In Vitro Effects of Zinc 2+ Ions
Chapter 2. - In Vitro Effects of Zn2+ Ions

Chapter Executive Summary

Although the exact mechanism by which zinc shortens common colds may never be determined, the Zn2+ ion has multiple, biochemical effects in vitro, almost certainly acting synergistically in common cold therapy. According to Charles A. Pasternak of the St. George's Hospital Medical School at the University of London, hydrated Zn2+ ion at 6 to more than 60 times normal zinc serum concentration has multiple beneficial effects, increasing as extracellular Zn2+ ion concentration increases.(1)

As discussed in detail below, the beneficial effects observed in treating colds with lozenges releasing Zn2+ ions are caused by 1) astringent drying action on all cell membranes including mucus-secreting goblet cells; 2) inhibition of rhinovirus replication above 0.05 mMol Zn2+ ion; 3) immediate protection and stabilization of cell membranes from all cytotoxic agents including cytolysin (perforin), with a strength equal to interferon; 4) induction of interferon-gamma within 24 hours; 5) immediate cell and capillary membrane pore closure and prevention of transcapillary serous leakage, a function which promotes long-distance closed-circuit transport of ions; and 6) immediate inhibition of release of histamine and other vasoactive biochemicals from mast cells and catabolism of histamine. Serum zinc concentration is 0.015 mMol.

Elevated extracellular Zn2+ ion is beneficial and elevated intracellular zinc is cytotoxic, although recently some authors have suggested that slightly elevated extracellular zinc is cytotoxic. In some cells, rounding and refractile changes occur at about 0.1 mMol Zn2+ ion and at much lower concentrations for most lipophilic and strongly chelated zinc complexes. These observations were interpreted as cytotoxicity and suggested a low 1:2 or 1:4 antirhinoviral therapeutic index for zinc.

Researchers highly familiar with the unique appearance of cell plasma membranes after the astringent action of Zn2+ ions, place the index at 10 to 100 times higher; as extracellular Zn2+ ion is a newly recognized host defense according to Pasternak.(1) Hydrated Zn2+ ions protect cell plasma membranes against damage induced by cytotoxic agents of environmental origin long enough for other defense mechanisms to be brought into play. Up to 1 mMol, Zn2+ ions protect cell plasma membranes in vitro.(1)

No evidence of toxicity from lozenges releasing Zn2+ ions exists. The only adverse effect -- if it can be called that -- is mild, transitory oral irritation on occasion, even though lozenges used by Eby et al.(2) in 1984 produced initial salivary Zn2+ ion concentrations of 7.4 mMol. The highest recorded initial salivary Zn2+ ion concentration from zinc acetate lozenges is 21 mMol. Rather than being toxic to oral tissues, Zn2+ ions are associated with accelerated patient recovery. Oral irritation and strong taste sensation from strong lozenges are much less apparent in patients with colds than without colds, perhaps because of increased oral membrane permeability in patients with untreated colds.

Antiviral Effects of Zinc Ions

Positively charged Zn2+ ions from soluble, highly ionizable zinc compounds with very low stability constants were demonstrated to be highly effective antirhinoviral agents in vitro by Korant and others at Du Pont using HeLa cells (human epithelioid carcinoma of the cervix cells).(3) Many other metals were determined not to be antirhinoviral at nontoxic dosages. Korant and colleagues determined that Zn2+ ions inhibit cleavage of rhinovirus polypeptides. Addition of Zn2+ ions, at concentrations 0.1 mMol and above, at any time during rhinovirus replication immediately inhibited further formation of infective virions.(3) Antirhinoviral concentration is 6.67 times higher than normal zinc serum concentrations. Plaque-forming ability of rhinoviruses was reduced by 90 to 100 percent in eight of nine rhinoviruses tested.(3) Zinc ions rapidly inhibit virus production and lead to accumulation of rhinovirus precursor polypeptides cleaved predominantly to stable virus polypeptides upon removal of Zn2+ ion.(4) Zn2+ ions complex with rhinovirus coat proteins and alters them to prevent their function as substrates for proteases or as reactants in the assembly of virus particles.(4) Zn2+ ions were not shown to de-activate mature rhinoviruses.(3) The Du Pont team conducted an exhaustive five-year study of antiviral effects of Zn2+ ions on rhinoviruses and other picornaviruses.(3 - 10)

A. S. Prasad, who is regarded as the father of human zinc nutrition research, suggested two ways by which Zn2+ ions block cleavage of rhinovirus proteins. One is by activation of one or more proteases. The other is by binding to and altering substrate so it cannot be cleaved.(11) The latter model is preferred because (a) Zn2+ ion almost immediately blocks virus production, suggesting one of the components of the virion is affected directly by Zn2+ ions; (b) Zn2+ ions interact directly with rhinovirus capsids; (c) sufficient amounts of purified virus will produce crystals, but amorphous precipitates form in the presence of a small amount of Zn2+; and (d) results of cleavage inhibition indicate sensitive proteolytic reactions invariably involve precursors containing capsid protein sequences.(11) No information on changes in intracellular zinc concentration from zinc was presented by the Du Pont team, and none is likely since elevated extracellular Zn2+ ions strongly inhibit cellular Zn2+ absorption.(1) Zn2+ ions render the cell plasma membrane non-permeable so the rhinoviruses cannot enter or exit, thus terminating viral replication.

