REVIEWS FROM ASN EB 2012 SYMPOSIA
Discovery of Human Zinc Deficiency: Its Impact on
Human Health and Disease1–3
Ananda S. Prasad*
Wayne State University School of Medicine, Department of Oncology, Barbara Ann Karmanos Cancer Institute, Detroit, MI
The essentiality of zinc in humans was established in 1963. During the past 50 y, tremendous advances in both clinical and basic sciences of zinc
metabolism in humans have been observed. The major factor contributing to zinc deficiency is high phytate-containing cereal protein intake in
the developing world, and nearly 2 billion subjects may be zinc deficient. Conditioned deficiency of zinc has been observed in patients with
malabsorption syndrome, liver disease, chronic renal disease, sickle cell disease, and other chronic illnesses. Major clinical problems resulting from
zinc deficiency in humans include growth retardation; cell-mediated immune dysfunction, and cognitive impairment. In the Middle East, zinc-
deficient dwarfs did not live beyond the age of 25 y, and they died because of intercurrent infections. In 1963, we knew of only 3 enzymes that
required zinc for their activities, but now we know of >300 enzymes and >1000 transcription factors that are known to require zinc for their
activities. Zinc is a second messenger of immune cells, and intracellular free zinc in these cells participate in signaling events. Zinc has been very
successfully used as a therapeutic modality for the management of acute diarrhea in children, Wilson’s disease, the common cold and for the
prevention of blindness in patients with age-related dry type of macular degeneration and is very effective in decreasing the incidence of
infection in the elderly. Zinc not only modulates cell-mediated immunity but is also an antioxidant and anti-inflammatory agent. Adv. Nutr. 4:
In 1869, the essentiality of zinc for the growth of Aspergillus
niger was observed (1). In 1933, zinc was shown to be essen-
tial for the growth of the rats (2). Although by 1960, the es-
sentiality of zinc for growth in various animal species was
reported, it was considered improbable that zinc deficiency
in humans could lead to significant clinical problems.
I was trained as a clinical scientist at the University of
Minnesota, Minneapolis, MN, and after my training, a
strange set of circumstances brought me to Shiraz, Iran.
The story of zinc beganwhen an Iranian physician presented
to me at the medical center grand rounds, a 21-y-old man
who looked like a 10-y-old boy and who was severely ane-
mic. His genitalia were infantile. He had rough and dry
skin, mental lethargy, hepatosplenomegaly, and geophagia.
He ate only bread (whole wheat flour) and had no intake
of animal protein. He consumed 0.5 kg of clay daily. He
was severely irondeficient but had no blood loss. Later, Idis-
covered that this syndrome was common in the villages of
Shiraz, Iran (3).
Iron deficiency alone could not account for all the fea-
tures that we observed in this case because growth retarda-
tion and testicular atrophy are not seen in iron-deficient
experimental animals. An examination of the periodic table
suggested to me that deficiency of another transitional ele-
ment, perhaps zinc, may have also been present, which
may account for growth retardation and hypogonadism.
We hypothesized that a high phosphate content in the diet
and geophagia may have decreased the availability of both
iron and zinc, which resulted in deficiency of both elements
Our later studies in Egypt documented conclusively that
zinc deficiency occurred in humans and that zinc supple-
mentation resulted in 12.7–15.2 cm of growth in 1 y and
that genitalia became normal within 3–6 mo of zinc supple-
For nearly a decade, the idea that zinc deficiency occurred
in humans remained very controversial. Several reports,
1Presented at the symposium “Zinc Nutrition: From Discovery to Global Health Impact,” held
April 22, 2012, at the ASN Scientific Sessions and Annual Meeting at Experimental Biology
2012 in San Diego, CA. The symposium was sponsored by the American Society for
Nutrition. A summary of the symposium “Zinc Nutrition: From Discovery to Global Health
Impact” was published in the September 2012 issue of Advances in Nutrition.
2This project was supported in part by Labcatal Laboratories, Paris, France.
3Author disclosure: A. Prasad, no conflicts of interest.
*To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
ã2013 American Society for Nutrition. Adv. Nutr. 4: 176–190, 2013; doi:10.3945/an.112.003210.
by guest on December 28, 2015
however, confirmed our observation, and in 1974, the Na-
tional Research Council of the National Academyof Sciences
declared zinc as an essential element for humans and estab-
lished a recommended dietary allowance (RDA)4(7). In
1978, the FDA made it mandatory to include zinc in the total
parenteral nutrition fluids (8).
The details of the circumstances leading to the discovery
of human zinc deficiency in the Middle East have been pre-
sented in another paper.
In this paper, the nutritional deficiency of zinc, which is
present throughout the developing world, and a conditioned
deficiency of zinc, which may complicate many diseased
states, are discussed. Zinc has also been recognized to have
a therapeutic role in the management of certain diseases.
This is also presented.
Chronology of zinc-related observations in
Eggleton (9) in China demonstrated in 1939 that the zinc
contents of the toenails, fingernails, and skin were signifi-
cantly decreased in poor Chinese individuals with beriberi
compared with healthy subjects, and he suggested that
zinc deficiency may be a factor in beriberi. In another pub-
lication, Eggleton (10) analyzed zinc and copper in 13 differ-
ent organs and tissues from 26 Chinese subjects of both
sexes ranging in age from 156 d to 60 y who had died of mis-
cellaneous causes. He showed that both elements were pre-
sent in all the tissues examined. He also observed that
whereas zinc was concentrated in all organs and tissues, cop-
per was concentrated only in the liver, brain, kidney, and
hair, in descending order. The cerebellum contained more
zinc and copper than the cerebrum.
By using the dithizone technique, Lutz (11) analyzed zinc
in various human tissues and concluded that the total zinc
content of a 70-kg man was ~2.2 g. This figure is remarkably
close to what is accepted today (12). Absorption and excre-
tion of zinc were first investigated by McCance and Wid-
dowson (13). They showed that the principal route of zinc
excretion was in the feces, and only a small quantity of
zinc was excreted in the urine.
Vikbladh (14) analyzed serum zinc by the dithizone tech-
nique and reported that the normal level was 19.7 6 0.24
mmol/L, a value similar to what has been reported by mod-
ern techniques. In another paper, Vikbladh (15) reported
that serum zinc was decreased in many chronic diseases, in-
cluding liver disease. In 1956, Vallee et al. (12) reported that
the serum zinc concentration was decreased in patients with
cirrhosis of the liver and suggested that this represented a
conditioned deficiency of zinc due to hyperzincuria.
Nutritional deficiency of zinc
The first case of zinc deficiency in the United States was re-
with dwarfism, hypogonadism, hypogammaglobulinemia, gi-
ardiasis, strongyloidosis, and schistosomiasis. Zinc supplemen-
tation resulted in improved growth and development. In 1972,
Hambidge et al. (17) reported the occurrence of nutritional
zinc deficiency in Mexican-American children from Den-
ver, CO. They responded well to zinc supplementation.
In 1972, Halsted et al. (18) published the results of their
study involving a group of 15 men who were rejected at the
Iranian Army Induction Center because of “malnutrition.”
Two women, 19 and 20 y old, were also included in their
study. The unique feature was that all their subjects were
19 and 20 y old. Their clinical features were similar to those
reported by Prasad et al. in 1961 (3) and 1963 (4). They were
studied for 6–12 mo. One group received a well-balanced
diet containing adequate animal protein plus a placebo cap-
sule. A second group was given the same diet plus a capsule
of zinc sulfate containing 27 mg of elemental zinc. A third
group received the diet without additional supplement for
6 mo. The 2 women lived in the house of Dr. Ronaghy and
received the same treatment and observation program. The
zinc-supplemented group gained considerably in height and
showed evidence of early onset of sexual function as defined
by nocturnal emission in males and menarche in females
compared with those receiving only a well-balanced diet.
Severe deficiency of zinc
Acrodermatitis enteropathica. In1973,BarnesandMoynahan
(19) reported a 2-y-old girl with severe acrodermatitis enter-
opathica who was being treated with diiodohydroxyqui-
nolone and a lactose-deficient synthetic diet but was not
showing any satisfactory response to this therapy. The serum
zinc concentration was significantly decreased. They, there-
fore, administered oral zinc sulfate to correct this deficiency.
