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JAC Journal of Antimicrobial Chemotherapy (1997) 40, 475–483 Reviews Cross-protection by anti-core glycolipid antibodies: evidence from animal experiments Willem N. M. Hustinxa,b,c*, Kees Kraaijevelda, Andy I. M. Hoepelmana,c and Jan Verhoefa a Eijkman-Winkler Institute for Medical & Clinical Microbiology, bDepartment of Intensive Care & Clinical Toxicology and cDepartment of Medicine, Division of Infectious Diseases and AIDS, Utrecht University Hospital, The Netherlands The ability of antibodies against the core glycolipid (CGL) of endotoxin to protect experimentally infected animals against death from Gram-negative sepsis is reviewed. The limitations and confounding factors inherent to animal models of sepsis are also briefly discussed. This review considers 30 studies in mice and 12 in other animal species that investigated protection against heterologous challenge by passive immunization with anti-CGL antibodies. In 28 (67%) of the reviewed studies antibodies were found to be protective, either prophylactically (n = 17) or therapeutically (n = 11). With the possible exception of the type of antibody preparation that was used (monoclonal versus polyclonal antibodies), none of the many differences in the experimental protocols were clearly correlated with success. Convincing proof is still lacking for any of the hypothetical mechanisms of protection by anti-CGL antibodies. Moreover, the evidence that protection by these antibodies is attributable to their anti-CGL specificity is poor. The available data raise serious questions about the validity of the concept underlying the search for broadly cross-protective antibodies raised against the core region of endotoxin. However, continuing research suggests that endotoxin still is a valid target in devising new adjunctive treatment strategies to improve the outcome of serious Gram-negative infections. Introduction Infections caused by Gram-negative bacteria, especially when complicated by septic shock, continue to have a high associated mortality despite considerable improvements in antimicrobial and supportive care. The pathophysiology of these infections is thought to be governed by endotoxin, a cell wall constituent of all Gram-negative bacteria.1,2 The inner core glycolipid (CGL) oligosaccharide structure of endotoxin is highly conserved among these bacteria3 and its lipid A moiety has been shown to contain most of the toxicity.4 These findings supported the hypothesis, originally suggested by Chedid et al.5 and McGabe, 6 that ways to neutralize CGL/lipid A might provide a broadly cross-protective therapy. Research in the years that followed confirmed that anti- bodies to CGL epitopes were able to neutralize various biological actions of endotoxin in vitro and in vivo. However, the outcome of several recent clinical trials of anti-CGL monoclonal antibodies (mAb) as adjunct therapy for serious Gram-negative infections has been rather disappointing, as discussed recently.7 It has been argued that, in retrospect, the preclinical and clinical data available at the time provided insufficient scientific justification to proceed into clinical trials with anti-CGL immunotherapy.8 We here review studies conducted in animals that have investigated protection by anti-CGL antibodies against experimental Gram-negative infections. The focus of this review is on mouse models of infection because these animals are the most extensively studied species. Relevant studies in other species will be mentioned briefly. *Corresponding author. Present address: Department of Medicine, Diakonessen Hospital, PO Box 80250, 3508 TG, Utrecht, The Netherlands. Tel: + 31-30-2566566; Fax: + 31-30-2566606. 475 © 1997 The British Society for Antimicrobial Chemotherapy W. N. M. Hustinx et al. Data sources This review is based on English-language primary and secondary (i.e. review) articles dealing with protection by antisera or mAb in animal models of Gram-negative infection and is in part based on a search in MEDLINE (National Library of Medicine), using relevant key words. Only those studies that have investigated protection by passive immunotherapy against heterologous challenge (i.e. against infection with Gram-negative bacterial strains other than those used as antigens for antibody production) were selected for review. rats20,21 and dogs.22 In sharp contrast to the studies in mice, seven (58%) of these studies were conducted in neutropenic animals (rats and rabbits) and antibodies were administered therapeutically (i.e. after the onset of infection) in eight (67%) of the studies. mAb, used in seven of these studies (58%), were found to be protective in five of them (71%). As in mice, studies reporting protection (n 9) outnumbered those reporting failures (n 3). Is protection by anti-CGL antibodies specific? The specificity of protection by anti-CGL antibodies has been a mattter of considerable debate which concerns two interrelated questions: (i) can cross-protection be conferred only by antibodies with CGL/lipid A specificity?; Mouse models (ii) does the mechanism of protection indeed involve an 5 interaction of anti-CGL antibodies with the broadly conAfter the landmark study by Chedid et al., who reported served inner core of endotoxin? The first question refers to protection of mice against a lethal challenge with Kleb the methods used to investigate protection by anti-CGL siella pneumoniae by high-titre horse antiserum raised antibodies in experimental infections. Most studies that against a rough mutant of Salmonella typhimurium, a great tested protection by anti-CGL antisera used preimmune many comparable studies in mice followed (Tables I and sera as controls. Their inherent polyclonal nature, howII). The experimental protocols of these studies show ever, precludes a definite answer to the question of marked differences. At least ten different mouse strains specificity, as was demonstrated quite dramatically by were used in the 30 reports selected for this review (six 23 Greisman et al. These authors showed that preimmune reports did not indicate which mouse strain had been sera from rabbits, immunized with different rough Enteroused). Anti-CGL antibodies were given prophylactically bacteriaceae mutants to produce anti-CGL antisera, also in 83% and therapeutically in 17% of these studies. The had definite protective abilities. It was hoped that, when challenge inoculum ranged from LD50 to 100LD50, and was mAb became available, this issue would be resolved. Howgiven by the intravenous route (30%) or the intraever, a suitable control mAb was used in only four (29%) peritoneal route (60%). In four studies (10%) both routes were used simultaneously or in parallel experiments. The and five (50%) of studies that tested protection by antidose of mAb ranged from 2 g9 to 2000 g.10 No attempt CGL mAb in mice and other animals, respectively. The has been made to compare ‘doses’ of antisera (usually overall evidence that protection by anti-CGL mAb is expressed as reciprocal titres of anti-CGL antibodies attributable to their specificity therefore remains poorly against specified inner core epitopes). mAb were used documented. The debate on specificity is