In 1989, Merluzzi and others showed antirhinoviral effects of zinc to be directly related to amount of Zn2+ available and unrelated to the total amount of zinc complex available.(12) As zinc was complexed with ligands of increasing binding strength, antirhinoviral activity proportionately fell. Maximum antirhinoviral effectiveness was with zinc chloride (K1 = 0.0). Minor increases in lipophilicity using crypate complexes drastically affected their activity-to-toxicity ratios, with increases in toxicity associated with increases in lipophilicity, suggesting lipophilic zinc complexes should not be used in throat lozenges to treat common colds.

Cell rounding and refractile changes occur at about 0.1 mMol Zn2+ and at much lower concentrations for most lipophilic and strongly chelated zinc complexes.(12) These observations were interpreted as cytotoxicity by Merluzzi and co-workers.(12) The effect of 0.05 mMol Zn2+ from zinc chloride (one-half the concentration used by Korant and co-workers) was equal to human interferon-beta at its most effective concentrations of 100 to 1000 IU in its antirhinoviral activity and ability to protect infected cell monolayers.(12) No significant antirhinoviral activity for several zinc compounds was reported by Geist and associates (nor by Korant and others) at 0.03 mMol,(13) a Zn2+ ion concentration that does not produce cellular rounding and refractile changes. Antirhinoviral effects were similarly observed by Geist and co-workers at 0.10 mMol.

Korant and his co-workers at Du Pont have found refractile changes and cell rounding not to be an indication of zinc cytotoxicity. Korant and co-workers showed the effects noted by Geist and associates, as well as by Merluzzi and associates could be induced by many noncytotoxic agents, including slight changes in carbon dioxide concentration and very minor changes to culture medium.(14)

Other common cold-causing viruses inhibited by Zn2+ ions include herpes simplex viruses, reviewed by Eby and Halcomb,(15) and coxsackie(7) viruses. Zn2+ ions were shown by Merluzzi and co-workers not to have an antiviral effect on influenza-A.(12) Oxford and Perrin, and Cload and Hutchinson separately found Zn2+ ions to inhibit polymerase activity in influenza A and B,(16,17) although its effect on infectivity was not demonstrated.

Zn2+ ions, but not zinc complexes, have antiviral properties, and those properties are documented for a number of important viruses unrelated to common colds. Avian myeloblastosis,(18,19) bacteriophages,(20) calicivirus,(21) equine herpes,(22) herpes simplex I and II,(23-30) polio,(31,32) encephalomyocarditis,(33,34) enterovirus,(35) foot-and-mouth disease,(36-38) mengovirus,(39) Rous sarcoma,(40) Semliki Forest,(41) Sindbis,(42) SV40,(43) tobacco mosaic viruses,(44) vaccina,(45,46) viroids and prions,(47) are all reported to have features controlled by Zn2+ ion usually at concentrations between 0.1 and 2.0 mMol without harm to cells.

DNA is about 5000 times less susceptible to damage by Zn2+ ion than is RNA, suggesting zinc plays a role in predominant evolutionary selection of DNA, rather than RNA, as the bearer of the primary genetic information.(48)

Inhibitors of viral protein cleavage are considered a possible method to control HIV infection.(49-51) Zn2+ ions, inhibitors of viral protein cleavage, have been found to have effects on Human Immunodeficiency Viruses 1 and 2 (HIV-1 and HIV-2).(49) Its nucleocapsid binds zinc and forms retroviral-type zinc fingers, which may have significance in the development of vaccines.(52) HIV-1 and HIV-2 protease are easily inhibited by Zn2+ ions at nearly normal zinc serum concentration(49) but without inhibition of virus infectivity.(14) Several virologists, including Korant, suggest viral replication or inhibition of various viral functions by Zn2+ ions may occur in many other viruses.