Surprisingly, the skin lesions and gastrointestinal symptoms
cleared after zinc supplementation. When zinc was inadver-
tently omitted from the child’s regimen, the child suffered a
relapse; however, she again completely responded to oral
zinc therapy. The authors attributed zinc deficiency to the
synthetic diet that the patient received. The authors, how-
ever, soon realized that zinc might have been fundamental
to the pathogenesis of this rare inherited disorder and that
the clinical improvement reflected correction of the zinc sta-
tus in the patient. This original observationwas quickly con-
firmed in other patients with acrodermatitis enteropathica
(AE) throughout the world. The underlying pathogenesis
of the zinc deficiency in these patients is due to malabsorp-
tion of zinc caused by a mutation in ZIP4, an intestinal zinc
AE is a lethal, autosomal, recessive trait that usually oc-
curs in infants of Italian, Armenian, or Iranian lineage
4Abbreviations used: AB, b amyloid protein; AD, Alzheimer’s disease; AE, acrodermatitis
enteropathica; APP, amyloid precursor protein; CRP, C-reactive protein; DC, dendritic cells;
HAE, 4-hydroxyalkenal; HAEC, human vascular endothelial cell; HL-60, human promyelocytic
leukemia cell line; ICAM-1, intercellular adhesion molecule 1; IFN, interferon; IGF-1,
insulin-like growth factor 1; MDA, malondialdehyde; MNC, mononuclear cell; NF-kB, nuclear
factor kB; oxLDL, oxidized LDL; PHA-P, phytohemagglutinin P; PMA, phorbol-12 myristate 13
acetate; ra, receptor antagonist; RDA, recommended daily allowance; ROS, reactive oxygen
species; SCD, sickle cell disease; sIL-1 ra, soluble interleukin-1 receptor antagonist; THP-1,
human monocytic leukemia cell line; TK, deoxythymidine kinase; VCAM-1, vascular cell
adhesion molecule 1.
Zinc and health177
by guest on December 28, 2015
(21). The disease develops in the early months of life soon
after weaning from breastfeeding. The dermatologic mani-
festations of severe zinc deficiency in patients with AE in-
clude bullous pustular dermatitis of the extremities and
oral, anal, and genital areas around the orifices, paronychia,
and alopecia. Ophthalmic signs include blepharitis, con-
junctivitis, photophobia, and corneal opacities. Neuropsychi-
atric signs include irritability, emotional instability, tremors,
and occasional cerebellar ataxia. Weight loss, growth retar-
dation, and male hypogonadism are also prominent clinical
features. Congenital malformation of fetuses and infants
born of pregnant women with AE has been commonly ob-
AE patients have an increased susceptibility to infections.
Thymic hypoplasia, absence of germinal centers in lymph
nodes, and plasmacytosis in the spleen are seen consistently.
All T cell–mediated functional abnormalities are completely
corrected with zinc supplementation. Abnormal chemotoxis
is also corrected with zinc therapy in AE patients. The clin-
ical course is downhill with failure to thrive and complicated
by intercurrent bacterial, fungal, viral, and other opportun-
istic infections. Gastrointestinal disturbances are severe and
include diarrhea, malabsorption, steatorrhea, and lactose
intolerance. The disease, if unrecognized and untreated, is
fatal. Zinc supplementation, however, results in complete
The AE gene has been localized to a w3.5-cm region on
the 8q24 chromosome. The gene encodes a histidine-rich
protein, now referred to as ZIP-4, which is a member of a
large family of transmembrane proteins known as zinc
transporters. In patients with AE, mutations in this gene
have been documented (20).
Total parenteral nutrition. In 1975,Kay and Tasman-Jones
(23) reported the occurrence of severe zinc deficiency in
subjects receiving total parenteral nutrition for prolonged
periods without zinc. Okada et al. (24) and Arakawa et al.
(25) reported similar findings without zinc. These observa-
tions have been documented by several investigators, and,
indeed, in the United States, zinc is now being routinely in-
cluded in total parenteral fluids for subjects who are likely to
receive such therapy for extended period.
Patients on total parenteral nutrition with diarrhea may
lose up to 6 to 12 mg/d of zinc. The excessive loss of zinc re-
sults in severe deficiency of zinc. Manifestations such as der-
matologic, alopecia, neuropsychiatric, weight loss, and
intercurrent infections are commonly seen. There are a neg-
ative nitrogen balance and impaired carbohydrate utilization.
If zinc deficiency is not recognized and treated, the condi-
tion may become fatal (8). Zinc deficiency may also be com-
mon in very low birth weight infants because fortified
human milk and preterm formula may not contain zinc; as
such, physicians must be aware of this problem (26).
Penicillamine therapy. A severe deficiency of zinc has also
been observedin patients with Wilson’s disease who received
penicillamine therapy as decoppering agent. This treatment
may induce excessive zinc loss and cause severe deficiency of
In summary, the manifestations of severe zinc deficiency
in humans include bullous pustular dermatitis, alopecia,
diarrhea, emotional disorders, weight loss, intercurrent in-
fections due to cell-mediated immune dysfunctions, hypo-
gonadism in males, neurosensory disorders, and problems
with healing of ulcers. Severe deficiency of zinc, if untreated,
may become fatal.
Moderate deficiency of zinc
The manifestations of a moderate deficiency of zinc include
growth retardation, male hypogonadism in adolescents,
rough skin, poor appetite, mental lethargy, delayed wound
healing, cell-mediated immune dysfunctions, and abnormal
neurosensory changes. These manifestations have been re-
ported in subjects with nutritional deficiency of zinc as ob-
served originally in Iran and Egypt (3,4) and many subjects
with conditioned deficiency of zinc.
It is now apparent that a nutritional deficiency of zinc in
humans is fairly prevalent throughout the world, particu-
larly in areas where cereal proteins are primary in local diets.
In Turkey, geophagia is also a common problem, and the
majority of the adolescents in the villages in Turkey with ge-
ophagia exhibit both iron and zinc deficiencies (22).
Cavdar et al. (22) observed a decreased zinc level in al-
most 30% of low socioeconomic status pregnant women
in Turkey. Their diet consisted of mainly cereals. Maternal
zinc deficiency was associated with severe congenital malfor-
mation of the central nervous system in the fetuses, and ma-
ternal morbidity was increased.
Zinc and growth. Growth is the first limiting effect of zinc
deficiency in experimental animals (28). Zinc deficiency de-
creases circulating insulin-like growth factor 1 (IGF-1) con-
centration independent of total energy intake (29).
In humans, zinc deficiency decreases circulating IGF-
1 concentration (30,31). IGF-1 receptor possesses tyrosine
kinase activity (28). On activation of the receptor by IGF,
a cascade of phosphorylation occurs within the cell leading
to regulation of cell cycle and cell division. Tyrosine phos-
phorylation of the receptor is essential for its activation,
and I hypothesize that because zinc has been shown to in-
hibit various protein tyrosine phosphatases (32), phospho-
rylation of the tyrosine kinase receptor by zinc is perhaps
the most important critical step of zinc action on human
IGF-1 activation leads to stimulation of thymidine uptake
in cells (33). In our earlier studies, we showed that in zinc-
deficient rats, the activity of deoxythymidine kinase (TK), an
enzyme required for conversion of deoxythymidine to deox-
ythymidine 59-monophosphate, a precursor of thymidine
triphosphate, is considerably decreased in the implanted
sponge connective tissue, and this reduced activity of TK de-
creased DNA, protein, and collagen synthesis in rats (34).
Thus, it appears that zinc has multiple roles in growth. It
is required for IGF-1 generation, phosphorylation of IGF-1
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receptor, and upregulation of the activity of TK, all of which
are involved in cell division and growth.
Mild deficiency of zinc
Although the clinical, biochemical, and diagnostic aspects of
severe and moderate levels of zinc deficiency in humans are
well defined, the recognition of mild deficiency of zinc re-
mains very difficult. We therefore developed an experimen-
tal model of zinc deficiency in humans to define mild zinc
In a group of human volunteers, we induced a mild state
of zinc deficiency by dietary means. Adult male volunteers
were hospitalized at the Clinical Research Center of the Uni-
versity of Michigan Medical School Hospital, Ann Arbor,
MI. A semipurified diet that supplied ~3.0–5.0 mg/d of
zinc was used to induce zinc deficiency (35).
The volunteers were given a hospital diet containing an-
imal protein daily for 4 wk. This diet averaged 12 mg/d of
zinc, consistent with the RDA. After this, they received
3.0–5.0 mg/d of zinc while consuming a soy protein–based
experimental diet. This regimen was continued for 28 wk.
After this, the volunteers received 2 cookies containing 27
mg of zinc supplement. This supplementation was contin-
ued for 12 wk.
Throughout this study, the level of all nutrients including
protein, amino acids, vitamins, and minerals (both micro
and macro elements) were kept constant, meeting the
RDA except for zinc. By this technique, we were able to in-
duce a specific mild deficiency of zinc in human volunteers
As a result of mild deficiency of zinc, we observed a de-
creased serum testosterone level, oligospermia, decreased
natural killer cell lytic activity, decreased IL-2 activity of
T helper cells, decreased serum thymulin activity, hyperam-
monemia, hypogeusia, decreased dark adaptation, and
decreased lean body mass (36–38). This study clearly es-
tablished that even a mild deficiency of zinc in humans af-
fects clinical, biochemical, and immunological functions
Zinc and immune cells. Zinc is a second messenger for im-
mune cells, and its intracellular status is directly altered by
an extracellular stimulus and then intracellular zinc partici-
pates in signaling events (39). Hirano et al. (40) showed
that a decrease in intracellular free zinc is critical for LPS-
mediated CD4+ T-cell activation by dendritic cells (DCs).
