Can Zinc correction in SARS-CoV-2 patients improve treatment outcomes?

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic for which there is no established treatment available yet, has caused more than 68,000 deaths so far. Following the SARS-CoV outbreak in 2003, an Italian group described a hypothesis about the efficacy of two old drugs: Chloroquine (CQ) and Hydroxychloroquine (HCQ), against SARS-CoV and its future emergents. Later, this hypothesis was shown to be relevant in-vitro. Due to the high genetic similarity of SARS-CoV-2 and SARS-CoV, the hypothesis introduced by Savarino et al. and the further supportive in-vitro evidence served a rational ground for three different Chinese groups to test the efficacy of CQ or HCQ against SARS-CoV-2 in-vitro. These studies showed promising in-vitro efficacy of CQ and HCQ against SARS-CoV-2. Unfortunately, in the absence of sufficient clinical data on the (in)efficacy of CQ and HCQ in SARS-CoV-2 patients, the compassionate and off-label use of these medications is becoming politicized. Herein, we underline some critical features of the CQ/HCQ mechanism of action concerning SARS-CoV-2. Moreover, we put forward a hypothesis based on three lines of evidence on a probable link between zinc-deficiency/zinc correction and response to CQ/HCQ- and possibly other SARS-CoV-2 treatments.

Both CQ and HCQ are reported with such an effect, but Zhou et al. recommend HCQ over CQ for SARS-CoV-2 treatment due to a better safety profile 14,16,17 .
Interestingly, Zhou et al. have also highlighted another mechanism by which HCQ/CQ interferes with coronavirus binding to the host cells, a feature that can contribute to their anti-SARS-CoV-2 effects 14 .

Hypothesis: Zinc correction in SARS-CoV-2 patients improves HCQ/CQ and other treatments outcomes
Since the discovery of the first reported case with zinc-deficiency in Iran 24 by Prasad et al. in 1961, we have learned a lot about Zinc, and we have much more left to learn.
Zinc is the second most abundant common trace mineral in the human body, with vital biological functions from cell growth and development to cell homeostasis and immune response 25,26 .
Up to a fifth of the global population is estimated to suffer from different degrees of Zinc deficiency 27 . In the western world, Zinc deficiency is more prevalent among the geriatric population 26 and vegans/vegetarians as well as among people with certain underlying conditions 27 .
Notably, the early reports show that the elderly SARS-CoV-2 patients are among those with a higher fatality rate 28 .
Herein, we summarize three lines of evidence that underscore the significance of Zinc and a possible link with the response to any anti-SARS-CoV-2 treatment, including CQ/HCQ therapy (figure 1).

Zinc deficiency hampers antiviral and antibacterial immune response
Zinc deficiency has been associated with increased risk of infectious diarrhea and pneumonia among children, while Zinc supplements were shown to have a corrective impact on both complications 29 .
Moreover, zinc deficiency, whether mild or severe, can negatively impact different adult human organs, including the immune system 25,26,30 .
Zinc deficiency is associated with increased risk of developing Staphylococcus aureus pneumonia and streptococcus pneumonia tonsillitis infections 30 among the elderly population.
This risk can reduce, upon Zinc correction in the geriatric population 25 . Markedly, both of these respiratory system infections are reported among the fatal co-infections of SARS-CoV-2 31 .
Additionally, zinc is associated with antiviral immune response in general, involving various viruses 27 .
Not surprisingly, yet there is no data available on such a correlation concerning SARS-CoV-2.
It would be essential to learn whether, in particular, zinc-deficient SARS-CoV-2 patients are at higher risk of mortality due to a weak or inefficient cellular and/or humoral immunity in response to SARS-CoV-2.
Further, the potential of combinatorial zinc correction and anti-SARS-CoV-2 treatments such as CQ/HCQ could be clinically explored.