Antibacterial and Antifungal Activity

Zinc also has antibacterial activity in the mouth regarding bacteria unrelated to common colds. Zinc and other metal salts temporarily inhibited growth of Streptococci and Actinomyces as well as other dental caries producing bacteria.(53) Zinc chloride solutions of 20 mMol reduced plaque formation in heavy plaque formers(54) and reduced acidogenicity of dental plaque for several hours but were associated with undesirable taste and excessive oral drying.(55,56)

Other reported therapeutic activity of zinc complements the record. Growth of Chlamydia trachomatis, a frequent cause of sexually transmitted diseases, was inhibited at 0.01 - 0.1 mMol Zn2+ ion in vitro.(57) Antibiotic activity of amniotic fluid is dependent upon zinc.(58) The list of zinc compounds used for topical antiseptic, antifungal, or astringent purposes in human beings or animals in the Merck Index includes zinc bacitracin, zinc acetate, zinc chloride, zinc carbonate, zinc citrate, zinc iodate, zinc oxide, zinc permanganate, zinc peroxide, zinc p-phenolsulfonate, zinc propionate, zinc salicylate, zinc stearate, zinc sulfate and zinc tannate.(59) Calamine lotion is mainly zinc oxide and has been used for many years as a topical protectant and astringent.(60) Zinc pyrithione is an active ingredient found in antibacterial, antifungal, and antiseborrheic shampoos.(61)

Astringency

Zn2+ ions are best known for their astringency. The basic effect of Zn2+ ions on many cells, including the immune system cells, seems related to their effects on cell plasma membranes, perhaps in a manner linked to and dependent upon astringency. Hydrated Zn2+ ions from loosely bound zinc complexes such as zinc chloride and zinc acetate (but not most tightly bound or lipophilic zinc complexes) are astringent. Zinc oxide is topically astringent but releases hardly any Zn2+ ions in a nonacidic medium. For Zn2+ ions, but not for other more tightly bound zinc compounds, cell rounding and refractile changes appear to result from its astringent, protective action on cell membranes, not from cytotoxicity. These changes often occur at about 0.1 mMol Zn2+ ions and at much lower concentrations for most lipophilic and strongly chelated zinc complexes.(12) These observations were interpreted as cytotoxicity for zinc and suggested a low 1:2 or 1:4 antirhinoviral therapeutic index for Zn2+ ions.(12,13)

Other researchers, including those much more familiar with the appearance of the astringent action of Zn2+ ion on cell plasma membranes, place the index 10 or more times higher in vitro. Extracellular Zn2+ is a newly recognized host defense acting to stabilize and protect cell membranes.(1,62-67) All astringents, including Zn2+ ions, are locally applied protein precipitants having such low cell penetrability that action is essentially limited to cell surfaces and interstitial spaces (contraction, rounding, wrinkling and blanching in vitro). Permeability of cell membranes is reduced by astringents including Zn2+, but cells remain viable. Astringents harden the cement substance of capillary epithelium, inhibiting pathologic transcapillary movement of plasma protein and reducing local edema, inflammation, and exudation. Additionally, astringents reduce mucus and other secretions in tissues containing goblet cells and other secretory cells, causing affected areas to become drier and heal faster.(62)

Many astringents, including Zn2+ ions, are irritants or caustic in high concentrations.(62) Astringents are used therapeutically to arrest hemorrhage by coagulating blood (styptic action) as well as to check diarrhea, reduce inflammation of mucous membranes, promote healing, toughen skin, and decrease sweating.(62) The astringent effects of zinc lozenges on immune system cells and mucosal tissues are harmless, and normal cellular appearance and function returns upon removal of Zn2+ ions. The astringent effects of Zn2+ on immune system cells and particularly its effects on mast cells as well as other immune cells and functions is important in understanding how Zn2+ affects common colds. Recommended concentrations of zinc acetate for use as a topical astringent are 0.2 to 2.0 percent (9 to 90 mMol Zn2+),(62) suggesting concentrations from zinc lozenges to be appropriate.

From these observations, hydrated Zn2+ ions are observed not to enter or damage cells, and beneficial effects of Zn2+ ions occur exclusively at the cell membrane. By inference, oral absorption of topical Zn2+ ions through the oral mucosal membrane from zinc lozenges relies upon a non-lipophilic mechanism.

Non-Specific Membrane Protection

Perhaps the most consistently reported effect of Zn2+ ion on mammalian cells is membrane stabilization.(63) Zinc is present in many mammalian cell membranes, and stabilizing actions of certain steroid hormones on lysosomal membranes may be secondary to the effects of zinc.(63) The exact mechanism by which zinc stabilizes cell membranes is not clear and may be different for different membranes.(63) Hemolytic viruses, bacterial and animal toxins, components of activated complement, cytolysin (perforin), cationic proteins, and detergents have all been shown to induce a sequence of permeability changes at the plasma membrane that are in every case beneficially sensitive to changes in Zn2+ ion concentrations from normal levels up to 100 times normal serum concentration.(64-67) Membrane damage induced by a wide variety of hemolytic agents can be prevented by zinc ions at normal to 100 times the normal concentration of zinc found in human serum without harm to cells.(67) Within a range, as Zn2+ ion concentration is increased, the strength of protection also increases.