further compliin ten (53%) studies reporting successful protection and cated by the observation that ascitic fluid (as opposed to preparations) may contain polyin four (36%) studies that failed to do so. Virulence- affinity-purified mAb 24,25 reactive IgM mAb. Furthermore, lipopolysaccharide enhancing agents (mucin, haemoglobin or silica) were (LPS) contamination of prophylactically administered used in 34% of the studies. On aggregate, differences in mAb preparations (regardless of their specificity) has been the experimental protocols, other than those concerning claimed to induce host resistance to endotoxin and thereby the type of antibody preparation that was used (i.e. mAb generate false-positive data on antibody efficacy.26 versus polyclonal antiserum), were not obviously associThe second question has been extensively addressed in ated with success or failure to protect and therefore do not a previous review by Baumgartner,27 who concluded that readily explain different outcomes. With mortality or the change of the LD50 inoculum as the study endpoint, 16 anti-CGL antibodies had not been clearly shown to (64%) of the prophylaxis studies and four (80%) of the neutralize endotoxin in Limulus amoebocyte lysate (LAL) therapeutic studies found anti-CGL antibodies to be pro- assays or haemolysis assays, or enhance opsonophagocytotective. It is not known, however, to what extent a reluct- sis, or to be bactericidal in the presence of complement, or ance to publish negative findings has biased this overall to reduce blood endotoxin concentrations and/or levels of bacteraemia under appropriate conditions. These in-vitro picture. studies, therefore, failed to confirm any of the presumed mechanisms of cross-protection by anti-CGL antibodies. Other animal models Finally, doubts about the ability of anti-CGL antibodies to Protection has been reported in rats,11–14 guinea pigs,15 confer cross-protection are further strengthened by the rabbits16–18 and dogs.19 Failures have been reported in observation that these antibodies bind poorly, if at all, Cross-protection by anti-CGL antibodies in experimental models of infection 476 Cross-protection by anti-core glycolipid antibodies 477 W. N. M. Hustinx et al. 478 Cross-protection by anti-core glycolipid antibodies to clinically relevant (smooth) Gram-negative isolates. This is explained by masking of CGL epitopes by long O-polysaccharide side-chains and capsular envelope structures.28,29 In vitro, antibiotics have been shown to enhance binding of anti-CGL mAb to smooth strains.30–34 In vivo , several studies showed that protection by anti-CGL mAb was enhanced by12 or even vitally dependent on14,19,35,36 concurrent administration of antibiotics. In only four (21%) of the studies in mice that reported protection were antibiotics used concurrently with anti-CGL antibodies (Tables I and II). These observations then beg the question, how do anti-CGL antibodies protect if not by means of neutralization of free endotoxin1 or by a mechanism involving enhanced binding to cell-bound inner core epitopes, thought to be inaccessible in smooth strains? Modification of LPS uptake and of the response to tumour necrosis factor by human monocytes37 and enhanced clearance of LPS, complexed with mAb and complement, via the CR1 receptor on erythrocytes38 have been proposed as their mechanisms of action. Problems and limitations of animal models The animal models of infection most frequently used to evaluate protection by anti-CGL antibodies fall into one of the following categories: intravenous or intraperitoneal bolus administration of live bacteria, caecal ligation and puncture, or oral feeding of virulent bacteria. In addition, these models can be modified by compromising the host response or by enhancing bacterial virulence in several ways: by inducing neutropenia (e.g. by pretreatment with cyclophosphamide), by embedding the challenge inoculum in a fibrin clot (thereby shielding bacteria from macrophage activity) and by mixing the inoculum with mucin (compromising the host defence lines)39 or with haemoglobin (promoting bacterial growth). 40 A discussion of the relative merits and limitations of the various models is beyond the scope of this review and the reader is referred to overviews by others. 41–43 Some of their conclusions are, however, pertinent to the present context. (i) Intravenous bacterial challenge, because of the relatively large inoculum required, probably constitutes a model of (endotoxin) intoxication rather than of evolving infection. Intraperitoneal challenge typically requires 100- to 1000-fold fewer bacteria. Virulence enhancement and neutropenia allow challenge inocula to be reduced further and are thought to help avoid false-negative findings that might be attributable to the overwhelming infection and endotoxaemia associated with large challenge inocula.42,44 (ii) Endotoxin preparations can to some extent mimic the pathophysiology of Gram-negative infection, and endotoxin challenge therefore has come to be regarded as a near-perfect, simple and cheap surrogate for bacterial challenge. This may, however, be a grave misconception in view of observations that endotoxin may induce cardio- vascular, metabolic and cytokine responses quite different from those observed in bacterial sepsis.43,45,46 Models that take mortality from sepsis as their primary study endpoint, ideally use animal species that are inherently susceptible to the challenge organism (i.e. without previous or concomitant sensitization measures). In addition, infection and its sequelae should be inducible with a small inoculum and evolve over days rather than hours. Finally, the animal should be large enough to permit easy intravascular access (for repeated blood sampling and administration of fluids and other supportive therapy) and other forms of instrumentation. Mice and rats can hardly be considered to fulfil these requirements. Undoubtedly, their widespread use is driven by practicality (relatively low cost, easy availability, genetic standardization and ease of handling) rather than by scientific arguments. It should be realized that the success or failure of immunotherapy with anti-CGL mAb may be critically, but unpredictably, dependent on such variables as inoculum size,47,48 treatment timing,49 the degree of antigenic heterogeneity within a given bacterial population,50–53 growth conditions,54 the characteristics of the inflammatory response in the animal that is being studied55 and, possibly, also by antibiotic-induced excess endotoxin release.56–58 Given these various confounding factors, it is not surprising that the outcome of animal experiments, investigating protection by anti-CGL antibodies, has proven to be so variable and poorly reproducible. To quote Fink et al.,42 “That there are so many models of sepsis and septic shock is tacit evidence that none of them is perfect”. Is endotoxin still a suitable target candidate for experimental adjunctive therapy? Some authors 8,27 have queried whether endotoxin should still be considered an appropriate target candidate in devising new treatment strategies for Gram-negative sepsis. A positive answer to that question is encouraged by results of recent animal experiments