These are some thoughts addressing whether Cu(I)NA₂  could be used as a medical food to treat various infections in addition to COVID-19 that is making the news as of this posting.   This “proper copper thought was inspired by the study of Qin(2020) study that showed an increase in the neturophil to lymphocyte ratio in severe cases of COVID-19.   Early studies of  Cu(I)NA₂  show that it decreases neutrophils and increases lymphocytes in humans and fish.  The ratio of neutrophils to lymphocytes has gained popularity as a prognostic marker in infectious diseases.   Don’t we need both?  The following is a Devil’s Advocate examination of copper and its deficiency in sepsis.  Your comments are invited.

Does the neutrophil to lymphocyte ratio matter?

Dr Lagunas-Ramal (2020) has called for use of the neutrophil to lymphocyte ratio as a metric.   of hyper-inflammation to predict which cases of COVID-19 are likely to progress to becoming severe.  Most hospitals in places like Mexico have the instrumentation to perform such tests.  Cu(I)NA₂ has been shown to decrease neutrophils and increase lymphocytes in a human clinical trial and in two fish studies. Neutrophils are not only the first line of defense of the innate immune system but also a source of reactive oxygen species (Kircher 2012).

Neutrophils have gotten a bad reputation in the prognostic value of the neutrophil to lyphoxyte ratio. Kirchner and coworkers (2012) demonstrated that NADPH oxidase generated superoxide and myeloperoxidase generated hypochlous (bleach) are involved in production of the neutrophil extracellular trap (NET). Myeloperoxidase produces hypochlorous acid from the Cl^– anion and hydrogen peroxided, H₂O₂. NET is composed of DNA and a variety of antimicrobial proteins.

As of 15 April 2020 (Velavan review) low lymphocyte counts were still considered to be a predictor of severe COVID-19 cases.  T- and B-cell differentiation factor  interleukin 6 (IL-6), C-reactive protein (CRP), and fimbrin degradation product D-dimer were added to the list of predictors of severe COVID-19.

Do NETs catch viruses?

Do NETs catch viruses? We do not currently have information on the Corona virus family but have the influenza study of Tang (2019).

These authors started with 720 patients who met the World Health Organization’s criteria for influenza. Influenza cases were confirmed with PCR. Messenger RNA was extracted from whole blood from confirmed cases in order to determine which mRNA best differentiate severe cases that require a ventilator versus those that do not. Healthy controls were also added to the analysis.

• Each mRNA message was identified and quantitated with microarray analysis.
• Messages were grouped according to protein function in the disease process.
• Of the six modules with predictive power, the “neutrophil module” had the best ability to differentiate severe from moderate cases of influenza.
• The NET protein myeloperoxidase was one of the top proteins expressed more in severe cases of influenza compared to mild cases.

Ni and coworkers (2019) found that septic patients admitted to the hospital with a high neutrophil to lymphocyte ratio were less likely to die in the hospital. Similar use of the NLR has yet to take hold in the literature as a prognostic test for yeast pathogens like Candida species. A recent review (Patricio 2019) has compared differences in the immune response to Candida which may involve
outer layer of mannose rich N-linked glyco proteins
an inner layer of chitin
activation of host NADPH oxidase, an iron containing generator of supreoxide
activation of NET along with enzymes that attack the fungal pathogen

The Patricio review (2019) goes into great detail of the ways in which the innate immune and adaptive immune systems communicate with one another by way of cytokines. These authors argue that if the balance shifts to the innate immune system, persistent inflammation-immune suppression, catabolism syndrome (PICS) can occur. Compensatory anti-inflammatory response syndrome (CARS) occurs when anti-inflammatory cytokines, some of which are secreted by T and B lymphocytes, turn off the immune reaction prematurely. These authors suggested the value of prognostic tests. We need innate and adaptive arms our immune systems. From early studies we know that neutrophils need copper. These early studies supplied copper in the Cu(II) oxidation state. We are proposing that copper in the Cu(I) oxidation state may bring better balance.

and COVID-19 in particular?

A review published in April of 2020 (Barnes) reported finding evidence of NET deposits in a lung biopsy of a deceased COVID-19 patient.  This prompted the authors to review the literature.  The authors made some interesting points.