The large dietary requirement for zinc cannot be explained by its known cellular and enzyme requirements, but it can easily be explained by requirement for high plasma membrane Zn2+ ions as protection against damage induced by cytotoxic agents of environmental origin. Zinc, present in human extracellular fluid at approximately 0.015 mMol, is likely to prevent damage only at rather low concentrations of cytolytic agent, and a higher concentration is required for protection against higher amounts of various cytolytic agents. Increasing concentration of extracellular divalent zinc may be useful in augmenting host defenses against a wide variety of cytotoxic agents and is considered by Pasternak to be a newly recognized host defense.(1,67)

Similarly, calcium ions have been known since 1914 to protect cell membranes in vitro, but calcium ions are not useful in vivo since their concentration is under tight endocrine control. Elevated calcium ion concentration leads to adverse effects in vivo in excitable cells like heart, nerve, and muscle.(1) Concentrations of Zn2+ ions are not under endoctrine control, and their levels can be raised in tissues in a beneficial manner protecting tissues from some viruses and other cytotoxic agents. According to Pasternak, such action may well be the responsible mechanism for ameliorating effects of common colds as shown by Eby and others.(67)

Cytolysin

Cytolysin (perforin) is a strong pore-forming agent released by natural killer T-cell lymphocytes in response to virally injured cells.(65) The molecular structure of cytolysin has been described.(68) Cytolysin is released into virally infected tissues where it can cause cell membrane damage or cell death. Some believe cytolysin to be responsible for the increase in serous nasal drainage in common colds. In some respects cytolysin is analogous to the C9 component of complement. Zn2+ ion prevents pore formation by cytolysin between 0.01 and 0.1 mMol Zn 2+ ion, and blocks leakage from cells containing preformed cytolysin. The beneficial response from Zn2+ ions occurs within minutes. Removal of Zn2+ ions by chelation with EDTA in the presence of cytolysin restores some cytolysin pore-forming activity. The concentration of Zn2+ ions required to stop hemolysis of cells containing preformed pores is somewhat higher than the concentration required to prevent pore formation when all agents are introduced at the same time.(65)

Complement

The C9 component of complement has cell membrane-damaging properties capable of lysing or damaging cells. In the presence of 0.1 mMol Zn2+ ions, red blood cells do not lyse when complement C9 is added. The inhibitory action of Zn2+ ions appeared to be on the reaction or reactions occurring between the insertion of C9 and the damage-producing step and is completely reversed upon Zn2+ ion chelation.(69)

Mast Cells

Mast cells and basophils are commonly known to be mediators of Type I allergy and possibly also several symptoms collectively known as the common cold. Mast cells are discussed because of their historic association with common colds, because of their ubiquitous and mysterious nature, because mast cells are the current subject of extensive zinc research, and because mast cells are theorized by the present author to be potent antiviral and T-cell function-modulating cells. Mast cells have been implicated in allergy and common colds as causing tissue redness, inflammation, nasal congestion, release of mucus from goblet cells, nose and throat pain, tickling and itchiness, and, indirectly, coughing and sneezing.

Mast cell derived reactions result from release of histamine, heparin, prostaglandins, SRS-A, and various powerful vasoactive amines from granules on the surface of mast cells, possibly including kinins. Mucosal mast cells are widely distributed in nasal, throat, and tracheal tissues and are believed to respond to viral antigens by degranulation. However, research shows histamine does not play a part in common colds.

One product of mast cell-induced inflammation in response to rhinoviral attack is fever. One chemical mediator generated is endogenous pyrogen which increases concentration of prostaglandins from the hypothalamus, resetting the body's temperature to cause chills and fever.

Hydrated Zn2+ ions prevent induced histamine release from mast cell granules(70) as well as release of all mast cell granule contents as Zn2+ ions stabilize mast cell plasma membranes.(1,64,67,70 ) Several receptors at plasma membranes might function as gates for transmitting information to intracellular space. In the case of mast cells, histamine-releasing agents appear to work through specific receptors at the cell membrane or by calcium antagonism. Masking receptor sites by membrane- impermeable zinc compounds could explain inhibition of release action,(70) but at least four mechanisms could also be operational. The granules of both basophils and mast cells also contain Zn2+ ions stored combined with histamine and heparin.(71) Concentrations are in the 4 to 20 mMol range for mast cell secretory granules,(72) which is 400 to 2000 times greater than Zn2+ ion concentration in human serum and identical to concentrations of Zn2+ ion in saliva from zinc gluconate or zinc acetate lozenges. Both human and rat tissue mast cells contain high concentrations (2.1 mg/million cells) of granule-associated zinc.(73) Perhaps some zinc binds histamine with heparin,(74) although research has shown this idea does not hold true in mast cells of rats.(75)