Zinc deficiency is associated with cytokine storm
Zinc-deficiency not only hampers our immune system in terms of lack of efficient response when required 30 , but it can also be associated with an irrelevant and damaging response.
Zinc levels among patients suffering from different auto-immune disorders appear to be lower than the healthy individuals 32 .
Importantly, Zinc deficiency is associated with the excessive and tissue-damaging proinflammatory release of cytokines, including increased tumor necrosis factor-alpha (TNF-α) 33,34 and IL-6 35 .
As previously mentioned, so far, the TNF-α and IL-6 have been reported among the usual suspects responsible for lung-damaging and fatal "Cytokine Strom" in a group of SARS-CoV-2 patients.
Notably, Zinc correction is associated with the down-regulation of tumor necrosis factor-alpha 30,33,34 .
Concerning IL-6, however, the impact of Zinc might be rather qualitative than quantitative. One study reports that zinc deficiency is associated with increased IL-6 gene expression in mice 36 , while another study on a human experimental model reports that zinc deficiency does not decrease IL-6 levels 37 . Nevertheless, it is reported that among the geriatric female population, a gene polymorphism that leads to an increased immune response-mediated release of Zinc is associated with decreased IL-6 levels 38 . On the other hand, zinc supplementation can even lead to the up-modulation of IL-6 39 .
However, it seems to be a consensus that zinc correction inversely affects the IL-6-mediated response, which is, by nature, a pro-inflammatory response [35][36][37]40 .
Interestingly, zinc deficiency is reported to increase the risk of ventilator-induced injury in mice 41 .
Moreover, zinc deficiency is associated with an increased risk of acute respiratory distress syndrome (ARDS) in human 41 .
These associations merit special attention concerning SARS-CoV-2 patients management 15 .
Therefore, we suggest that zinc deficiency should be investigated in a subgroup of SARS-CoV-2 patients at risk for developing hyperinflammatory complications 15 .
Additionally, we suggest that zinc correction might cooperate with HCQ in reducing the risk of SARS-CoV-2-related tissue-damaging cytokine storm, and we believe that such a probable synergy worth clinical exploration.
This large piece of the evidence inspired Velthuis et al. . 50 to test a similar possibility in two RNA viruses, including SARS-CoV.
The group discovered that both a candidate zinc ionophore (pyrithione) and Zinc itself could inhibit SARS-CoV replication as singe agents and better in combination. However and possibly due to low cellular uptake of Zinc, the optimal zinc concentration to efficiently inhibit the SARS-CoV replication was relatively high 50 .
Notably, Velthuis et al. . 50 could show that Zinc can directly disrupt the initiation of RNA synthesis by the SARS-CoV RNA-dependent RNA polymerase (RdRp) in-vitro, possibly via interacting with two zinc-binding pockets present in the SARS-CoV RdRp 50 .
It deserves to be mentioned that a Chinese group who has unraveled the SARS-CoV-2 RdRp genome, could not observe zinc ions being chelated at the two candidate pockets within the RdRp of SARS-CoV-2 51 . The group addresses the discrepancy in their technical approach compared to the precedent works on SARS-CoV 51 .
Thanks to cancer research efforts, we learn a more exciting feature of CQ associated with Zinc. In 2014, a Chinese cancer study by Xue et al. reported that CQ increases zinc uptake in ovarian cancer cells and mediates zinc accumulation into the lysosomes of these cells 52 .
In contrast, a Korean study conducted by Seo et al. . 53  In contrast to the conclusion of Xue et al., Seo et al. . 53 reported that Chloroquine decreases the free zinc levels in lysosomes. However, they still observed some increased intracellular zinc levels upon CQ treatment compared to the control group.
Therefore, we acknowledge that whether CQ/HCQ are global zinc ionophores mediating intracellular uptake of Zinc by cells of different origins, at this stage, should remain an open question and the subject of further investigation.
On the other hand, even if CQ or HCQ does not turn out to be zinc ionophore, it would still be possible that Zinc can exert an anti-SARS replication effect independent of CQ/HCQ. Patients with zinc deficiency would likely be deprived of this additive effect.
If further data suggests that CQ/HCQ are zinc ionophores mediating zinc uptake into the SARS-CoV-2 infected cells, one can postulate combining zinc supplements with CQ/HCQ or at least zinc correction in zinc-deficient patients could be beneficial.
However, if the new data suggest that CQ/HCQ is interfering with zinc uptake into the SARS-CoV-2 infected cells or in an organelle such as lysosomes-in line with findings of Seo et al. combining zinc correction or zinc supplementation with CQ/HCQ might be even highly essential.

Practicality
There is no consensus and widely accepted guideline concerning zinc deficiency diagnosis and correction. Radioisotopes are a decent diagnostic tool, but they are costly. New biomarkers such as Linoleic Acid are emerging [54][55][56][57] . However, considering the vast population involved with SARS-CoV-2 and the high economic Burdon of such approaches, we propose a simple and cheap method to select zinc-deficient SARS-CoV-2 patients and include zinc correction in their treatment package.