with anti-CGL mAb,13,14,59 human recombinant bactericidal permeabilityincreasing factor27,60–62 and other agents, such as non-toxic lipid A-derivatives, non-toxic polymyxins and polyvalent anti-O-side-chain antibody preparations, that antagonize, neutralize or induce tolerance to endotoxin.63 We are still far from understanding the pathophysiological events shaping the diagnostic entities currently defined as systemic inflammatory response syndrome (SIRS) and sepsis.64 Although these entities are used to screen patients for entry in phase II and III trials of adjunctive treatment for sepsis, it is increasingly recognized that they select groups of patients that are highly heterogenous in terms of predicted mortality risk. We do not know when and why the effects of endotoxin and other activators of the host defence system are vital and beneficial to one patient but harmful to another.65 As Cohen66 has stated, 479 W. N. M. Hustinx et al. “. . . to demonstrate that these mediators [endotoxin and cytokines] play a part in the pathogenesis of sepsis is not the same as showing that neutralizing or removing them is necessarily beneficial.” This problem is well illustrated by several reports suggesting that certain experimental adjunctive treatments may have adverse or divergent effects.67–71 11. Collins, H. H., Cross, A. S., Dobek, A., Opal, S. M., McClain, J. B. & Sadoff, J. C. (1989). Oral ciprofloxacin and a monoclonal antibody to lipopolysaccharide protect leucopenic rats from lethal infection with Pseudomonas aeruginosa. Journal of Infectious Diseases 159, 1073–82. Conclusions 13. Bhattacharjee, A. K., Opal, S. M., Palardy, J. E., Drabick, J. J., Collins, H., Taylor, R. et al. (1994). 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Immunotherapy of Gram-negative bacterial sepsis: a single murine monoclonal antibody provides cross-genera protection. Archives of Surgery 121, 58–62. 78. McCabe, W. R., DeMaria, A., Berberich, H. & Johns, M. A. (1988). Immunization with rough mutants of Salmonella minnesota: protective activity of IgM and IgG antibody to the R595 (Re chemotype) mutant. Journal of Infectious Diseases 158, 291–300. 79. Salles, M.-F., Mandine, E., Zalisz, R., Guenounou, M. & Smets, P. (1989). Protective effects of murine monoclonal antibodies in experimental septicemia: E. coli antibodies protect against different serotypes of E. coli. Journal of Infectious Diseases 159, 641–7. 80. Appelmelk, B. J., Cohen, J., Silva, A., Verwey-van Vught, A. M. J. J., Brade, H., Maaskant, J. J. et al. (1990). Further characterization of monoclonal antibodies to lipopolysaccharide of Salmonella minnesota strain R595. Advances in Experimental Medical Biology 256, 319–30. 81. Silva, A. T., Appelmelk, B. J., Buurman, W. A., Bayston, K. F. & Cohen, J. (1990). Monoclonal antibody to endotoxin core protects mice from Escherichia coli sepsis by a mechanism independent of tumor necrosis factor and interleukin-6. Journal of Infectious Diseases 162, 454–9. 82. Lun, M. T., Amatucci, A. M., Raponi, G., Filadoro, F., Bartolazzi, A., Fraioli, R. et al. (1994). Murine monoclonal antibody elicited with antibiotic-exposed Escherichia coli exerts protective capacity in experimental bacterial infections. Journal of Medical Microbiology 41, 179–83. 69. Luce, J. M. (1993). Introduction of new technology into critical care practice: a history of HA-1A human monoclonal antibody against endotoxin. Critical Care Medicine 21, 1233–41. 83. Mullan, N. A., Newsome, P. M., Cunnington, P. G., Palmer, G. H. & Wilson, M. E. (1974). Protection against Gram-negative infections with antiserum to lipid A from Salmonella minnesota R595. Infection and Immunity 10, 1195–201. 70. Netea, M. G., Blok, W. L., Kullberg, B.-J., Bemelmans, M., Vogels, M. T. E., Buurman W. A. et al. (1995). Pharmacologic inhibitors of tumor necrosis factor production exert differential effects in lethal endotoxemia and in infection with live microorganisms in mice. Journal of Infectious Diseases 171, 393–9. 84. Ng, A.-K., Chen, C.-I. H., Chang, C.-M. & Nowotny, A. (1976). Relationship of structure to function in bacterial endotoxins: serologically cross-reactive components and their effect on protection of mice against some Gram-negative infections. Journal of General Microbiology 94, 107–16. 482 Zinc ion availability and common colds 85. Peter, G., Chernow, M., Keating, M. H., Ryff, J. C. & Zinner, S. H. (1982). Limited protective effect of rough mutant antisera in murine Escherichia coli bacteremia. Infection 10, 228–32. 86. Trautmann, M. & Hahn, H. (1985). Antiserum against Escherichia coli J5: a reevaluation of its in vitro and in vivo activity against heterologous Gram-negative bacteria. Infection 13, 140–5. 87. Appelmelk, B. J., Verweij-van Vught, A. M. J. J., Maaskant, J. J., Schouten, W. F., de Jonge, A. J. R., Thijs, L. G. et al. (1988). Production and characterisation of mouse monoclonal antibodies reacting with lipopolysaccharide core region of Gram-negative bacilli. Journal of Medical Microbiology 26, 107–14. 88. Baumgartner, J.-D., Heumann, D., Gerain, J., Weinbreck, P., Grau, G. E. & Glauser, M. P. (1990). Association between protective efficacy of anti-lipopolysaccharide (LPS) antibodies and suppression of LPS-induced tumor necrosis factor and interleukin 6. Journal of Experimental Medicine 171, 889–96. Received 7 January 1997; accepted 30 April 1997 JAC Journal of Antimicrobial Chemotherapy (1997) 40, 483–493 Zinc ion availability—the determinant of efficacy in zinc lozenge treatment of common colds George A. Eby* George Eby Research, 2109 Paramount Avenue, Austin, TX 78704, USA This is a re-analysis of reports from 1984 to 1992 of double-blind, placebo-controlled, clinical trials of zinc lozenges in the treatment of common colds. This re-analysis was performed to test the hypothesis that major variations in daily zinc ion availability (ZIA) between chemically different lozenge formulations caused differing results in these clinical trials. Solution chemistry computations determined the bioavailability of Zn2 + ions at physiological pH from the lozenges used in these clinical trails. ZIA was derived from Fick’s laws of diffusion in a bio-electric field. Lozenges that released Zn2 + ions at physiological pH (positive ZIAs) shortened colds. Lozenges that released negatively charged zinc species at physiological pH (negative ZIAs) lengthened colds. Lozenges having a zero ZIA had no effect on common colds. Lozenges with ZIA = 100 shortened colds by 7 days while ZIA = – 55 lozenges lengthened colds by 4.4 days. A linear dose–response relationship exists between ZIAs of zinc lozenges and changes in duration of common colds. It is concluded that: prospective efficacy of zinc lozenges can be predicted based upon readily determined ZIA factors and ZIAs; chemically different zinc lozenge formulations having greatly different ZIAs resulted in greatly differing results in clinical trials; mast cell granule-derived Zn2 + ions are the foundation of the primary immune system; and high ZIA zinc acetate lozenges are beneficial for common colds. Introduction In 1984, the present author and co-workers1 reported that lozenges releasing 23 mg zinc (from zinc gluconate) shortened the duration of