Hydrated zinc ions stabilize cell plasma membranes and prevent induced histamine and vasoactive amine release from tissue mast cells.(1,64,67,72,76-78) Inhibition of histamine release begins at about 0.001 mMol concentration of Zn2+ ions in human beings and is greatest (80 to 100 percent inhibitory) at 0.1 mMol concentration.(76-79) This concentration of zinc is about 6.67 times higher than normally found in human serum. When histamine is released from mast cells, Zn2+ ions are also released in large amounts into surrounding tissues, perhaps acting as a source of local antiviral activity and T-cell immunostimulation. Zn2+ ions from zinc gluconate act to inhibit 100 percent of rat mast cell histamine release at 0.1 to 1.0 mMol concentration in typical or connective tissue mast cells, while much more is required for leukemic basophil cells.(80) Physiologic concentrations of Zn2+ ions inhibit release of histamine from human basophils and lung mast cells presumably by blocking calcium uptake induced by anti-IgE activation.(78) Zn2+ ions are a competitive antagonist of action of calcium ions in histamine secretion induced by anti-IgE. The zinc- histamine stability constant is log K1= 5.0 for both basophil and mast cell histamine,(78) but stability is sensitive to pH and is reported for pH 7.4. Similarly, Martell reports log K1= 5.4 for zinc and histamine.(81)

Zn2+ ion has been proposed to be involved in catabolism of histamine, as zinc has been proven to interfere with pharmacologic effects of histamine leading to anaphylactic shock.(82-84) Berthon and others showed the complexes of histamine formed with Zn2+ ion and several naturally occurring amino acids in plasma to be neutral and unlike histamine itself. Histamine is inherently polar and exists mainly in a charged form because of protonation under the prevailing physiologic 7.4 pH. These neutral, mixed ligand complexes are thus thought to be capable of passively diffusing through lipid membranes into tissue where histamine can be catabolized.

Dicarboxylic acid complexes of zinc, malate, malonate, tartarate, and maleate (log K1 = 2.0 to 2.8), were shown to be effective in catabolism of histamine,(83) but zinc aspartate and zinc glutamate (first stability constants log K1= 5.9 and 5.45 respectively) were not.(84) Berthon and Germonneau found zinc aspartate concentration in vitro needed to be raised 1000 times over normal levels to be more efficient than Zn2+ ion alone to favor zinc-mediated histamine diffusion into tissues.(84) Considering the stability constant of zinc and histamine is log K1 = 5.0, zinc complexes with stability constants above 5.0 cannot readily react with histamine to catabolize it. Zinc complexes with stability constants lower than 5.0 may reduce massive surges of histamine suddenly released into plasma in response to antigens, a variety of local stimuli, or general toxins. Mast cell Zn2+ ion complexation of histamine might explain absence of histamine from nasal lavages in colds (see below - Histamine or Kinins in Colds?).

Hydrated zinc ions (Zn2+ ions) in mast cell granules are chemically or physically protected from strong zinc chelators, thus denying access to Zn 2+ ions by serum proteins and other zinc-complexing agents.(73) Such a unique property allows zinc to be delivered as Zn2+ ions or very lightly complexed zinc and not as strongly bound complexes of serum proteins to local virally infected tissues upon mast cell degranulation for use as a local, natural antiviral agent, an interferon-inducing agent, and a T-cell lymphocyte immunoactivator as well as a preventive of allergic responses, when sufficiently present in mast cell granules.

Introduced nasally, zinc compounds including zinc chloride, sulfate, sulfocarbolate, oxide, stearate, and borate have been used to treat nasal catarrh for about 100 years.(85-91) Reports show intranasal zinc to be a mild, short-term nasal decongestant. Zinc electrically driven into nasal tissues provides beneficial relief from nasal allergy for up to one year, although treatment is painful (without cocaine pre-treatment) and may cause sloughing of nasal epithelium.(87-89)

Histamine or Kinins in Colds?

Histamine appears active in common colds, considering the widespread use of antihistamines to treat common cold symptoms, but such may not be true. The British Medical Research Council Common Cold Unit first reported histamine to be uninvolved in common colds in 1951, and antihistamines were worthless in common cold treatment.(92) Even so, nasal secretions from histamine-stimulated goblet cells in colds and allergy have been poorly studied until fairly recently.

A wide-ranging amount of histamine (5 to 1,519 ng/ml, with a mean 91 ng/ml for non-allergic patients and 51/ng/ml for allergic patients) is found in nasal secretions, with the histamine amount being four times higher in men than women.(93) During symptomatic rhinovirus infections, analysis of nasal lavages shows kinins to be generated, vascular permeability increased, mast cells not participating, and neutrophils entering nasal secretions.(94)

To test the hypothesis that viral respiratory infections cause symptoms by activating nasal mucosal mast cells to release mediators active on vasculature and mucosal glands, the presence of histamine in nasal secretions was assessed during natural colds and in laboratory-induced rhinoviral infections. Infection with rhinovirus and with influenza did not change these concentrations significantly. Histamine tended to be lower during viral infections.(95) Increase in kinins, but not histamine, in nasal lavages occurred in symptomatic common colds, suggesting mast cells and basophil activation do not occur during rhinovirus colds.(96) Increases in kinins correlated with increased vascular permeability, as monitored by increased concentrations of albumin in nasal lavages.(96) More information implicating kinins and not histamine in common cold symptomology has been obtained. (97-101) 2001 UPDATE: More on these important findings here.