Zinc uptake and absorption:
The Recommended Dietary Allowance (RDA) of zinc intake is 11mg and 8mg/day for adult males and females, respectively 58 .
According to an analysis of the National Health and Nutrition Examination Survey (NAHNES III), 35%-45% of adults above 60 years old have daily zinc intake below the recommended average 59 .
The zinc transport protein family members that are apically positioned on the cell surface mediate zinc uptake. Foods with a good source of Zinc include meat, fish, shellfish, legumes, nuts, seeds, eggs, and whole-grain cereals 60 . Phytate, the hexaphosphate ester of inositol, due to its high polarity, strongly binds to divalent Zinc, thereby preventing its absorption 61,62 . Due to phytate-rich content, the zinc-bioavailability of vegan/vegetarian diets are less compared to omnivore diets 62 .
Nevertheless, unrefined phytate containing food such as whole-grain bread, despite higher phytate content, has higher zinc-bioavailability compared to refined ones like white bread, which have poor zinc content 62 .

Evaluation of zinc status: Serum or plasma Zinc
Plasma and serum zinc concentration are the most frequently used biomarkers to establish zinc status 63 . Clinical effects of zinc deficiency can be existing despite normal zinc plasma concentration 63 . Conditions that lead to hypoalbuminemia also reduce plasma zinc concentrations, as Zinc is bound to the albumin in the plasma 64 . Therefore, it is essential to include albumin along with plasma zinc test and consider albumin-normalized zinc values to determine the zinc cut-off 65 .
Infections, fever, contraceptives, and pregnancy lower the plasma zinc while starvation and catabolism increase it 63 .
In particular and concerning SARS-CoV-2 patients, the status of two widely used biomarkers of inflammation 31,66 ; c-reactive protein and procalcitonin, is linked to zinc status.
Interestingly, both these markers are inversely associated with serum zinc levels 67,68 , other evidence that zinc might have anti-hyper inflammatory effects in SARS-CoV-2 patients.
The interpretation of the concentration of plasma zinc must, therefore, take into account all the confounding factors 69 . Plasma zinc concentrations dose-dependently respond to supplementation in patients with a low or moderate baseline, independent of gender and age 69 .
On the other hand, Zinc deprivation results in a reduction in the plasma zinc concentrations.
The fact that plasma zinc concentrations are normally-distributed among healthy populations makes it useful as a zinc-deficiency marker 63 .
Frank T. et al. concluded that as plasma and urinary zinc concentrations are probably more precise indicators currently available, as opposed to indirect ones such as stunting, anemia, or iron deficiency. They suggest that direct indicators can be used to estimate the prevalence of zinc deficiency in populations 57 .

Cutoffs:
A revisit of the NHANES II data suggests cutoffs of serum zinc concentrations for assessing the fasting zinc status at 70 and 74 μg/dL for females and males over ten years old, respectively 70 .

Treatment:
Long term zinc supplementation should be very well-weighed 71 as the therapeutic window of chronic zinc supplementation can relatively narrow.
In contrast, short term zinc supplementation may be less of a concern 26 . The proposed dose of zinc (Znso4) injection for parenteral nutrition in metabolically stable adult patients is 3 mg/day 72 .
The maximum level of the excipient used in FDA-approved oral zinc tablets (excluding extended-release forms) is 25 mg 73 .
The recommended oral daily dose for zinc correction in adults is 10-30 mg/day 26,74 .
Various forms of zinc supplements exist, including zinc gluconate, zinc citrate, zinc sulfate, and zinc acetate. Rita Wegmüller et al. study demonstrated that zinc citrate could be as active as zinc gluconate in the prevention of zinc deficiency.
The high zinc content of zinc citrate, acceptable quality perception for the patients, the comparable bioavailability, and its low cost 75 may make it a relevant form to be considered for zinc correction in zinc-deficient SARS-CoV-2 patients.

Conclusion
Taken all together, we believe there is a large piece of evidence that links antiviral response, zinc deficiency, and in particular, HC/HCQ therapy. At this stage, this evidence is far from being conclusive. However, such an indispensable and intriguing hint in the precedent literature and azithromycin in 80 COVID-19 patients with at least a six-day follow up: an observational study. 8,.