common colds by an average of 7 days in a double-blind, placebo-controlled clinical trial. No other soluble excipients were present in the unsweetened lozenges. The original report was confirmed by Al-Nakib et al.2 at the Medical Research Council (MRC) Common Cold Unit in 1987 using 23 mg of zinc from zinc gluconate in flavoured fructose tablet. *Tel: 1-(512)-442-2933; Fax: Other clinical research using zinc gluconate and food acid additives, low zinc gluconate dosages and other zinc compounds was published from 1987 to 1992. These reports showed conflicting results. Potter & Hart3 reviewed all the published literature in 1993. Unfortunately, they did not consider (i) solution chemistry analyses of the lozenges used in the reviewed trials, (ii) unpublished data available only from lozenge manufacturers and authors of the original reports and (iii) analyses of the bioavailability of Zn2 ions to the oral mucosa at physiological pH. This re-analysis includes that omitted information, and shows that the bio- 1-(512)-442-2933; E-mail: coldcure bga.com 483 © 1997 The British Society for Antimicrobial Chemotherapy G. A. Eby availability of Zn2 ions at physiological pH is associated with positive results and that the absence of bioavailable Zn2 ions at physiological pH is associated with negative results. Common cold aetiology New data obtained by polymerase chain reaction techniques indicates that about 60–70% of upper respiratory viral infections (common colds) are caused by rhinoviruses.4 Common cold symptoms are generally believed to be caused by over-reaction of the immune system to these relatively harmless viruses. In-vitro effects of Zn2 ions on rhinoviruses Zn2 ions at a 0.10 mM concentration were demonstrated by Korant, Butterworth and their co-workers5–9 to be highly effective antirhinoviral agents inhibiting cleavage of rhinovirus polypeptides in vitro. Merluzzi et al.10 showed that the effects of zinc on rhinovirus replication were related to the concentration of Zn2 ions and unrelated to the total amount of zinc. They also showed that the antirhinoviral effect of Zn2 ions was as strong as the antirhinoviral effect of interferon at its most effective concentration10 No invitro effects were found by Geist et al. 11 below 0.03 mM, a finding in agreement with those of the others.5–10 Other researchers have found that Zn2 ions at about 0.10 mM induce the production of large quantities of interferon and are mitogenic to T-cell lymphocytes in vitro.12–14 Changes in cell appearance suggested toxicity of Zn2 ions to some authors,10,11 but many others strongly assert that Zn2 ions lack cytotoxicity and that, rather, they cause potent stabilization of cell membranes, drying effects, astringency and anti-inflammatory action.15–23 Astringency versus pseudo-astringency Zn2 ions are well known for their astringency. Pasternak15 has shown that elevated Zn2 ion serum concentrations are a form of host defence acting to stabilize and protect cell membranes. All astringents, including Zn2 ions, are locally applied protein precipitants having such low cell penetrability that their action (contraction, rounding and blanching in vitro) is essentially limited to cell surfaces and interstitial spaces. Permeability of cell plasma membranes is reduced by astringents, including Zn2 , but the cells remain viable. Astringents harden the cement substance of capillary epithelium, inhibiting pathological transcapillary movement of plasma proteins, and reduce local inflammation, oedema, and exudation. Additionally, astringents reduce mucus and other secretions in tissues containing goblet cells and other secretory cells, causing affected areas to dry and heal faster.16 Pseudo-astringents include anionic metal complexes in solution, acids, alcohols, phenols and other substances that precipitate proteins. They may seem astringent to the mouth but they readily penetrate cells and promote tissue damage.16 Food acid flavoured zinc lozenges caused sufficient oral tissue irritation in several trials to imitate the oral tissue irritation reported in the successful studies by Eby et al.1 and Al-Nakib et al.2 and this pseudo-astringency incorrectly convinced several common cold researchers of the availability of zinc ions from their research lozenges. Only analysis by solution chemistry methods enables the correct determination of availability of metallic ions at physiological pH. Clinical trials re-analysed Contact with lozenge manufacturers and clinical researchers to obtain critical missing lozenge solution chemistry data and trial data was essential in order to provide a valid re-analysis. All clinical trials re-analysed here1,2,24–29 used accepted, double-blind, placebo-controlled, clinical research methods. All known reports of zinc lozenges as therapy for common colds are included. With the exception of two clinical trials,26,28 all patients were prohibited from using other common cold medications. No patients suffering from allergies or other diseases or conditions that might mislead the results were included in these studies. No trial permitted enrolment of pregnant women. All but two trials 2,29 reported results from natural colds. The demographics of patients in each trial were found to be well randomized. The number of patients in the clinical trials varied from 55 to 146. Methods The zinc ion availability (ZIA) method of analysis was developed to evaluate the amount of Zn2 ions available at physiological pH for absorption into the oral and oropharyngeal tissues, and to determine if the in-vivo dose–response to Zn2 ions was similar to the in-vitro dose–responses shown by Merluzzi et al.10 The review also evaluated the hypothesis that Zn2 ions in saliva diffuse into the infected tissues of the nose from the membranes of the oral cavity via oral–nasal tissues. Fick’s laws of diffusion were used to derive both the ZIA method of analysis and diffusion theories. Fick’s laws of diffusion Fick’s first law of diffusion is dm/dt DA dc/dx, where m is the quantity of drug or solute diffusing in time t, dm/dt is the rate of diffusion, D is the diffusion constant, A is the cross-sectional area of the membrane, dc is the change in concentration, and dx is the thickness of the membrane.30 Fick’s second law of diffusion is similar and relates to diffusion through solids. The rate of drug transport over time can be altered by a change in any of the above variables. If both the cross-sectional area of the oral cavity and the rate 484 Zinc ion availability and common colds of mouth-to-nose diffusion is held constant and lozenge dissolution times are introduced, ZIA can be calculated.26 Zinc ion availability Zinc ion availability is the term used to identify the potential for absorption of Zn2 ions at physiological pH 7.4 into oral and oropharyngeal mucosal membranes. It is given by ZIA KZiT, where K is 0.7697 (the constant used to set ZIA value from the Eby et al. trial1 to the reference value of 100), Zi is the initial concentration (mM) of Zn 2 ions and T is the daily duration of oral contact time. For comparative purposes, ZIA can be determined by multiplying the constant 0.7697 by the zinc dosage (mg) in the lozenge the fraction of zinc available as Zn2 ion at pH 