Rhinoviral illness of the respiratory tract enhances airway reactivity and predisposes allergic patients to develop late asthmatic reactions, which may be an important factor in virus-induced bronchial hyper responsiveness.(102) In infants with respiratory syncytial virus (RSV) infection, histamine and RSV-specific IgE were far more common in wheezing infants than in non-wheezing infants and adversely affected the outcome of RSV infections.(103)

T-Cell Lymphocytes

B- and T-cell lymphocytes are the main warriors in the defense of the nose. B-cells produce immunoglobins, antibodies that plug up cold-virus receptor sites and prevent viruses from attaching to cells of the nose and throat. Some cells become memory cells helping to prevent the recurrence of infection. T-cell lymphocytes, when summoned to the site of infection, turn into "killer cells" (perhaps with help from Zn2+ ions released by mast cells) which release cytolysin (perforin), opening pores in cell membranes and attacking and killing virus-infected cells. Other T-cells ("helper/inducer" cells) stimulate B-cells to produce more antibody. Still other T-cells become "suppressor" cells to shut off antibody production and killer-cell activity after viruses have been defeated. T-cells also release several other substances to help the body conquer infection, including macrophage activation factor, macrophage inhibition factor, interleukin 1 and 2, and interferon. Interferon prevents cell death caused by viruses and prevents viral replication.(92)

In the common cold, Zn2+ ions have effects on local T-cell function not yet completely elucidated, and which are too complex for full discussion here. Emphasis here is on local mucosal tissue and cutaneous effects, as zinc serum level does not rise with administration of zinc lozenges in common colds. Zinc deficiency has been studied more than zinc excesses. Zinc deficiency is universally accepted as being harmful to the T-cell lymphocyte system and is potentially lethal in humans(63,104-107) because zinc transferrin is the body's only T-cell lymphocyte mitogen.(108) Its necessity in thymic T-cell lymphocyte function is well known.(109) Zinc is also necessary for transferrin synthesis(110) and a deficiency in either transferrin or zinc can cause profound T-cell immunosuppression. Zinc deficiency during prenatal life causes persistence of immunodeficiency for three generations in mice,(111) with predictably dire consequences for human beings. Golden and associates showed zinc-deficient children to have greatly increased susceptibility to severe infection, and restoration of thymic function and regrowth occurs only when large doses of zinc (2 mg zinc/kg body weight) are administered.(112) Whether the condition needing immunostimulation by zinc is malnutrition, HIV infection, AIDs, acute lymphocytic leukemia (ALL) or any other T-cell lymphocyte zinc immunodeficiency, daily dosage should be as described by Golden and associates for thymic stimulation and regrowth in malnutrition. Both the young and the elderly receive T-cell immunologic benefit with supplemental zinc in the 150 mg/day range.(113,114)

At concentrations of 0.1 to 0.4 mMol zinc, a mitogenic response is induced in normal T-cell lymphocytes but not in leukemic lymphocytes.(115) This difference in response has been used to stimulate normal T-cell function in leukemia.(116) Zinc supplementation has been proposed to rectify T-cell anergy in pediatric Hodgkin's disease.(117) As little as 15 mg zinc from zinc gluconate after three weeks beneficially changes the helper (OKT4) to suppressor (OKT8) ratio by normalizing the suppressor population without increasing the absolute number of helper cells in healthy people, with no effect on leukemic T-cells.(115) One explanation for modulation of T-cell subset ratios is histamine induction of suppressor T-cells,(118) and Zn2+ ion inhibition of release of histamine, and consequently its effect on suppressor proliferation. As activation of T-cell lymphocytes requires 4 to 6 days for full effect (10 - 15 percent activated T-cells),(115) T-cell activity is a delayed response and comes into play late in common colds.

T-cell activity is impaired after one month when healthy people take 300 mg zinc per day.(119) Large excesses of zinc are known to compete with copper and manganese for intestinal binding sites adversely driving down their absorption, resulting in several blood abnormalities.

The zinc lozenge technique induces identical results in both normal people and in children immunosuppressed by chemotheraphy for treatment of acute T-cell lymphocytic leukemia (ALL). This similarity in response suggests zinc lozenges may help shorten colds in patients with other lymphocyte diseases including HIV infection and AIDS --- perhaps with prevention of life-threatening sequela. Much of the preliminary (pre-1984 Eby et al trial) research with zinc gluconate lozenges in the treatment of common colds was done in a small child suffering from ALL. The results appeared to be at least as good when treating colds in the ALL patient as when treating non-leukemic volunteers.