• Histones in NET also bind to phospholipids in platelets triggering the clotting cascade by activating platelets.  This might explain blot clots and strokes seen in younger COVID-19 patients.
• Barnes (2020) proposed a loop that involves NET  ⇒ IL1β⇒accelerated
• Barnes (2020) made brief mention of neutrophil elastase, a protease that degrades the extracellular matrix.   This damage may be repaired by lysyl oxidase (LOX),a copper containing amine oxidase that cross links extracellular matrix proteins elastin and collagen.

Another very recent study from Yu Zuo and collaborators (2020) found myeloperoxidase-DNA complexes and citrinullated histones in the sera of COVID-19 patients.   NETs were found to be associated with COVID-19 cases requiring mechanical ventilation. Further correlation was found with NET components cell-free DNA- myeloperoxidase.    COVID-19 sera trigger control neutrophils to release NETs.

Early Studies: Neutrophil and Macrophage activity in Cu deficiency

Jones and Suttle (1981) compared the ability of leukocytes ( presumably macrophage and neutrophils) from copper deficient (< 8 µM in plasma ) and copper sufficient (> 8 µM in plasma ) ewes and lambs to kill the yeast pathogen Candida albicans. Copper deficiency in ewes and lambs decreased the candidacidal activity in ewes and lambs. Copper deficiency further resulted in a decrease in superoxide dismuase activity in erythrocytes and leukocytes.

Babu and Failla (1990) studied the affect of dietary copper on macrophage and neutrophil activity in weaned male rats for a period of five weeks.

• adequate (7 mg/kg diet; +Cu)
• deficient (0.7 mg/kg diet; -Cu)

The following were found to decrease as a result of five week copper deficiency

• Cellular Cu concentration
• activity of Cu,Zn superoxide dismutase (Cu,Zn-SOD),
• neutrophil generation of superoxide anion (O₂^–) in response to opsonized zymosan, phorbol myristate acetate
• candidacidal activity

While phagocytic activity was independent of copper, survival of Candida albicans injected into the peritoneal cavity was greater in Cu-deficient rats than in controls. The authors tested a gradient of dietary copper to differentiate between the double edge sword of superoxide to kill the yeast and SOD to prevent the super oxide from killing the rat host. Candidacidal activity were significantly lower in neutrophils from rats fed diets with less than or equal to 2.7 mg Cu/kg feed compared to control cells.
Reduced erythrocyte Cu,Zn-SOD activity was observed only when dietary Cu was less than or equal to 2.0 mg/kg feed. A follow up study (Balla 1994) noted an increase in T cell proliferation in male mice on copper sufficient versus copper deficient diets in response to a mitogen that seems to operate via an Interleukin pathway.

Of the Cu carrier protein and neutrophils

and coworkers were interested in the role of Cu(I) bound ceruloplasmin protects neutrophils from apoptosis, programmed cell death, as a means of ending the acute phase of a neturophil mediated immune reaction. Several lines of evidence led them to this interest

• Ceruloplasmin (CP) is a Cu and Fe carrier protein P is a ferro:O2-oxidoreductase.
• CP is found in the blood plasma as well as interstitial fluid.
• CP concentration increases 4x in response to inflammation.
• CP is an anionic protein that interacts with and inhibits cationic proteins of the NET such as lactoferrin and myeloperoxidase.

The authors were primarily interested in an in vitro model of chronic inflammation induced by the cytokine TNF-α . They compared Cu replete with holo CP as well as some proteolytic fragments. The Cu replete versus apo (copper deficient) are the most relevant for Cu(I)NA2 as a medical food for sepsis.

Some findings of this study are

• Cu CP, but not apo CP, reduced intracellular super oxide generation by neutrophils.
• This was observed n the presence and absence of TNFα.
• Intracellular reactive species, that include H2O2, were increased by Cu(I) CP but not by apo CP.
• Intact, Cu CP promoted neutrophil survival.

The observation that superoxide decreased and H₂O₂ increased suggested that intracellular Cu/Zn SOD1 might have been Cu deficient.

And there are more Cu-ceruloplasmin interactions…

Kostevich (2015) demonstrated that Cu is necessary for CP binding to macrophage migration inhibitory factor (MIF). MIF is considered pro-inflammatory. Increased MIF levels are correlated with low survival in sepsis patients.  This is not the end of the list.  If Cu(I)NA₂ is to be used as a medical food in patients with sepsis, perhaps a good index of dosing would be measuring Cu content of ceruloplasmin in the patients serum.  Neutropils are good first responders.  They just need to be kept in check with copper containing enzymes so that second ponderers can take over.