7.4 the oral dissolution time (min) of each lozenge the number of lozenges used per day, and dividing by the volume of saliva (mL) generated during each oral dissolution. For example, the ZIA calculation for the lozenges used by Eby et al.1 is: 0.7697 23 (mg zinc/lozenge) 0.30 (fraction of zinc as Zn2 ion at pH 7.4) 30 (dissolution time in minutes) 9 (doses per day), divided by 14.34 (mL saliva—which numerically equals the total saliva weight minus lozenge weight). Specific gravity is considered for soluble lozenges. Initial Zn2 ion concentrations are 50–100 times greater than the concentration needed to have pharmacological activity in vitro. For example, the successful Eby et al.1 lozenges produced a salivary Zi concentration of 7.4 mM, while the pharmacologically active in-vitro concentration is only 0.1 mM. However, many Zn2 ions are lost during their diffusion through tissues from interactions with diverse ligands found in saliva, blood, lymph and tissues. stability constant for the metal and its associated ligand. The basic solution chemistry equation, [CM] [M] [ML], where [CM] is the total metal concentration, [M] is the concentration of metallic ions and [ML] K [ML][L] (where [L] is the free concentration of ligand and K is the stability constant of the metal–ligand complex), results in the equation [CM] [M](1 K[L]). Consequently, [M] [CM]/(1 K[L]), which shows that [M] depends upon [CM], K and [L] which in turn depend upon corresponding pK and pH values (personal communication, G. Berthon, Director of Research, INSERM, U-305, Toulouse, France). Figure 1 shows that 30% of the zinc from zinc gluconate is available as Zn 2 ions at physiological pH. 33 Because of the extremely low affinity of sweet carbohydrates for Zn2 ions,34 Figure 1 also applies to aqueous solutions of most sweet carbohydrate-based zinc gluconate lozenges. Figure 2 shows an absence of Zn2 ions at each pH greater than 6 in the system with a 1:1.33 ratio of zinc gluconate to citric acid and the presence of negatively charged zinc citrate species at physiological pH. Figure 3 shows an absence of Zn2 ions at each pH greater than 6 in the system with a 1:10 ratio of zinc gluconate to glycine and presence of mainly neutrally charged zinc glycinate at physiological pH 7.4. Similarly, the bioavailability of Zn2 ions for other commonly used zinc compounds is shown in Figure 4. Measuring common cold treatment responses Half-lives of common colds and weighted average durations of exponentially decaying common colds have been Physiological pH The only relevant pH the for purposes of determining the availability of Zn2 ions in tissue, lymph and blood is physiological pH 7.4. This results from all acids and bases being instantly buffered by the bicarbonate, phosphate and protein buffer systems in tissue, lymph and blood during homeostatic regulation of acid–base balance.31,32 The Zn2 ion is a Lewis acid capable of existing at physiological pH 7.4.7 Salivary pH was 5.5 when zinc gluconate lozenges having no other zinc ligands were used. Salivary pH ranged from pH 4.3 to 7.0 in lozenges containing other zinc salts.26 In instances when the salivary or infected respiratory secretion pH is lower than physiological pH, Zn2 ion concentration is higher. However, these higher concentrations are reduced as a result of being instantly buffered to pH 7.4 upon absorption.26 Bioavailability of metallic ions Bioavailability of metallic ions, including Zn2 ions, occurs only at physiological pH 7.4 and is dependent upon the first Figure 1. Distribution of zinc ionic species in the Zn2 and gluconic acid (L) system. Curves were constructed from pK values shown after the reactions: Zn2 L ZnL (1.62) and ZnL OH ZnL(OH) 0 (8.14) at a concentration of 30 mM zinc. The pK values are courtesy of Gerritt Bekendam, Akzo Chemicals BV Research Centre, Deventer, The Netherlands. The Zn2 fraction over pH 6 is strongly affected by the second pK value. Precipitates of hydroxides of zinc result in supersaturated solutions above pH 7.4. (Figure reproduced with permission.33) 485 G. A. Eby Figure 2. Distribution of Zn2 species plotted against pH for solution with Zn2 and excess citric acid (H3L). At any pH, the curves sum to unity. Curves were constructed from the stability constant logarithms in parentheses after each reaction: Zn2 HL2 ZnLH (3.0), Zn 2 L3 ZnL (4.8), and ZnL 4 3 L ZnL2 (1.7). Except for the ratio of the last two complexes, the general shapes of the curves are independent of the specific concentrations of 18 mM Zn2 and 23 mM citric acid. Successive pKa values for citric acid deprotonations are pK1 3.0, pK2 4.4 and pK3 5.8. Not included in the analysis is a small amount of ZnLH2 of low stability likely to form near pH 3. Figure reproduced with permission.38 Figure 3. Distribution of Zn2 , positively charged zinc gluconate (ZnL ), and various zinc glycinate and zinc hydroxide species in the 1:10 M zinc gluconate–glycinate system. Zinc and gluconate are present at 30 mM and glycine is present at 300 mM concentration. Distribution of zinc ionic species is courtesy of G. Berthon, INSERM U 305, CNRS, Toulouse, France.26 shown to be acceptable analytical mechanisms.1 Half of untreated rhinovirus colds resolve in 1 week, threequarters resolve in 2 weeks, while seven-eighths last 3 weeks or less,35 which indicates an exponential decay rate, with a half-life (T1) of 7 days. By integrating, the average duration is T1/ln 2.1,26 If the observed T1 of untreated colds is 7 days, then the calculated average duration of those same colds is 10 days. The accepted model for calculating treatment efficacy is to subtract the T1 of actively treated Figure 4. Percentage of zinc released as biologically available Zn2 ions at pH 7.4 from zinc compounds used in common cold clinical trials plotted against the logarithm of the first stability constant K1. colds from the T1 of placebo-treated colds, and likewise for the average duration of colds. Methods of obtaining the average duration of colds differed slightly among trials because the researchers’ methods of reporting were not uniform. Average duration data and half-life data were published for the Eby et al. zinc gluconate trial. 1 Mean clinical score end-points were used to calculate the average duration of colds in the Al-Nakib et al. zinc gluconate trial.2 In the Smith et al. zinc gluconate trial,24 average durations were calculated from half-lives obtained from published figures and data. Average durations of colds were stated by the authors in the Weismann et al. zinc gluconate,25 Eby et al. zinc orotate,26 McCutcheon et al. zinc aspartate,26 Douglas et al. zinc acetate–tartarate– carbonate,27 and Godfrey zinc gluconate–glycine28 trials. In the case of the zing gluconate–glycine trial,28 this reanalysis showed that the claimed reduction in duration was erroneously reported, because data were obtained by subtracting observed half-lives of colds from historic ‘average duration’ of colds. Average duration of colds in the zinc gluconate–citrate trial29 were determined from day 7 symptom scores. Lozenge side-effects and safety Documented side effects and safety data were taken directly from the reports of clinical trials of zinc lozenges. Statistical methods Statistical methods used in each trial’s research report were cited as originally reported. Spearman’s rank difference correlation method was used in analysis of the relationship between lozenge ZIA values and their effects on common colds. 