LPS binds to Toll-like receptor 4 on DCs and initiates
Myd88 and TRIF [a domain containing adapter–inducing
interferon (IFN)-b]-mediated signaling (39). TRIF-mediated
signaling increases the ZnT (a solute carrier )-5 mRNA and
decreases ZIP-6 mRNA, thus resulting in a decrease in the in-
tracellular free zinc in DCs. Reduction in intracellular free
zinc increases surface expression of major histocompatibility
complexclass II molecules, which is important for the activa-
tion of CD4+ T cells (39,40).
Zinc affects the activity of monocytes/macrophages in
several ways. Zinc is involved in monocyte/macrophage
development (41–43) and regulates its functions such as
phagocytosis and proinflammatory cytokine production.
LPS stimulation of zinc-sufficient monocytes results in
downregulation of inflammatory cytokines such as TNF-a,
IL-1b, IL-6, and IL-8 (37–39). Zinc inhibits the membrane
phosphodiesterase, leading to elevated levels of the second
messenger guanosine 39,59 cyclic monophosphate, which is
followed by a subsequent suppression of the nuclear factor
kB (NF-kB)–dependent mRNA of TNF-a, IL-1b, and other
inflammatory cytokines (40–42). Additionally, zinc induces
A-20, which inhibits NF-kB signaling via TNF receptor–
associated factor pathways, resulting in downregulation of
the mRNA of inflammatory cytokines (44–46). Based on
this, we propose that zinc is an important anti-inflammatory
Zinc deficiency affects T helper subset 1 cell function ad-
versely in humans (36–38,43). Serum thymulin activity and
generation of T helper subset 1 cell cytokine, IL-2, and IFN-g
were affected within 8–12 wk of institution of a zinc-
restricted diet (3–5 mg/d) in human volunteers, whereas
plasma zinc decreased after 20 to 24 wk of the institution
of the experimental diet. This suggests that T helper subset
1 cells are very sensitive to zinc restriction. T helper subset
2 cell cytokines were not affected as a result of the institution
of zinc-deficient diet in humans.
In the human malignant lymphoblastoid cell line of Th0
phenotype cells, a human malignant lymphoblastoid cell
line of the Th0 phenotype, we showed that in zinc-sufficient
cells, mRNA levels of IFN-g, IL-12 receptor b2, and T-bet (a
transcription factor involved in T-cell differentiation) in
phorbol-12 myristate 13 acetate (PMA)/phytohemaggluti-
nin-P (PHA)–stimulated cells were increased compared
with zinc-deficient cells (47). Although intracellular free
(noncovalently bound zinc) zinc increased only slightly in
PMA/PHA-stimulated cells, in concanavalin A–stimulated
cells in zinc-sufficient medium, there was an increased sus-
tained level of intracellular free zinc compared with the zinc-
deficient cells (47). We concluded that stimulation of cells by
concanvalin Avia T-cellreceptor, therewas areleaseof intra-
cellular free zinc that functioned as a signal transduction
molecule for the generation of IFN-g, T-bet, and IL-12 re-
ceptor b2mRNA required for T helper subset 1 cell differen-
Conditioned deficiency of zinc
GI disorders. A moderate level of zinc deficiency has been
observed in many gastrointestinal disorders. These include
malabsorption syndrome, Crohn’s disease, regional ileitis,
In 1968, MacMahon et al. (48) were the first to report
zinc deficiency in a patient who had steatorrhea. Zinc de-
ficiency in patients with malabsorption syndrome is now
well recognized, and most physicians are aware of this
Liver disease. Low serum and hepatic zinc and, paradoxi-
cally, hyperzincuria were reported in patients with cirrhosis
Zinc and health 179
by guest on December 28, 2015
of the liver many years ago (12). Some patients with cirrho-
sis of the liver who had night blindness did not respond to
vitamin A supplementation; however, they responded to
zinc administration (21).
Elevated blood ammonia levels are known to be a factor
in the development of hepatic coma. It is known that zinc-
deficient rats have a defect in the metabolism of sulfur-
containing amino acids. Zinc deficiency also affects urea
synthesis and thus an abnormality related to metabolism
of amino acids and ammonia may act in concert to produce
hepatic coma. We reported earlier that dietary zinc restric-
tion may lead to hyperammonemia (49). Rabbani and Prasad
(49) observed a decrease in hepatic ornithine transcarba-
moylase activity and an increase in plasma ammonia levels
in zinc-deficient rats. Additionally, increased activity of the
purine nucleotide enzyme adenosine monophosphate de-
aminase as a result of zinc deficiency has also been observed,
which mayalso contribute to increased ammonia levels (50).
Zinc therapy has been reported to be beneficial in sub-
jects with hepatic encephalopathy (51). More studies are
needed in this important area. It is likely that some of the
clinical features of cirrhosis of the liver, such as loss of
body hair, testicular hypofunction, poor appetite, mental
lethargy, difficulty in healing, abnormal cell–mediated im-
munity, and night blindness, may indeed be related to the
secondary zinc-deficient state induced by hyperzincuria.
Renal diseases. Mahajan et al. (52,53) were the first to doc-
ument that patients with chronic renal failure showed de-
creased concentration of zinc in plasma, leukocytes, and
hair; increased plasma ammonia levels; and increased activ-
ity of plasma ribonuclease. Uremic hypogeusia improved af-
ter zinc supplementation. Impotence is common in uremic
males and is not improved by hemodialysis. A double-blind
clinical trial of zinc supplementation showed that zinc defi-
ciency was a reversible cause of sexual dysfunction in uremia
Zinc deficiency in sickle cell disease. Our studies have
documented the occurrence of zinc deficiency in adult sickle
cell disease (SCD) patients (54,55). Growth retardation, hy-
pogonadism in males, hyperammonemia, abnormal dark
adaptation, and cell-mediated immune dysfunction in
SCD patients have been related to a deficiency of zinc. The
biochemical evidence of zinc deficiency in SCD patients
was decreased levels of zinc in the plasma, erythrocytes,
and hair; hyperzincuria; decreased activity of certain zinc-
dependent enzymes such as carbonic anhydrase in erythrocy-
tes, alkaline phosphatase in the neutrophils, deoxythymidine
kinase activity in newly synthesizing skin connective tissue
and collagen; and hyperammonemia (54,55). Inasmuch as
zinc is known to be an inhibitor of RNA, increased activity
of this enzyme in plasma was considered to also be evidence
of zinc deficiency. Zinc supplementation in SCD patients
resulted in significant improvement in secondary sexual
characteristics, normalization of plasma ammonia level,
and correction of dark adaptation abnormality. Zinc
supplementation also increased zinc levels in plasma, eryth-
rocytes, and neutrophils. The expected response to zinc
supplementation in enzyme activities was also observed. In-
creased longitudinal growth and body weight in 14- to 18-y-
old SCD patients were observed. Zinc supplementation also
corrected impaired delayed-type hypersensitivity reaction
and decreased natural killer cell lytic activity in SCD patients
A 3-mo placebo-controlled zinc supplementation trial
(25 mg zinc as zinc acetate 3 times a day) in 36 SCD patients
showed that zinc-supplemented subjects had a decreased in-
cidence of infections, increased hemoglobin and hematocrit
levels, and increased plasma zinc and antioxidant power
compared with the placebo group (55). Plasma nitrite
and nitrate, lipid peroxidation products, DNA oxidation
products, and soluble vascular cell adhesion molecule
1 (VCAM-1) decreased in the zinc-supplemented group
compared with the placebo group. Zinc-supplemented sub-
jects showed significant decreases in LPS-induced TNF-a
and IL-1b mRNA and TNF-induced NF-kB DNA binding
in mononuclear cells (MNCs) compared with the placebo
group (55). Zinc supplementation also increased relative
levels of IL-2 and IL-2 receptor a mRNA in PHA-stimulated
Therapeutic impact of zinc
Zinc and infectious diseases. Acute diarrhea in children.
Supplementation with zinc has been shown to prevent and
treat diarrhea in childrenyounger than 5 y of age, decreasing
both diarrhea morbidity and mortality (56,57). Zinc defi-
ciency is also correlated with the risk of respiratory tract in-
fections, but the benefit of supplementation appears to be
limited to more severe episodes and in populations with a
high incidence of zinc deficiency (57).
Diarrhea causes the breakdown of absorptive mucosa, re-
sulting in poor absorption of nutrients, including zinc. Stud-
ies conducted earlier linked diarrheal illness to the loss of
endogenous zinc (57). Children with low plasma zinc were
observed to be more susceptible to diarrhea pathogens, prop-
agating a cycle of deficiency and infection.