References

Babu U, Failla ML. Respiratory burst and candidacidal activity of peritoneal macrophages are impaired in copper-deficient rats. J Nutr. (1990) 120:1692–9. 10.1093/jn/120.12.1692

Babu U, Failla ML. (1990) Copper status and function of neutrophils are reversibly depressed in marginally and severely copper-deficient rats. J Nutr. 120:1700–9. 10.1093/jn/120.12.1700

Bala S, Failla ML, Lunney J. (1990) Alterations in Splenic Lymphoid Cell Subsets and Activation Antigens in Copper-Deficient Rats. Ann N Y Acad Sci 1990;587:283–5. Link

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Cintia PG, Leonardo M, Israel OR, Andrea S, Beatriz VL, Elena DM.(2016) Superoxide Dismutase Activity, Hydrogen Peroxide Steady-State Concentration, and Bactericidal and Phagocytic Activities Against Moraxella bovis, in Neutrophils Isolated from Copper-Deficient Bovines. Biol Trace Elem Res. 171(1):94-100.

Golenkina EA, Viryasova GM, Galkina SI, Gaponova TV, Sud’ina GF, Sokolov AV. (2018) Fine Regulation of Neutrophil Oxidative Status and Apoptosis by Ceruloplasmin and Its Derivatives. Cells. 2018 Jan 12;7(1). pii: E8 Link

Jones DG, Suttle NF.(1981) Some effects of copper deficiency on leucocyte function in sheep and cattle. Res Vet Sci.31(2):151-6.

Kamiya T, Takeuchi K, Fukudome S, Hara H, Adachi T. (2018) Copper chaperone antioxidant-1, Atox-1, is involved in the induction of SOD3 in THP-1 cells. Biometals.31(1):61-68.

Kirchner T, Möller S, Klinger M, Solbach W, Laskay T, Behnen M. (2012) The impact of various reactive oxygen species on the formation of neutrophil extracellular traps. Mediators Inflamm. 2012;2012:849136

Kostevich VA, Sokolov AV, Grudinina NA, Zakharova ET, Samygina VR, Vasilyev VB.(2015)Interaction of macrophage migration inhibitory factor with ceruloplasmin: role of labile copper ions. Biometals. 28(5):817-26

Lagunas-Rangel FA. (2020) Neutrophil-to-Lymphocyte ratio and Lymphocyte-to-C-reactive protein ratio in patients with severe coronavirus disease 2019 (COVID-19): A meta-analysis J Med Virol. 2020 Apr 3. doi: 10.1002/jmv.25819. [Epub ahead of print]

Ni J, Wang H, Li Y, Shu Y, Liu Y. (2019) Neutrophil to lymphocyte ratio (NLR) as a prognostic marker for in-hospital mortality of patients with sepsis: A secondary analysis based on a single-center, retrospective, cohort study. Medicine (Baltimore). 98(46):e18029. Link

Patricio P, Paiva JA, Borrego LM. (2019) Immune Response in Bacterial and Candida Sepsis. Eur J Microbiol Immunol (Bp). 9(4):105-113. Link

Tang BM, Shojaei M, Teoh S, Meyers A, Ho J, Ball TB, Keynan Y, Pisipati A, Kumar A, Eisen DP, Lai K, Gillett M, Santram R, Geffers R, Schreiber J, Mozhui K, Huang S, Parnell GP, Nalos M, Holubova M, Chew T, Booth D, Kumar A, McLean A, Schughart K. (2019) Neutrophils-related host factors associated with severe disease and fatality in patients with influenza infection. Nat Commun. 31;10(1):3422. Link

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Qin C, Zhou L, Hu Z, Zhang S, Yang S, Tao Y, Xie C, Ma K, Shang K, Wang W, Tian DS (2020) Dysregulation of immune response in patients with COVID-19 in Wuhan, China.Clin Infect Dis. 2020 Mar 12. pii: ciaa248 Link

Zuo Y, Yalavarthi S, Shi H, Gockman K, Zuo M, Madison JA, Blair CN, Weber A, Barnes BJ, Egeblad M, Woods RJ, Kanthi Y, Knight JS.(2020)Neutrophil extracellular traps in COVID-19. JCI Insight. 2020 Apr 24. pii: 138999 Link