486 Zinc ion availability and common colds Figure 5. Relationship of zinc ion availability (ZIA) values and reduction in duration of common colds in days. 0.96. The regression equation for non-negative values is y 0.077x 0.16. Straight line projection using this equation and increases in duration data provided estimates for negative ZIAs. There is no 1:10 M zinc gluconate–glycine data point28 because it is a horizontal line in the upper left quadrant. Results The main finding of this re-analysis is the dose–response linearity shown in Figure 5. Evidence of dose–response linearity is sufficient to reject the null hypothesis that there is no relationship between ZIAs and changes in duration of common colds. ZIAs—not total zinc—provide a means of evaluating zinc lozenges of different formulations. Each of the reports listed in Tables I and II and represented in Figure 5 is briefly summarized below. In the original double-blind, placebo-controlled clinical trial by Eby et al.,1 23 mg zinc (175 mg zinc gluconate) unsweetened lozenges reduced the duration of natural colds by an average of 7 days compared with placebo. The reduction in average duration of colds was highly significant (P 0.10 at 12 h, P 0.008 at 24 h, and P 0.0005 at day 7). These zinc gluconate lozenges were assigned a ZIA of 100 and hence, using the equation ZIA KZiT, the value of the constant K, was determined to be 0.7697. Al-Nakib et al. reported the effects of pleasant-tasting, highly flavoured, 23 mg zinc (175 mg zinc gluconate), fructose-based lozenges. As a treatment, zinc gluconate lozenges shortened experimentally-induced rhinovirus-2 colds by a statistically significant average of 4.8 days compared with placebo. Although Zn2 ion release was the same as in the 1984 Eby et al. trial,1 the Al-Nakib et al. lozenges dissolved faster and, thus, these zinc gluconate lozenges had a calculated ZIA of 44. In sharp contrast, Smith et al. in 198924 reported the effect of bitter, 11.5 mg zinc (87.5 mg zinc gluconate) sucrose-, fructose-, mannitol- and sorbitol-based lozenges in a double-blind, placebo-controlled, clinical trial of natural colds in 174 patients. On day 7 there were 12% fewer ill patients in the zinc-treated group than in the placebo group (P 0.09), and the severity of zinc-treated colds on days 5–7 was reduced by 8% (P 0.02). Compared with placebo, the average duration of common colds was reduced by 1.6 days. These mildly astringent lozenges had a calculated ZIA of 25 because of low zinc gluconate content, rapid lozenge dissolution and bitterness-induced deviation from the clinical trial protocol. In 1990, Weismann et al .25 found no reduction in common cold duration from very low dose zinc gluconate lozenges in a double-blind placebo-controlled trial of natural colds involving 130 patients in Denmark. These non-astringent, maltitol-based, hard-boiled candy lozenges had a low ZIA of 13.4 because they contained only 4.5 mg zinc (31.3 mg zinc gluconate). Very low zinc dosage was used in an effort to avoid the extreme bitterness of zinc gluconate in maltitol-based lozenges. Non-astringent 37 mg zinc (zinc orotate) lozenges in conjunction with 10 mM zinc gluconate nasal spray were found to have no effect on the duration of colds in a 1984 doubleblind, placebo-controlled trial of natural colds in 77 patients by Eby et al.26 At physiological pH 7.4, zinc orotate is an insoluble, non-ionizable compound having a first stability constant of log K1 6.42 at 25°C. 36 The ZIA was zero because no Zn2 ions were released. Non-astringent 24 mg zinc (zinc aspartate) lozenges and paracetamol produced no significant differences in responses compared with placebo and paracetamol in a double-blind trial of natural colds in 100 patients (personal communication, M. L. McCutcheon, Student Health Director, University of Minnesota, Duluth, MN, USA).26 Zinc aspartate tastes pleasant and has a log K1 at 37°C of near 5.9.37 Because of the protonated ammonium group, the 487 G. A. Eby Table I. Zinc ion availability (ZIA) factors26 Trial lozenge/reference (formulation) Zinc gluconate 23 mg1 (non-soluble compress) Zinc gluconate 23 mg2 Zinc gluconate 11.5 mg24 (soluble multi-ingredient compress) Zinc gluconate 4.5 mg25 (soluble maltitol candy) Zinc orotate26 (non-soluble compress) Zinc aspartate26 (soluble multi-ingredient compress) Zinc gluconate-citrate29 (soluble corn syrup–sucrose candy) Zinc acetate–tartarate–carbonate27 (soluble mannitol compress) Zinc gluconate–glycine28 (soluble corn syrup–sucrose candy) Zinc dosagea (mg) Fraction of zinc as Zn2 at pH 7.4 Dissolution timeb (min) Doses per dayc Total salivad (mL) Lozenge weighte (g) 23.0 0.3 30 9 15.0 0.66 23.0 11.5 0.3 0.3 19 15 9 10 22.0 17.5 1.00 1.56 4.5 0.3 15 12 17.0 3.00 37.0 0.0 40 6 18.0 3.00 24.0 0.0 14 9 18.0 3.00 23.0 0.0 15 9 35.0 4.50 10.0 0.0 10 6 44.0 3.00 23.7 0.0 15 9 26.3 4.50 a Amount of zinc per lozenge. Mean time required for lozenge to dissolve in the mouth. Oral dissolution times of lozenges are considerably greater than those found using USP disintegration tests. c Mean number of doses actually taken, rather than intended number. d Total volume of saliva generated from use of a lozenge. e Actual weight of the lozenges tested. Specific gravity of soluble lozenges is 1.5. b Table II. Trial lozenges, ZIA, Zn2 ion concentration, electronic charge of zinc, and efficacy of treatment26 Trial lozenges/reference Zinc gluconate 23 mg1 Zinc gluconate 23 mg2 Zinc gluconate 11.5 mg24 Zinc gluconate 4.5 mg25 Zinc orotate26 Zinc aspartate26 Zinc gluconate–citrate29 Zinc acetate–tartarate–carbonate27 Zinc gluconate–glycine28 ZIA m/Mol Zn2 ions at pH 7.4 (Z1) 100 44 25 13.4 0 0 11a 55a Indeterminate 7.4 5.0 3.3 1.5 0 0 0 0 0 Electronic charge of zinc species at pH 7.4 2 ,1 2 ,1 2 ,1 2 ,1 0 0 0, N 0, N 0, N ,0 ,0 ,0 ,0 Change in duration of common colds 7 day reduction 4.8 day reduction 1.6 day reduction none none none 1 day increase 4.4 day increase 1.27 day reduction a Negative ZIA values were estimated using increase in duration data and straight-line projection of non-negative values using formula shown in Figure 5. conditional first stability constant is log K1 2.9 at pH 6.8.38 The high stability constant prevented significant Zn2 ion release at pH 7.4; therefore, ZIA was barely positive. Zinc gluconate (23 mg zinc) candy lozenges with 90 mg citric acid (1.33 moles citric acid to 1 mole zinc gluconate) as a flavour-mask were tested by Farr et al. in 1987 in trials of clinically induced human rhinovirus-13 and -39 colds in 55 individuals.29 Compared with placebo lozenges, zinc 488 Zinc ion availability and common colds gluconate–citrate lozenges worsened cold severity and lengthened them by a day. The log K1 of zinc citrate is 4.7 at 37°C.38,39 Zinc, lightly bound to gluconate, readily dissociates and strongly and instantly binds to citrate. Figure 2 shows that the reaction produces only negatively charged zinc species at physiological pH 7.4.38,39 Pseudo-astringency and a mild