There is extensive evidence supporting the efficacyof zinc
supplementation for the prevention of childhood diarrhea
(57). In 2004, WHO issued a global recommendation for
the daily supplementation with 20 mg zinc in children 6
mo of age and older and 10 mg of zinc in infants younger
than 6 mo for 10–14 d on diarrheal onset.
Meta-analysis of routine supplementation for as long as 3
mo in 7 studies providing 1–2 times the RDA of elemental
zinc 5–7 times per week found an 18% reduction in diar-
rheal incidence, a 25% decrease in diarrhea prevalence,
and a 33% reduction in persistent diarrhea episodes among
supplemented children compared with children who re-
ceived placebo (57).
A meta-analysis of 3 randomized, controlled trials pro-
viding short-course zinc supplementation with 2–4 times
the daily RDA for 2 wk after the onset of an episode of acute
or persistent diarrhea was reported. The pooled analysis
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showed an 11% decrease in diarrhea incidence and a 34%
decrease in diarrhea prevalence during 3-mo observation
Severe infection and zinc in children. Serious bacterial in-
fections are a major cause of death in early infancy in devel-
oping countries. In 1 study, the effect of zinc as an adjunct to
antibiotics in infants with probable serious bacterial infec-
tion was assessed. A randomized, double-blind, placebo-
controlled zinc supplementation trial in infants 7–120 d of
age with probable serious bacterial infections at 3 hospitals
in New Delhi, India, was conducted (58). The patients
were stratified according to whether they were underweight
or had diarrhea at enrollment, and they received either 10
mg zinc or placebo orally every day in addition to standard
antibiotic treatment. The primary outcome was treatment
failure, which was defined as the need to change antibiotics
within 7 d of randomization, the need for intensive care
management, or death at any time within 21 d. Significantly
fewer treatment failures occurred in the zinc group (10%)
compared with the placebo group (17%). Ten infants receiv-
ing zinc died compared with 17 receiving placebo (58).
Zinc for the treatment of the common cold. The common
cold is one of the most frequently occurring diseases in the
world (59,60). More than 20 viruses cause the common
cold, and these include rhinovirus, coronavirus, adenovirus,
respiratory syncytial virus, and parainfluenza virus. Annu-
ally adults in the United States may develop a common
cold 2–4 times in a year, and children may develop colds
6–8 times in a year. The morbidity and subsequent financial
loss resulting from absenteeism from work are substantial.
Previously prescribed treatments have not provided a con-
sistent relief of symptoms.
Eby et al. (61) in 1984 were the first to show in a double-
blind, placebo-controlled trial that zinc gluconate lozenges
administered every 2 hwere effective in decreasing the sever-
ity and duration of common cold. We tested the efficacy of
zinc acetate lozenges in the common cold in 50 volunteers
who were recruited within 24 h of symptoms of the com-
mon cold developing and conducted a randomized, double-
blind, placebo-controlled trial(59). Participants took1 lozenge
containing 12.8 mg zinc (as acetate) or placebo every 2 to
3 h while awake as soon as common cold symptoms de-
veloped. Subjective symptom scores for sore throat, nasal
discharge, nasal congestion, sneezing, cough, scratchy
throat, hoarseness, muscle ache, fever, and headache were
recorded daily for 12 d. Plasma zinc and proinflammatory
cytokines were assayed on day 1 and after participants were
Twenty-five in the zinc group and 23 in the placebo
group completed the study. Compared with the placebo
group, the zinc group had shorter overall duration of cold
symptoms (4.5 vs. 8.1 d, P < 0.01), cough (3.1 vs. 6.3 d,
P = 0.01), and nasal discharge (4.1 vs. 5.8 d, P = 0.02), and
decreased total severity scores for all symptoms (P < 0.002).
In another study, we recruited 50 ambulatory volunteers
within 24 h of common cold symptoms developing for
randomized, double-blind, placebo-controlled trial of zinc
(60). Participants took 1 lozenge containing 13.3 mg of
zinc (as zinc acetate) or placebo every 2–3 h while awake.
The subjective scores of clinical symptoms were recorded
daily. Plasma zinc, soluble IL-1 receptor antagonist (ra), sol-
uble TNF receptor 1, soluble ICAM-1 were assayed on days
1 and 5 (60).
Compared with the placebo group, the zinc group had a
shorter mean overall duration of the cold (4.0 vs 7.1 d, P =
0.001), cough (2.1 vs 5.0 d, P < 0.001), and nasal discharge
(3.0 vs 4.5 d, P = 0.02). Blinding of subjects was adequate,
and adverse effects were comparable in the 2 groups. Symp-
tom severity scores were significantly decreased in the zinc
group (P = 0.002). The mean changes between zinc and pla-
cebo groups (before vs after therapy) showed significant dif-
ferences in soluble IL-1-ra (P = 0.033) and soluble ICAM-
1 level (P = 0.04). Both decreased in the zinc group, and
the mean changes in zinc and placebo group (before vs after
therapy) showed a significant difference (P < 0.001)
Our results suggest that common cold viruses increase
oxidative stress, which activates macrophages and mono-
cytes and results in increased production of both the inflam-
matory cytokines and the anti-inflammatory product
soluble IL-1ra; thus, a decrease in soluble IL-1ra in the
zinc group suggests that zinc decreased the activation of
monocytes and macrophages by decreasing oxidative stress.
We previously showed that zinc functions as an antioxidant
Human rhinovirus type 24 “docks” with ICAM-1 on the
surface of the somatic cells (59,60). Thus, zinc may act as an
antiviral agent by reducing ICAM-1 levels. We previously
showed that zinc functions as a downregulator of NF-kB ac-
tivity involved in the gene expression of adhesion molecules
such as ICAM-1 (46).
We conclude that zinc acetate lozenges given within 24 h
of the onset of common cold in proper doses are very effec-
tive in decreasing the duration and severity of the common
cold. We propose that the beneficial effects seen in the zinc
group were due to the antioxidant and anti-inflammatory
effects of zinc. We also suggest that a decrease in plasma
ICAM-1 levels due to zinc therapy may have decreased the
docking of the cold viruses on the surface of somatic cells.
A meta-analysis selected randomized, double-blind, pla-
cebo-controlled trials using zinc for at least 5 consecutive
days to treat or at least 5 mo to prevent the common cold
were included for analysis (62). Thirteen therapeutic trials
(966 participants) and 2 preventive trials (394 participants)
were included for analysis. Studies reporting the duration
and severity of cold symptoms suggest that zinc significantly
reduced the overall duration and severity of common cold
symptoms if the therapy was started within 24 h of the onset
of the cold. Zinc supplementation for prevention of the
common cold showed that the incidence of the common
cold, school absenteeism, and use of antibiotics were de-
In another meta-analysis, 13 placebo-controlled studies
examined the therapeuticeffect of zinc lozenges on common
Zinc and health181
by guest on December 28, 2015
cold symptoms (63). Five of the trials used a total daily zinc
dose of <75 mg and uniformly found no effect. Three trials
used zinc acetate in daily doses of >75 mg; the pooled results
showed a 42% reduction in the duration of colds. Five trials
used zinc salts rather than acetate in daily doses of >75 mg;
the pooled results showed a 20% reduction in the duration
of common colds.
In another report, the investigators included 17 random-
ized, controlled trials to analyze the effect of zinc on the
common cold (64). Although the authors observed an in-
creased heterogeneity due to age, zinc dose, and chemical
formulation, they concluded that zinc may shorten the du-
ration of the common cold (64).
To optimize the therapeutic effect of zinc lozenges on the
common cold, one must pay attention to several issues.
First, zinc therapy must begin within 24 h of the onset of
cold symptoms. Second, the total daily dose of elemental
zinc should be >75 mg. Third, the chemical formulation
should be optimal so that zinc is ionized in the oral cavity
at pH 7.4. Zinc acetate and zinc gluconate are good salts
to use; however, if citric acid, glycine, tartarate, and other
binders are used, zinc is prevented from ionization. Thus,
it is critical that the solution chemistry of the preparation
is proper. Physicians and health practitioners must realize
that one cannot treat common cold symptoms by swallow-
ing zinc tablets, zinc syrup, or zinc lozenges. Zinc lozenges
must be used orally and allowed to dissolve slowly in the
mouth, which will then allow ionic zinc to be released, ab-
sorbed, and transported to the virally infected nose.
Zinc therapy for Wilson’s disease
Wilson’s disease is an inherited autosomal disorder of cop-
per accumulation. The excretion of liver copper in the bile
is decreased. This leads to failure of proper copper excretion
in the stool and to the accumulation of copper in the liver.
