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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). Affinity-purified Escherichia coli
J5 lipopolysaccharide-specific IgG protects neutropenic rats
against Gram-negative bacterial sepsis. Journal of Infectious
Diseases 170, 622–9.
The available animal experimental data discussed in this
review mostly support the notion that preparations of antibodies, raised against CGL epitopes of endotoxin, can be
cross-protective. Proof, however, that such protection is
attributable to the CGL-specificity of these antibodies is,
at best, meagre. These observations, combined with results
of extensive in-vitro research, which was unable to confirm
any of the hypothetical mechanisms of protection, at least
give serious reason to question the validity of the concept
underlying the search for broadly cross-protective antibodies raised against the core region of endotoxin.
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482
Zinc ion availability and common colds
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S. H. (1982). Limited protective effect of rough mutant antisera in
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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. Special thanks are also
extended to Dr Ananda S. Prasad, Director of Research,
Department of Internal Medicine, Wayne State University
School of Medicine, Detroit, MI, USA; Dr David A. J.
Tyrrell, retired Director of the British Medical Research
Council Common Cold Unit, Salisbury, UK; and Dr
Charles A. Pasternak, Professor of Biochemistry, St
George’s Hospital Medical School, London, UK, for their
unique contributions in support of this research.
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