Interferon Induction

Working with mice in 1987, Reardon and Lucas reported Zn2+ ions to have T-cell mitogenic activities, inducing lectin-dependent cellular cytotoxicity of target cells and having interferon- inducing properties. Medical interests in zinc in immune function have been related to its requirements in the lymphocyte cell cycle, especially noted in zinc immunodeficiency diseases. Zn2+ is chemically the simplest compound known to activate lymphocytes to undergo cellular proliferation, and Zn2+ ions are the only ubiquitous cellular component functioning as lymphocyte mitogens in animals and as T-cell lymphocyte-specific mitogens in humans.(120,121) Splenic and lymph node lymphocytes from mice were activated with Zn2+ ions in vitro, as noted by several-fold increases in 3H-thymidine incorporation after 144 hours of culture. Optimal mitogenic concentration was 0.20 mMol Zn2+ ions. Lymphocyte responses were inhibited at 0.80 mMol concentration of Zn2+ ion.

To determine whether Zn2+ ion could activate lymphocyte functions other than mitogenesis, interferon production was assessed. Splenic lymphocytes were stimulated by Zn2+ ions to produce interferon after an incubation period of 96 hours, but interferon was not produced at 48 hours. Interferon was interferon-gamma as the interferon was acid labile. Zn2+ ions produced 16 units of interferon, compared with 256 units of interferon when cells were stimulated by Concanavalin A. Optimal concentrations for interferon induction were 0.20 mMol for Zn2+ ion and 20 mcg/ml for Concanavalin A.(120,121)

In agreement with the murine studies, Salas and Kirchner found Zn2+ ions to induce large amounts of human interferon in vitro in a study at the Institute of Virus Research, German Cancer Research Center.(122) Salas and Kirchner also confirmed Zn2+ ions to have a mitogenic effect on human lymphocytes, and Zn2+ ions from 0.05 to 0.5 mMol concentration (antirhinoviral concentration) to have the ability to induce large amounts of interferon- gamma. Using blood from several healthy human volunteers, maximum interferon induction was 120 and 128 IU/ml at 0.1 to 0.2 mMol Zn2+ ions. By comparison, 5 mcg/ml PHA induced 729 IU/ml interferon and 314 IU/ml respectively.(122) Concentrations considerably higher than 0.5 mMol Zn2+ ion appeared toxic for certain cells (as measured by the trypan blue test), and concentrations of Zn2+ ion significantly below 0.05 mMol did not induce interferon, although mitosis occurred. Kinetic experiments showed production of interferon to occur as early as 24 hours and reach its optimum by 96 hours. Adherent cells were required for the effect. Zn2+ ions were thus suggested to play a role in interferon induction in vivo explaining some human immune disorders characteristic of zinc deficiency, such as a decrease in natural killer cell activity and an increase in susceptibility to infection.(122) In mice, Gainer found impaired interferon action with zinc deficiency.(123)

Anti-Inflammatory Action

Although common colds are essentially viral infections of the nose, colds are characterized by tissue inflammation, large amounts of thin, serous nasal drainage, and thick goblet cell-derived nasal mucus. The effect of zinc on mast cells in vitro suggests zinc could impact upon mast cells in common cold therapy. Likewise, the effects of zinc on all mucosal immune system cells suggests zinc has immunomodulating effects which remain only partially understood. Most of the research on zinc and the immune system has focused upon deficiencies of zinc. In the case of treating common colds with zinc lozenges, zinc may be present in nasal and nasopharyngeal tissues in concentrations up to 50 times higher than normally found in serum, which may result in temporary, localized, mucosal immunologic functional changes. Zinc deficiency is often found in dermatologic diseases where inflammation is characteristic; and treatment with zinc (usually as zinc oxide) to control inflammation is common.

With elevated concentrations of Zn2+ ions, the cement substance of capillary endothelium of all cells is known to become hardened so pathologic transcapillary movement of plasma protein is inhibited and local edema, inflammation, and exudation are thereby reduced. Mucus and other secretions are reduced in tissues containing goblet cells and other secretory cells, and the affected area dries and heals faster with added zinc ions.(62)

Paradoxically, and beneficially in common colds, the functions of monocytes, neutrophils, and macrophages are inhibited by elevated concentrations of zinc(63,124,125) through cell plasma membrane stabilization, and these cells function maximally in moderate-to-severe zinc deficiency. Their antibacterial function is maximized automatically in vivo by the leukocyte endogeneous mediator (LEM) role of the liver, as bacterial growth typically is increased with normal zinc serum status.(126,127) Severe bacterial infections (and several severe viral infections) cause a rapid decline in body zinc pools by LEM to effectuate action by macrophages and polymorphonuclear cells (PMN), often accompanied by endogenous pyrogen (EP) -induced fever.(126) However, the functions of PMNs are greatly inhibited in cases of extreme malnutrition and these patients respond well to supplemental zinc.(128)