Eventually not only the liver but also brain and other organs
are damaged due to excess copper accumulation. Patients
typically present with liver disease, neurological disease
(movement disorder), or psychiatric disturbances in the sec-
ond to fourth decades of life. In many cases, the diagnosis is
either missed or delayed (65,66).
The gene for Wilson’s disease has been now identified.
The genetic mutation leads to a defective production of a
protein called ATP7B (membrane-bound copper-binding
ATP), which is responsible for key step in biliary excretion
of copper (65,66). The disease is recessive; thus, both copies
of the ATP7B gene have to be mutated to cause a failure in
biliary excretion of copper and produce the disease. The
gene for Wilson’s disease codes for a membrane-bound cop-
per-binding ATP-type protein that probably acts as a copper
pump, in either the plasma membrane or the intracellular
membrane. A large number of mutations in this gene causing
Wilson’s disease have been identified. This complicates the de-
velopment of an easy DNAtestfor thediagnosis of this disease.
It is important to establish an early diagnosis of Wilson’s
disease because effective therapeutic measures may prevent
accumulation of copper and serious damage to organs
such as the liver and brain. Ninety percent of the Wilson’s
disease patients have low levels of ceruloplasmin, and ceru-
loplasmin-bound copper and nonceruloplasmin-bound
copper are elevated in the plasma. Measurement of the 24-h
urinary copper is a good diagnostic test because this is con-
sistently elevated in these patients (65,66). Urinary copper,
however, may be elevated in patients with obstructive liver
disease also who do not have Wilson’s disease.
A slit-lamp examination for corneal copper deposits
(Kayser-Fleischer rings) is a very useful noninvasive diag-
nostic test for Wilson’s disease. They are positive in only
50% of the hepatic cases and are invariably present in neu-
rologic cases of Wilson’s disease.
The initial treatment objective is to decrease copper levels
or otherwise affect copper such that new copper toxicity is
avoided. It is also desirable to prevent copper from shifting
from one pool to the other while decoppering is being done.
Initial copper control treatment may take 2–4 mo (65,66).
Several years ago, we were using 150 mg elemental zinc in
6 divided doses for the treatment of sickle cell disease (SCD)
patients (67,68). We had observed that zinc was an effective
antisickling drug. We observed that at this level of zinc ther-
apy, our treatment resulted in inducing copper deficiency in
these patients (69). This led Brewer et al. (65,66) to develop
zinc as an effective anticopper drug for Wilson’s disease.
Zinc competes with copper for similar binding sites, and
oral zinc decreases uptake of copper very efficiently (70).
Zinc may also act by induction of intestinal cell metallothi-
onein. Metallothionein T, once induced, has a high affinity
for binding copper and prevents the serosal transfer of cop-
per into the blood. The intestinal cells turnover rapidly and
take the complexed copper into the stool for final excretion.
Zinc not only blocks food copper but also the copper that is
endogenously excreted via salivary, gastric, and other gastro-
intestinal juices. Thus, zinc is effective in producing a
chronicnegative copper balance.Fifty milligrams of elemen-
tal zinc (as acetate) is given orally 3 times a day for the man-
agement of Wilson’s disease patients. Zinc is given in a
fasting or postabsorptive state. The only side effect is that
10% of the subjects mayhave gastricdiscomfort. This is usu-
ally observed after the first morning dose and can be avoided
if zinc is administered between breakfast and lunch or after
dinner before going to bed.
For maintenance therapy, zinc is the drug of choice
(65,66). Relatively speaking, zinc has no toxicity and is non-
teratogenic; thus, it can be given to subjects of all ages and
even the pregnant women. Zinc has been approved by
FDA for the treatment of Wilson’s disease patients.
Zinc and age-related macular degeneration
Age-related macular degeneration (AMD) affects nearly
25% of individuals older than 65 y of age, and late-stage dis-
ease accounts for nearly 50% of legal blindness in Europe
and North America (71). Newsome et al. (72) demonstrated
that concentrations of zinc are reduced in human eyes with
signs of AMD and suggested that zinc deficiency may lead to
oxidative stress and retinal damage.
by guest on December 28, 2015
The Age-Related Eye Disease Study Group, supported by
National Eye Institute/NIH, conducted an 11-center double-
blind clinical trial in patients with dry-type AMD (73). A to-
tal of 3640 participants were enrolled. Their ages ranged
from 55 to 80 y, and the average follow-up period was 6.3
y. Participants were randomly assigned to receive daily orally
one of the following: 1) antioxidants (vitamin C 500 mg, vi-
tamin E 400 IU, and b-carotene 15 mg); 2) zinc 80 mg as zinc
oxide and copper 2 mg as copper oxide to prevent copper
deficiency induced by zinc; 3) antioxidants plus zinc; or 4)
In the group taking the antioxidant plus zinc supple-
ments, the risk of advanced AMD developing was reduced
by w25% and vision loss by w19%. In the group taking
zinc alone, the risk of advanced AMD developing was re-
duced by w21% and vision loss by 11%. In the group taking
the vitamins alone, the risk of advanced AMD developing
was decreased by 17%, and vision loss was decreased by
10%. No significant side effects were noted in subjects
who received high levels of therapeutic zinc (73). This study
confirmed the antioxidant effect of zinc in humans. Interest-
ingly, only the zinc-supplemented group showed increased
longevity (74). The risk of mortality was reduced by 27%
in the Age-Related Eye Disease Study Group studies in sub-
jects 55–81 y of age who received only therapeutic zinc daily.
At present, most ophthalmologists throughout the world are
using zinc and vitamins as supplement for the treatment of
the dry type of AMD.
Zinc supplementation in the elderly decreases the
incidence of infection
The daily intake of zinc in elderly subjects in the Western
world including the United States is only w8–10 mg,
whereas the RDA is 15 mg. The elderly frequently do not
eat the usual 3 meals a day and skip either breakfast or
lunch. Many live alone and do not cook a proper meal for
themselves. Our study in the Detroit area showed that
35% of the well-off ambulatory elderly subjects may have
a deficiency of zinc based on their plasma zinc levels. Zinc
deficiency and susceptibility to infections due to cell-
mediated immune dysfunction have been reported to occur
in the elderly (75,76). The third NHANES (1988–1914) also
found that elderly persons (older than 71 y) were at the
greatest risk of inadequate zinc intake (77).
Oxidative stress and increased inflammatory cytokines
have been recognized as important contributing factors for
several chronic diseases attributed to aging, such as athero-
sclerosis and related cardiovascular disorders, mutagenesis
and cancer, neurodegenerative disorders, type 2 diabetes
mellitus, and Alzheimer’s disease (AD). Together, O$2-,
H2O2, and OH radicals are reactive oxygen species (ROS),
and excessive generation of ROS causes oxidative stress. In-
flammatory cytokines such as TNF-a and IL-1b, generated
by activated monocytes, are also known to generate greater
levels of ROS. Chronic inflammatory processes have been
implicated in high cardiovascular mortality in elderly sub-
Our previous studies showed that zinc supplementation
in individuals 20 to 50 y of age decreased oxidative stress
(HAEs), and 8-hydroxydeoxyguanine in the plasma; down-
regulated the ex vivo induction of TNF-a and IL-1b mRNA
in MNCs; and provided protection against TNF-a–induced
NF-kb activation in isolated MNCs (44). We also showed
previously that in the human promyelocytic leukemia cell
line (HL-60), which differentiates to the monocyte and mac-
rophage phenotype in response to PMA, zinc upregulates
the expression of A20 and the binding of A20 transactivating
factor to DNA, which results in the inhibition of NF-kB ac-
Inasmuch as zinc deficiency and susceptibility to infec-
tions due to cell-mediated immune dysfunctions have been
observed in the elderly, we conducted a randomized, pla-
cebo-controlled trial of zinc supplementation in 50 healthy
elderly subjects (55–87 y) of both sexes and all ethnic groups
from St. Patrick’s senior citizen center, Detroit, MI. One
subject in zinc group dropped out on the second day;
thus, we had complete data for 49 subjects (24 in the zinc
group and 25 in the placebo group). Exclusion criteria
were as follows: life expectancy of <8 mo, progressive neo-
plastic disease, severe cardiac dysfunction, significant kidney
disease, significant liver disease, and subjects who were not
competent mentally. Zinc supplementation consisted of 45
mg elemental zinc (as gluconate) daily for 12 mo.
A comparison of the baseline data between the younger
subjects (ages 18–54, n =31) and the elderly subjects showed
that the plasma zinc was lower and the percentage of cells
producing IL-1b and TNF-a and the generated cytokines
were significantly higher in the elderly subjects (76). VCAMs,
the plasma also were significantly higher in the elderly. IL-10
generated by T helper subset 2 cells, which is known to reg-
ulate negatively IL-2 generation from T helper subset 1 cells,
was significantly higher in the elderly. A similar observation
with respect to IL-10 generation by Th2 cells in the elderly
has been also reported by Cakman et al. (79). The oxidative
stress markers also were significantly higher in the elderly
compared with the younger adults (76).