acidic taste were caused by anionic zinc species at physiological pH16 and the ZIA was estimated to be 11. Douglas et al.27 reported that effervescent zinc acetate lozenges containing 10 mg zinc increased the average duration of natural colds by 4.4 days in 70 patients. Several hundred milligrams of tartaric acid and sodium bicarbonate were included to produce effervescence (personal communication, R. J. E. Williams, Development Scientist, F. H. Faulding & Co., Adelaide, Australia). Tartaric acid was present in considerable excess relative to zinc, and it has a high log K1 of 5.00.40 Zinc dissociates from acetate and binds instantly to tartarate. Few Zn2 ions remain available at physiological pH 7.4 and negatively charged zinc species predominate. Anionic zinc species caused pseudo-astringency. The ZIA for tartarate- and carbonate-complexed zinc acetate lozenges was estimated to be 55 because of negatively charged zinc tartarate species. Godfrey et al.28 reported results of a double-blind, placebo-controlled, clinical trial involving 87 patients. Active lozenges contained 23 mg zinc with 10 mol glycine/ mol of zinc gluconate as flavour-mask in 4.5 g sucrose and corn syrup hard-boiled candy lozenges. Both active- and placebo-treated groups were also given paracetamol. Reanalysis of data and figures provided by the authors suggests a reduction in the mean duration of natural common colds by 1.27 days by zinc compared with placebo.28 Published results calculated the reduction in mean duration of colds incorrectly by comparing half-lives of colds with the historical mean duration of colds. Due to this error, the authors’ conclusions that the lozenges shortened the mean duration of colds by 42% and the placebo was efficacious, are incorrect. Log K1 values for zinc–glycine complexes are slightly higher than for zinc–citric acid: 4.8 at 37°C,41 and 4.7 at 37°C.38,39 Figure 3 shows that zinc is largely dissociated from gluconate and bound to glycine in this 1 M zinc gluconate and 10 M glycine solution. Only neutral zinc glycinate, minor amounts of negatively charged zinc glycinate species and traces of negatively charged zinc hydroxide exist at pH 7.4. Because of these computational errors and the lack of positively charged zinc species at physiological pH, a ZIA cannot be determined. ZIAs ZIAs derived from the clinical trials are shown in Table II. The initial concentration of Zn 2 ions at pH 7.4 (Z i), electronic charges of zinc species at pH 7.4, and treatment efficacy of lozenges are presented in Table II. Details of ZIA calculations, previously unpublished manufacturers’ lozenge formulas, methods and manufacturing procedures of lozenges having a major impact upon lozenge ZIAs, and other data too complex or lengthy for presentation in this re-analysis have been published elsewhere.26 Dose–response linearity between ZIAs and common cold duration Non-negative data from Table II were analysed to ascertain the correlation between ZIA and reduction in common cold duration. The reduction in duration (response) and ZIA (dose) were linearly related with 0.96 (P 0.02, two-tailed test). Regression analysis of these data points yielded the equation y 0.077x 0.16 as shown in Figure 5. Safety of zinc lozenges None of the zinc or placebo lozenges were harmful. Side effects varied with each clinical trial due to differing formulations.1,2,24–29 Discussion The central finding of this report is linearity in the dose–response relationship between studies using zinc lozenges having positive ZIAs, when ZIA is used as a measure of zinc dosage and the duration of symptoms of common colds as the response. Linearity demonstrates that, through consideration of ZIA, divergent results are completely reconciled and the correlation is consistent with the findings of Merluzi et al.10 of activity of Zn2 ions against rhinovirus replicationin vitro. Although the linear relationship between ZIA and clinical efficacy of trials is clear, the evidence for efficacy relies upon three zinc gluconate trials.1,2,24 Null results from trials of lozenges having low, zero, and negative ZIA values show that Zn2 ions are needed for efficacy, and these findings are not in conflict with positive findings. Indeed, they clearly support the in-vivo Zn2 ion antirhinoviral activity theory presented by Eby et al .1 and they strongly support the theory that lozenges containing any zinc compound having the same ZIA value will have the same effect on the duration of common colds. The zinc gluconate lozenges having a molar proportion of glycine tested by the Cleveland Clinic Foundation were reported in 1996 to reduce the T1 of natural common colds by about one-half.42 Unpublished zinc speciation calculations by Guy Berthon showed that most zinc was in the form of zinc glycinate1 at physiological pH. Using Figure 5 and the relationship between the T1 of common colds and their average duration, a ZIA of 70 for these lozenges can be deduced when used six times per day. Prasad, in his accompanying editorial,43 suggested that the ligand 489 G. A. Eby gluconate was the likely cause of bad taste associated with zinc gluconate lozenges, and such problems might have been avoided had zinc acetate been used instead. Generally, carbohydrates do not bind zinc. For example, dextrose has a first stability constant for zinc of log K1 0.0.34 This lack of stability results in essentially no competition by dextrose as a zinc binding agent compared with gluconate (log K1 1.70) or acetate (log K1 1.0).44 However, zinc chloride and dextrose have identical first stability constants, resulting in reactions that discolour white lozenges. On the other hand, zinc chelators, also known as metal binding agents or sequestrants, tightly bind Zn2 ions at physiological pH. Zinc ions are consequently rendered biologically unavailable if zinc chelators are present in sufficient amount. Dependent upon the amount, ascorbic acid, citric acid, EDTA, salicylic acid, tartaric acid, amino acids and other food acids, acacia (gum arabic) and other vegetable gums, alkalis and their carbonates, oxalates, phosphates, sulphides, caustic lime, lake food colours, histamine, histidine, proteins, porphyrins, peptides and other macromolecules can be incompatible with release of Zn2 ions from zinc compounds at physiological pH.45,46 All water-soluble anionic chemicals bind zinc to some extent and should be excluded from zinc lozenges. The validity of negative estimates of ZIA might be questioned, but the highly significant fact remains that lozenges releasing negatively charged zinc species (ZnLN ) increased the duration of common colds in a dose–response manner compared with placebo. Negative ZIAs are possible only because Zn2 ions are released by the human immune system mast cell granules in their natural fight against viruses that cause common colds. In mast cells, which are plentiful in the respiratory tract, and basophil granules, Zn2 ions are highly concentrated (4–20 mM) and unsequestered.47 In inflammation Zn2 ions are released during degranulation of these cells.48 ZnLN from lozenges preferentially bind with positively charged substances, primarily Zn2 ions released from these cells, and neutralize them to a zero electrical charge. If