these patients. Future clinical trials are needed to unravel the role of zinc and copper supplementation in severe COVID-19 patients.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Restrictions apply to the availability of data generated or analyzed during this study to preserve patient confidentiality or because they were used under license. The corresponding author will on request detail the restrictions and any conditions under which access to some data may be provided.

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