The mean incidence of infections per subject in 12 mo
was significantly lower (P < 0.01) in the zinc-supplemented
group (0.29 6 0.46) than in the placebo group (1.4 6 0.95;
effect size 1.46). The plasma zinc increased, and ex vivo gen-
eration of TNF-a and IL-10 decreased significantly in the
zinc group compared with the placebo group (76). Oxidative
stress biomarkers in the plasma also decreased significantly
in the zinc group compared with the placebo group (76).
In MNCs isolated from zinc-deficient elderly subjects,
zinc supplementation increased the ex vivo PHA-induced
IL-2 mRNA expression and plasma zinc concentration com-
pared with the zinc-deficient subjects who received placebo.
Thus, our study showed that zinc supplementation
(45 mg/d elemental zinc) in the elderly subjects decreased
the incidence of infection by nearly 66%. After supplemen-
tation, we also observed that oxidative stress markers and
Zinc and health183
by guest on December 28, 2015
the generation of inflammatory cytokines that were in-
creased before supplementation, decreased significantly.
These are highly significant effects of zinc supplementation
in the elderly, and it may imply that zinc may also prove to
be an excellent agent for the prevention of some of the
Future implications of therapeutic use of zinc
Oxidative stress and increased generation of inflammatory
cytokines have been implicated in the initiation and progres-
sion of manychronicdiseases. These include atherosclerosis,
diabetes mellitus type 2, neurodegenerative disorders, AD,
and some malignancies. Inasmuch as zinc is not only re-
quired for cell-mediated immunity, it is also an effective an-
tioxidant and anti-inflammatory agent, I hypothesize that
zinc will prove to be an effective therapeutic agent in the
management of some of these disorders. In this section,
the present rationale for the use of therapeutic zinc in the
management of atherosclerosis, diabetes mellitus type 2,
and AD is presented.
Atherosclerosis and zinc
Atherosclerosis is a slowly progressive chronic inflammatory
disease characterized by focal arterial lesions that ultimately
block the blood vessels, which leads to angina, myocardial
infarction, cerebrovascular ischemia and stroke, and even
death (46,78). Inflammation, oxidative stress, and/or endo-
thelial dysfunction caused by known risk factors such as age,
sex, smoking, hypertension, diabetes, and obesity are in-
volved in the development and progression of atherosclero-
sis. Our previous studies showed that zinc deficiency
increases the generation of inflammatory cytokines, in-
creases oxidative stress, and induces endothelial cell dys-
Our studies also showed that zinc deficiency may affect
nearly 30%–40% of the well-off healthy ambulatory elderly
subjects in the Detroit area (75,76). In these subjects, de-
creased plasma zinc and increased plasma lipid peroxidation
products and endothelial cell adhesion molecules compared
with the healthy zinc-sufficient younger adults were observed.
In a randomized, placebo-controlled zinc supplementa-
tion trial in elderly subjects, we examined the effect of sup-
plementation on plasma C-reactive protein (CRP), IL-6,
macrophage chemoattractant protein 1, VCAM, and oxida-
tive stress markers (46). Additionally, we examined the effect
of zinc supplementation on A20, PPAR-a, and NF-kB ac-
tivation in HAEC and monocytic cell lines [HL-60 and
human monocytic leukemia cell line (THP-1)]. The zinc-
supplemented group received 45 mg/d zinc as gluconate
for 6 mo. At this level of zinc supplementation in the elderly,
we have not observed any decrease in copper status (46).
The plasma zinc increased in the zinc-supplemented
group, and no change was observed in the placebo group.
We observed a significant decrease in lipid peroxidation
product and a significant increase in antioxidant power [rep-
resented by ascorbate equivalent units (U/mL)] in the zinc-
supplemented group compared with the placebo group.
Plasma high-sensitivity CRP in the zinc-supplemented
group decreased significantly compared with the placebo
group. Plasma IL-6, macrophage chemoattractant protein
1, secretory phospholipase A2, soluble VCAM-1, and soluble
E-selectin decreased after zinc supplementation, and the
mean changes before and after between the 2 groups were
statistically significant (46). Plasma zinc concentrations in
the elderly subjects inversely correlated with the changes
in plasma concentrations of high-sensitivity CRP, VCAM-1,
macrophage chemoattractant protein 1, and MDA+HAEs
after 6 mo of supplementation (46).
Zinc decreased the generation of TNF-a, IL-1b, VCAM-1,
and MDA+HAEs in HL-60 and THP-1 and HAECs after in-
cubation with oxidized LDL (oxLDL) for 24 h compared
with zinc-deficient cells. Zinc increased A20 and PPAR-a
proteins in oxLDL-stimulated THP-1 cells and HAECs com-
pared with zinc-deficient cells.
The effect of zinc on NF-kB activation in THP-1 cells and
HAECs is shown in Figures 1 and 2. There was no signifi-
cant difference in NF-kB activation by either NF-kB–driven
luciferase reporter gene assay or electrophoretic mobility
shift assay between the nonstimulated THP-1 cells and
HAECs incubated in zinc-deficient and zinc-sufficient me-
dia. However, after 24 h of oxLDL stimulation, zinc-suffi-
cient THP-1 cells and HAECs showed a significant
decrease in NF-kB activation compared with zinc-deficient
A high plasma CRP concentration is a risk factor that is
independent of other risk factors such as total cholesterol,
LDL, age, smoking, BMI, diabetes, and hypertension (46).
CRP is a widely used marker for atherosclerosis and its clin-
ical course, complication, and prognosis (46). In prospective
studies, healthy men and women with an increased baseline
level of CRP were at greater risk of coronary artery disease
Our study showed that zinc supplementation (45 mg el-
emental zinc as gluconate) daily was effective in lowering
plasma CRP concentration. This is the first documentation
to show that zinc is effective in downregulating the plasma
CRP level in the elderly.
The increased production of ROS and the activation of
redox-dependent signaling cascades are involved in athero-
sclerosis (46,78). ROS itself can initiate NF-kB–mediated
transcriptional activation of inflammatory genes, thereby
potentially acting as independent triggers of atherosclerosis.
Zinc deficiency increased oxidative stress and zinc supple-
mentation decreased oxidative stress in cell culture models
and humans. We confirmed in this study that zinc supple-
mentation decreased oxidative stress in elderly subjects and
human vascular endothelial and monocytic cells. Thus, de-
creased oxidative stress by zinc may decrease the LDL oxida-
tion and exhibit an atheroprotective effect.
NF-kB is one of the major immune response transcrip-
tion factors involved in the initiation and development of
atherosclerosis (46,78). Zinc plays an important role in
NF-kB activation. However, the regulation of NF-kB activa-
tion by zinc is cell specific. Zinc is required for NF-kB DNA
by guest on December 28, 2015
binding in purified or recombinant NF-kB p50 protein of
the T helper cell line. Additional studies also showed that
zinc decreased LPS-, ROS-, or TNF-a-induced NF-kB acti-
vation in endothelial cells and cancer cells (46). We reported
previously (44) that compared with controls, healthy volun-
teers who were supplemented with zinc (45 mg/d) had a sig-
nificant decrease in TNF-a and IL-1b mRNA and TNF-a
induced NF-kB DNA binding in isolated peripheral blood
mononuclear cells, and zinc upregulated the expression of
A-20 in HL-60 cells. In this study, we observed that zinc de-
creased oxLDL-induced generation of TNF-a, IL-1b, and
VCAM-1, oxidative stress markers, and activation of NF-kB
and increased A20 and PPAR-2 proteins in human monocytic
and vascular endothelial cells. We propose that zinc inhibited
NF-kB activation by inducing A20, a transactivating factor
that played a role in reducing IL-1b– and TNF-a–induced
NF-kB activation. A20 inhibits NF-kB signaling via TNF re-
ceptor–associated factor pathways in endothelial cells (45).
Our concept of the mechanism by which zinc may be
beneficially regulating various pathways involved in the de-
velopment of atherosclerosis is presented in Figure 3. In-
flammation generates ROS, resulting in oxLDL. OxLDL
activates the NF-kB inducible kinase/1kB kinase/NF-kB sig-
naling pathway and upregulates the downstream target
genes such as inflammatory cytokines, CRP, adhesion mol-
ecules, inducible nitric oxide synthase, cyclooxygenase 2, fi-
brinogen, and tissue factor. These cytokines and molecules
attract blood cellsand platelets leading to coagulation, which
initiates the development of atherosclerosis. We showed that
zinc supplementation increased the plasma concentration of
antioxidant power and decreased the plasma concentration
activated receptor a (PPAR-a) in the human monocytic leukemia
cell line (THP-1) (A) and human aortic endothelial cells (HAECs)
(B and C) after oxidized LDL (oxLDL) stimulation. The cells were
incubated either in zinc-deficient (Zn2, 1 mmol/L) or zinc-
sufficient (Zn+, 15 mmol/L) medium for 8 d (for the THP-1) and for
6 d (for HAECs), followed by 24 h of stimulation with 50 mg
oxLDL/mL. A20 and PPAR-a proteins were measured by Western
blot analysis. *P , 0.05 for Zn2 compared with Zn+ (Values are
SD; n = 3). GADPH, glyceraldehyde 3-phosphate dehydrogenase.