release of Zn2 ions from cells during inflammation in common colds inhibits viral replication,5–10 stabilized cellular plasma membranes,12–20 activates T-cells,21–23 catabolizes histamine,37,40 regulates mast cell homeostasis48 and stimulates massive interferon production21–23 as they do in vitro, then one must infer: (i) common colds would be worsened by ZnLN by neutralization of native mast cell originated Zn2 ions to a neutrally charged, biologically useless species at physiological pH, and (ii) mast cell derived Zn2 ions are important in the mucosal primary immune system. Therefore, Zn2 ions from ZIA-positive lozenges would amplify natural immunity and limit the duration of common colds. As rhinovirus infection occurs in the nose, logic might suggest that Zn2 ions should be applied to the nose using nose drops or nasal sprays, not to the oral mucosa by lozenges or to the stomach by pills. Furnace analysis atomic absorption spectrophotometry demonstrated that zinc was not found in nasal mucus as result of zinc gluconate lozenge oral dissolution (personal communication, J. M. Gwaltney, University of Virginia School of Medicine, VA, USA) suggesting lozenge inactivity and, perhaps, that nose drops would be preferable to lozenges. Since 1903, a range of zinc compounds have been used intranasally as a mild, short-acting decongestant but have shown no evidence of a reduction in the duration of common colds. Zinc gluconate nasal spray used every 15–30 min in conjunction with zinc orotate lozenges has been reported to have a decongestant effect but no effect upon the duration of the common cold.26 Frequent nasal application of Zn 2 ions (0.1% w/v zinc sulphate) did not reduce the duration of the common cold but did show a mild decongestant effect (personal communication, D. BryceSmith, University of Reading, UK). Thus, more than exposing nasal tissue to Zn2 ions must be involved in reducing the duration of common colds. The transport of metallic ions long distances through naturally occurring biologically closed electrical circuits (BCECs) was described in great detail by B. E. W. Nordenström of the Karolinska Institute in Stockholm in 1983.49 Among his findings, Nordenström showed that electrical potentials unrelated to nervous system electrical activity are generated by infected, injured, and cancerous tissues as well as healthy muscles. Nordenström also showed that metallic ions adhered to the inside of capillaries, thus changing the charge of capillary walls from negative to positive, thereby providing a conduit for other positively charged metallic ions to move long distances. He also demonstrated that Fick’s laws of diffusion apply for metallic ions in a bio-electric field. Nordenström’s observations suggested the existence of a BCEC between the mouth and nose as a possible explanation for the Zn2 ion lozenge effect on colds. Evidence of a BCEC was found when an 80–120 mV potential difference was observed between the interior of the mouth and the interior of the nose in 19 unrelated, healthy volunteers.26 Placement of one electrode of an ohmmeter on to an oral cavity surface and the other on to a nasal turbinate usually registered either a 10,000 reading or a reading over 15,000 , dependent only upon electrode placement (polarity). The lowest resistance values found were 1000 and 1500 in an individual with chronic nasal drainage and frequent colds, while an individual extremely resistant to upper respiratory infections had the highest mouth–nose differential resistance readings of 50,000 and 100,000 . A significant resistance change always resulted when electrodes were reversed, mimicking a diode effect. Electron flow produced by the ohmmeter was either retarded or assisted by the electron flow in the mouth–nose BCEC producing the different resistance readings. The mouth–nose BCEC moves Zn2 ions through the oral mucosa and into the infected superficial columnar cells of the nasal turbinate epithelium and nasopharynx, result- 490 Zinc ion availability and common colds ing in efficacy from oral cavity Zn2 ion application. Conversely, inefficacy of nasal administration of Zn2 ions is due to the outflow of electrons from the surface of nasal tissue as well as outflow of nasal mucus and action of cilia. Is Prasad43 correct in suggesting that zinc acetate should be preferred over zinc gluconate? The log K1 of zinc acetate is 1.0.44 Hacht & Berthon50 demonstrated that 100% of zinc acetate is released as Zn2 ions at any pH between 2.8 and well above 7.4. In addition, zinc acetate is sufficiently stable to be non-reactive with sweet carbohydrates. Korants demonstrated the rhinovirus replication inhibition properties of zinc acetate in 1974.5 Anecdotal evidence—evinced by strong repeat purchases of ZIA 100 zinc acetate lozenges in the USA— supports Prasad’s suggestion. However, only carefully conducted clinical trials will prove or disprove efficacy of zinc acetate lozenges against common colds, and those trials are of immediate international importance. Unlike some unhelpful zinc gluconate lozenges, zinc acetate lozenges are flavour-stable and do not become bitter regardless of time or storage conditions. 51–53 Properly prepared ZIA 100 zinc acetate lozenges have no objectionable taste or aftertaste in common cold treatment. They do not seem to produce side-effects previously associated with zinc gluconate lozenges, perhaps because much less zinc acetate is required to produce identical results than zinc gluconate or any other suitable zinc compound.26 One simple way to determine a priori the likely efficacy of commercial ‘zinc lozenges’ is to taste-test them. Astringent lozenges suggest efficacy is likely. However, oral astringency is less noticeable in people with colds than in well people. This difference could be due to oral, facial and nasal tissues being more permeable in illness, resulting in more rapid absorption and removal of Zn2 ions from the oral mucosa. On the other hand, an acidic taste – usually from citric or ascorbic acid – in some commercial zinc gluconate lozenges available throughout the USA in 1997 is now being associated anecdotally with worsened colds, suggesting a negative ZIA. Additionally, there are zinc gluconate lozenges on the market in the USA that have very low positive ZIAs. Other commercial lozenges containing zinc aspartate have a very low positive ZIA and have essentially negligible effect on common colds. Variations in clinical trial results reviewed above have previously been attributed to placebo unblinding and faulty positive results. The necessity of preventing placebounbinding in clinical trials cannot be overstated. Complete details of taste testing results and placebo matching statistics must be published along with clinical results for positive results to be convincing. Clinical trials must be conducted in a statistically valid manner as demonstrated by Gwaltney et al.54 Acknowledgements Special thanks are extended to Dr Guy Berthon, Director of Research CNRS, INSERM Unit-305 Equipe ‘Bioréactifs: Spéciation et Biodisponibilité’, Toulouse, France, for definitive determinations of zinc speciation for several important zinc compositions. 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