LEM is not believed to be involved in common colds. Little fever or difference in zinc serum concentrations has been noted between zinc and placebo-treated patients in common cold clinical trials in relatively unstressed patients. Upon stabilization of cellular and lysosomal membranes of white blood cells with Zn2+ ion in common cold treatment, nasal inflammation quickly subsides. To assess LEM action in colds accurately, zinc serum levels of infected persons receiving zinc lozenges should be compared with an uninfected control group treated with zinc throat lozenges.

Zinc, often as zinc oxide, has long been used to inhibit inflammation of the skin (e. g., diaper rash, other dermatitis) in a safe and effective manner in over-the-counter preparations. Inhibition of nasal inflammation by zinc is not seen as especially different, and zinc oxide ointments applied to the nares are effective in temporarily drying nasal tissues in allergy or common colds without reduction in duration of the illness. Failure of zinc to inhibit release of mucus from nasal goblet cells when zinc is effective in other inflammatory responses would be viewed as an anomaly. Nasal mucus dries up quickly, and goblet cell membranes are also stabilized. Mast cells are known to be stabilized by zinc. Zn2+ ions prevent the release of histamine to trigger goblet cell's release of mucus.

Zn2+ ion has very potent and rapid-acting human prostaglandin metabolite-inhibiting properties (to 90 percent or more) at 0.01 to 0.1 mMol,(129) that may be responsible for reducing headaches, pain and inflammation in common colds.

Zinc, Stress, and Common Colds

Recently the relationship between stress and common colds has been a subject of interest. Patients with varying degrees of stress, from low to high, were administered rhinoviruses, and the results were compared to levels of stress in their daily lives.(130) Persons having more stressful lives were significantly more susceptible to colds. Zinc serum level has long been known to fall in highly stressful situations through LEM action, wherein the liver sequesters zinc from the blood within minutes,(126,127) and it should not come as a surprise that highly stressed persons have lower zinc serum levels and generally more infections.

Overview of Effects of Zn2+ Ions in Common Cold Therapy

Zn2+ ions have a number of effects in vitro of interest in common cold therapy. Their potent in vitro antirhinoviral effects are of foremost interest and may be observed in vivo in colds treated with zinc lozenges releasing Zn2+ ions. Zn2+ ions strongly stimulate interferon production within 24 hours and zinc lozenges may prompt interferon release in colds. Zn2+ ions are also necessary for the functioning of the immune system and the growth of immune cells. Zn2+ ions are strongly astringent and may appear harmful to immune cells to observers unfamiliar with its harmless astringent rounding and blanching effects.

Concentrations up to 100-fold normal serum concentration have been used in cell membrane stabilization studies, almost always without lasting harm. Addition of Zn2+ ions to cells undergoing hemolysis by many agents stops cell membrane leakage by closing membrane pores and has been shown to protect tissues in vivo. Mast cell plasma membranes are readily stabilized by Zn2+ ions, and the outflow of histamine, heparin, and all mast cell vasoactive agents, perhaps including kinins, is immediately halted. Zn2+ ions help metabolize histamine and may explain the relative absence of histamine in common colds. Increased nasal tissue concentrations of Zn2+ ions may occur naturally from mast cells in upper respiratory infection or exogenously by use of lozenges releasing Zn2+ ions in common cold therapy.

Pores in capillary walls are closed by Zn2+ ions, and Zn2+ ions may reduce or prevent movement of monocytes and lymphocytes into infected tissues. Closure greatly enhances the movement of Zn2+ ions over long distances through biologically closed electric circuits (see Chapter 3).(131) Once cell pores are closed by Zn2+ ions, pores tend to remain closed in vitro, appearing in vivo as a resistance to relapse. Even so, nasal mucus flow and tears caused by crying are neither stopped nor prevented, even with extensive use of zinc gluconate lozenges, suggesting two separate mechanisms of action on goblet cells.

Elevated concentrations of Zn2+ ions may inhibit activated PMN and macrophage mobility and function, but not their viability. Since fever subsides rapidly, interleukin-1 production may be inhibited by Zn2+ ions. Although lymphocyte movement into infected tissues may be impaired by Zn2+ ion capillary wall pore closure, both local B-cell and T-cell lymphocyte functions are believed to be beneficially affected, and perhaps T-cells are activated.


1998 ADDENDUM: More facts on the role of zinc ions in the biochemistry of immunology can be found in
Zinc in Leukemia.

Chapter 2 References


Chapter 3