Reproduced with permission from (46).
The effect of zinc on A20 and peroxisome proliferator–
(NF-kB) activation in the human monocytic leukemia cell line
(THP-1) after oxidized LDL (oxLDL) or phorbol myristate acetate
(PMA) stimulation. Zinc-deficient (Zn2) THP-1 cells and zinc-
sufficient (Zn+) THP-1 cells were used for the measurement of
NF-kB activation by electrophoretic mobility shift assay (EMSA)
(A) and luciferase reporter gene assay (B). Effect of zinc on NF-kB
activation in human aortic endothelial cells (HAECs) after oxLDL
stimulation. Zn2 HAECs and Zn+ HAECs were used for the
measurement of NF-kB activation by EMSA (C) and luciferase
reporter gene assay (D). *P , 0.05 for Zn2 compared with Zn+
(Values are SD; n = 3). AFU, arbitrary fluorescent unit/
b-galactosidase U/100 mg protein; P.C., positive control; C.C.,
competition control. Reproduced with permission from (46).
Effect of zinc on nuclear transcription factor kB
Zinc and health185
by guest on December 28, 2015
of inflammatory cytokines and lipid peroxidation bio-
markers in elderly subjects.
In view of these results, I recommend that a controlled,
prospective trial of zinc supplementation in elderly subjects
should be conducted to establish the atheroprotective effect
of zinc. This will have an immense impact on the manage-
ment of patients with coronary artery disease and patients
Role of zinc in diabetes
Diabetes mellitus is one of the most common chronic dis-
eases, and according to WHO, estimate nearly 300 million
individuals are affected by this disorder. Diabetes is one of
the major causes of blindness, increased risk of cardiovascu-
lar disorder, end-stage renal disease, and nontraumatic limb
Insulin, produced by the b cells of the pancreas, is essen-
tial for glucose clearance from the blood to muscle, fat, and
livercells. The hallmark of diabetes is a loss of control of gly-
cemia due to lack of insulin, which may be relative or abso-
lute depending on the type of diabetes.
Type 1 diabetes mellitus alone accounts for 5%–10% of
all cases of diabetes. It is caused by autoimmune destruction
of pancreatic b cells, resulting in virtually no production of
insulin. Without insulin, carbohydrate cannot be used for
energy; therefore, fats become the main intracellular source
of energy. This results in generation of ketone bodies leading
to ketoacidosis. Exogenous insulin administration is the
main treatment for this situation.
Type 2 diabetes mellitus accounts for >90% of the cases
of diabetes and is due to insulin resistance; there is no prob-
lem in insulin production initially by the b cells. Eventually,
however, islet b cell function also declines, leading to overt
diabetes. These patients ultimately also require exogenous
insulin for treatment.
Zinc is crucial for the pancreas and the regulation of
blood glucose. Insulin is stored in a crystalline form as a
zinc insulin complex. Hence, the zinc concentration of the
pancreatic b cells is among the highest in the body. Addition
of zinc to insulin in vitro extended the duration of insulin
action. In the 1930s, zinc ions were added in vitro to pro-
duce protamine zinc insulin to control the blood sugar in di-
In the presence of zinc ions, both insulin and proinsulin
dimers aggregate into hexamers containing bound zinc (80).
ZnT8 mRNA and protein has been shown to be almost ex-
clusively confined to pancreatic islets and to participate in
the regulation of insulin secretion (80). ZnT8 is believed
to be crucial for both zinc transport in the insulin granules
and insulin crystallization, which could not occur unless
zinc is present.
Zinc is a potent physiological regulator of insulin signal
transduction, mainly through the inhibitory effect on pro-
tein tyrosine phosphatase 1b, the key phosphatase that de-
phosphorylates the insulin receptor. An adequate supply of
zinc is crucial for insulin biosynthesis and storage, especially
when there is hyperglycemia. In zinc-deficient states, there is
a clear decrease in islet cell insulin content (80,81). Recent
studies by Jansen et al. (82) showed that zinc supplementa-
tion may be a potential treatment adjunct in type 2 diabetes
because zinc also promotes insulin signaling.
The decrease in total body zinc in diabetic patients may
be due to either hyperzincuria or decreased intestinal zinc
absorption. Decreased zinc in plasma, lymphocytes, granu-
locytes, platelets, and hyperzincuria has been observed in di-
abetic patients (83). Zinc deficiency contributes to diabetic
complications such as increased susceptibility to infections
due to immune dysfunction, increased generation of inflam-
matory cytokines, and increased oxidative stress. In strepto-
zotocin-induced diabetic rats, zinc supplementation (5 mg/kg
atherosclerosis in monocytes/macrophages and vascular
endothelial cells: a proposed hypothesis. Reactive oxygen
species (ROS) induced by many stimuli modifies LDL into
oxidized LDL (oxLDL) in macrophages and vascular endothelial
cells. oxLDL or ROS can activate the apoptotic pathway via
activation of proapoptotic enzymes and the nuclear
transcription factor kB (NF-kB) pathway via NF-kB inducible
kinase (NIK) activation, which eventually results in the
development and progression of atherosclerosis. Zinc might
have an atheroprotective function by the following
mechanisms: 1) inhibition of ROS generation via metallothionein
(MT), superoxide dismutase (SOD), and inhibition of NADPH
oxidase and 2) downregulation of atherosclerotic cytokines/
molecules such as inflammatory cytokines, adhesion molecules,
inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2),
fibrinogen, and tissue factor (TF) through inhibition of NF-kB
activation by A20-mediating TNF–receptor associated factor
(TRAF) signaling and peroxisome proliferator–activated receptor
a (PPAR-a)–mediating crosstalk signaling. The black arrows
indicate upregulation; arrows with a broken line indicate
downregulation or the inhibitory pathway. IKK, IkB kinase; MCP-
1, macrophage chemoattractant protein 1; CRP, C-reactive
protein; ICAM-1, intercellular adhesion molecule 1; VCAM-1,
vascular cell adhesion molecule 1. Reproduced with permission
Signaling pathway for zinc prevention of
by guest on December 28, 2015
zinc sulfate) attenuated diabetes-induced renal oxidative
damage and inflammation and prevented the kidney from
diabetes-induced proteinuria and pathological alterations
A positive correlation between hyperzincuria and glyco-
sylated hemoglobin has been observed (80,84). Inflam-
matory cytokines such as IL-1b, IL-6, and TNF-a play
important roles in the development and complications of
type 2 diabetes. Insulin signaling interference by adipokines
leads to insulin resistance, as demonstrated by serine phos-
phorylation of insulin receptor substrate by TNF-a (75,78).
CRP and IL-6 were significantly increased in the diabetic
group compared with the nondiabetic controls in 1 study
(80,84). In nested case-control and prospective trials, it
was shown that IL-1b, IL-6, TNF-a, and CRP were elevated
several years before the onset of type 2 diabetes (80,84).
In our limited trial, we supplemented 9 Type 2 diabetes
45 mg/d elemental zinc (as gluconate) and 7 subjects re-
ceived placebo for 3 mo. The plasma zinc increased signifi-
cantly and HbA1 C decreased in the zinc-supplemented
group (P = 0.03) Plasma IL-6, CRP, and ICAM-1 showed
a statistically nonsignificant decrease in the zinc group. Ob-
viously a large placebo-controlled trial is needed before any
firm conclusion can be drawn.
A prospective study of zinc intake and risk of type 2 dia-
betes was assessed among 82,297 women 33–60 y of age at
baseline in 1980 and followed until 2004 in the Nurses’
Health Study (85). During the 24 y of follow-up, they iden-
tified and confirmed 6030 cases of incident type 2 diabetes.
In an age-adjusted analysis, intake of total zinc but not die-
tary zinc from food sources was significantly associated with
a lower risk of type 2 diabetes. After adjustment for nondi-
etary risk factors, including age, BMI, smoking, and other
covariate, the highest quintiles of both total and dietary
zinc intake were significantly associated with an w20%
lower risk of type 2 diabetes.
At the cellular blood level, zinc increases total protein
phosphorylation of the insulin receptor b subunit of both
preadipocytes and adipocytes. Zinc inhibits tyrosine phos-
phatase 1B, leading to increased phosphorylation of the in-
sulin receptor b subunit. An intracellular release of free zinc
is a potent physiological regulator of insulin signal transduc-
tion through its inhibitory effect on tyrosine phosphatase
1B, the key phosphatase that dephosphorylates the insulin
receptor (80). Thus, this evidence implicates an important
role of zinc in diabetes management, and I recommend a
clinical trial of zinc in this disease.
Zinc and AD
The first description of AD was published by Alois Alz-
heimer in 1906 (86). AD now represents the most prevalent
form of dementia in modern society, accounting for 60%–
80% of all dementia cases (87). Individuals with AD have
a progressive cognitive decline that ultimately also erodes
all higher order executive functioning. At autopsy, they ex-
hibit a number of cardinal features within the brain. The
most pronounced features are the presence of extracellular
deposits known as plaques and intracellular accumulations
known as neurofibrillary tangles. These 2 features are diag-
nostic of AD. Other gross manifestations are the thinning of
the cortical gray matter, the enlargement of the ventricular
spaces, and generalized atrophy of the brain (87).
The potential role of zinc in dementia was first proposed
by Burnet (88). Since then, many reports have been pub-
lished concerning the role of zinc in AD. The concentrations
of zinc within the brain tend to increase from birth to adult-
hood and then remain stable throughout life. The serum/
plasma zinc in AD patients has been reported to be in-
creased, unchanged, and decreased (87,89,90); thus, there
appears to be a large degree of variations between different
Molina et al. (91) examined 26 AD and 28 control sub-
jects and found a significant decrease in cerebrospinal fluid
zinc levels in AD patients. Consistent with this, Kapaki et al.
(92) found a similar result in their study.
There is no difference in zinc level in frontal lobe tissue of
brain between AD and controls. However, when these tis-
sues were subfractionated, a significant decrease in zinc
levels in the nuclear fraction (but not the mitochondrial
or microsome fraction) of AD cases was observed (87). De-
creased zinc levels were also seen in the neocortex, superior
frontal and parietal gyri, medial temporal gyrus, thalamus,
and hippocampus in AD patients (87).
Studies showed that senile plaque cores of AD patients
consist primarily of b-amyloid protein (AB) derived from
a larger transmembrane protein amyloid precursor protein
(APP) with both zinc and copper coordinated to histidine
residues located at the N-terminal end of the protein (87).
It appears that a dysregulation in various metal storage and
transport proteins within the AD brain may contribute to a
involved in the regulation of zinc in the brain. These include
zinc transporting ZIPand ZNTand metallothionein. Further-
more, a number of these proteins such as ZNT family are
found not only in the neuropil but also within the plaques
APP is ubiquitously expressed. One function of APP is
to participate in the maintenance of metal ion homeostasis.
An abnormal expression of APP might result in dysregula-
tion of metal ion homeostasis. Recently, it was reported
that APP possesses iron-export ferroxidase activity that is
inhibited by zinc (87). A disturbance in zinc homeostasis
may result in decreased ferroxidase activity, resulting in
The processing of APP to generate AB peptides of
varying length involves different secretases, all of which
appear to interact with different metals including zinc
(87). After generation of AB, it too is capable of binding
zinc. It appears that metals promote the AB aggregation
Another important consequence of the binding of zinc to
AB is that it obscures the proteolytic cleavage site (87),
thereby inhibiting its ability to be degraded by matrix metal-
loproteinase. Removal of the zinc with clioquinol, however,
Zinc and health187
by guest on December 28, 2015
restored the sensitivity of AB degradation by matrix metal-
This discussion suggests a deleterious effect of zinc
binding to APP and AB; however, there are several studies
that suggest a potentially protective effect of zinc under cer-
tain circumstances (87). Lovell (93) reported that AB/Zn ra-
tios 1:0.1 and 1:0.01 result in a protective activity against AB
toxicity. This effect is mediated in part by a modulation of
Na+K+ATPase activity that prevents the typical calcium dys-
homeostasis and cell death associated with AB toxicity (87).
Garai et al. (94) suggested that, in vitro, very low concen-
trations of zinc may eliminate oligomeric AB, thereby limit-
ing the toxicity mediated by soluble oligomers. It has been
suggested that zinc-AB aggregates may form so as to inhibit
the reduction of Cu2+and the production of hydrogen per-
oxide that arises from AB-Cu interaction (87). This repre-
sents the role of zinc as an antioxidant, which results by
displacing pro-oxidant Cu2+.
Zinc as a therapeutic modality for AD. Studies using
Tg576 mice, which overexpress a mutant human form of
APP and develop cerebral AB plaques (87), and ZnT3
knockout mice have suggested that zinc may hold amyloid
load in a dissociable equilibrium and provide strong support
for a role of synaptic zinc in the metabolism of AB. The
study also showed a reduction in cerebrovascular amyloid
loads in these animals. Genetic ablation of ZnT3 may gener-
ate a phenocopy for the cognitive and synaptic deficits pre-
sent in transgenic mouse models of AD (87).
Recently, a number of metal-modulatory compounds
have been developed that normalize cerebral zinc ion ho-
meostasis (87). Clioquinol treatment in aged Tg 2576 mice
showed a significant reduction in plaque burden parallel
with a shift in brain content of AB toward more soluble spe-
cies (87). Analysis of cerebral metal levels did not reveal a re-
duction in metals content but showed a significant increase
in copper and zinc on treatment. These data obtained after
clioquinol and PBT2 (a metal-modulatory compound)
treatment thus showed that there is a redistribution of
metals in the brain and do not chelate metals out of the sys-
PBT2 showed profound effects in transgenic mouse
models of AD (87). It was shown that the administration
of PBT2 significantly decreased both insoluble and soluble
burden, decreased levels of phosphorylated tau, increased
levels of synaptophysin (a surrogate marker of synapses),
and rapidly improved learning and memory performances
in the Morris water maze (87). This drug is currently under-
going a clinical trial for the treatment of AD patients (87).
A placebo-controlled trial of zinc supplementation was
conducted for 6 mo recently in AD patients in Florida
(95). The subjects received 150 mg/d of elemental zinc
away from food. The new zinc product, Reazin, was devel-
oped by Adeona Pharmaceuticals, Ann Arbor, MI. The pro-
duct releases zinc slowly in the stomach and later in the
small intestine, which maintained a sustained elevation of
plasma zinc throughout the day. The sum of scores on 3
cognitive measures, Alzheimer’s Disease Assessment Scale-
Cognitive Subscale, Mini-Mental State Examination, and
Clinical Dementia Rating scale, were assessed.
The study showed that zinc had stabilized cognition,
whereas the placebo group continued to deteriorate. In
patients age 70 y and older, the study showed a statistically
significant effect of zinc supplementation on cognitive
Recent studies showed that neuronal stress and conse-
quent overexpression of proinflammatory proteins are the
likely instigators of neuropathological changes, including
both plaque and tangle formation in AD patients (96). It
is believed that microglia cells are essentially the immune
cells of the brain. It was shown that neurons were activating
microglia to release IL-1, which in turn activated astrocytes
and caused them to release S100, a soluble astrocyte Ca-
binding protein with proinflammatory effects. Thus, it ap-
pears that IL-1 and S100 are associated with AD (96). IL-1 was
shown to upregulate the production of bAPP in cord blood
and brain; thus, excess inflammatory cytokine IL-1 was a
driving force in neurodegeneration and genesis of amyloid
Brainwith IL-1 pellets had elevated levels of the mRNAof
a phosphorylating kinase protein mitogen-activated protein
kinase 38. It was shown that IL-1 was driving both the pro-
duction of tau protein and its hyperphosphorylation via IL-
1 induction of a specific kinase: mitogen-activated protein
kinase p38 (96). IL-1 may also contribute to the memory
deficits in AD by decreasing the levels of neurotransmitter
acetylcholine, a decrease often seen in AD patients. It was
observed that the enzyme acetyl cholinesterase, which de-
grades acetylcholine, is upregulated by IL-1 (96).
We reported in the past that zinc deficiency in humans
and in cell culture models activates macrophages and mon-
ocytes by increasing oxidative stress and via upregulation of
NF-kB activation generates mRNA of IL-1b and the cyto-
kine protein (37,44,46). I hypothesize that similar phenom-
enon may be taking place in zinc-deficient elderly subjects
such that microglia cells are being oxidatively stressed due
to zinc deficiency leading to upregulation of IL-1b in brain
cells. Increased IL-1b in turn could upregulate the genera-
tion of AB and tau proteins and hyperphosphorylation of
tau protein, leading to formation of neurofibrils and damage
to neuronal cells. If this hypothesis is correct, one would see
an effective prevention of AD in zinc-deficient elderly pa-
tients who are supplemented with zinc (45 mg/d elemental
zinc) to correct their deficiency. This hypothesis needs to
be tested. If correct, zinc supplementation may prove to
be a very important therapeutic adjunct of AD.
The sole author had responsibility for all parts of the
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