Zinc

B io l o g ic a l F u n c t io n o f Z in c
Is f a h a n U n iv e r s it y o f M e d ic a l S c ie n c e , S c h o o l o f
P ha rma c y
D e p a r t m e n t o f C lin ic a l B io c h e m is t r y


Z in c
B io lo g y

(An o v e r v ie w )

B y :e 2 6 ., N0 .1 2E m a m i R a z a v i
Ju n
A 2 T o t a l s l id e s : 7 8 2


Outlines

 Introduction
 Sources, requirement and homeostasis
 Zinc deficiency
 Zinc toxicity
 Biolochemical functions of zinc
 Molecular biology of zinc
 Immunological & Endocrinological Functions of zinc
 Case report (acrodematitis enteropathica)

Ju n e 2 6 , 2 0 1 2 T o t a l s l id e s : 7 8 3


Z in c

In t r o d u c t io n



 In ancient India the production of zinc metal
was very common. Many mine sites of Zawar
Mines, near Udaipur, Rajasthan;-Zawarmaala
were active even during 1300-1000 BC. There
are references of medicinal uses of zinc in the
Charaka Samhita (300 BC). The Rasaratna
Samuccaya (800 AD) explains the existence of
two types of ores for zinc metal, one of which is
ideal for metal extraction while the other is used
for medicinal purpose.



Pure metallic zinc was discovered by Andreas
Marggraf (Germany) in
1 746
 Atomic number: 30
 Atomic weight: 65.409
 Valency: +2
 Group #: 12
 Periodic #: 4
 State: solid metal at room
temperature
 Isotopes: 21 isotopes (5
stable and 16 unstable)



Appearance

Bluish pale gray



What is zinc?
 Zinc is an essential mineral that is found in
almost every cell. It stimulates the activity of
approximately 300 enzymes, which are
substances that promote biochemical reactions
in the body. Zinc supports a healthy immune
system , is needed for wound healing , helps
maintain the sense of taste and smell , and is
needed for DNA synthesis . Zinc also supports
normal growth and development during
pregnancy, childhood, and adolescence .



Z in c

S ourc e s ,
R e q u ie r m e n t &
H e m o s t a s is



Sources
Foods contain element zinc, much of it bound to
protein or DNA.

 Oysters (> 70 mg per serving).

 Meats (2-3 mg/100g).

 Shellfish (2.7 mg/100g)

 Other good food sources include:
 beans, nuts, certain seafood, whole grains,
fortified breakfast cereals, and dairy products .

Ju n e 2 6 , 2 0 1 2 T o t a l s l id e s : 7 8 1 0


 Zinc absorption is greater from a diet high in
animal protein than a diet rich in plant
proteins . Phytates, which are found in whole
grain breads, cereals, legumes and other
products, can decrease zinc absorption .



RDA



Absorption
 GIT modulates the quantity of exogenous dietary zinc absorbed
and the quantity of endogenous zinc excreted

 More than 70% of a small zinc dose (less than 3 mg) is absorbed
from the small intestine.

 Maximum absorption occurs in duodenum

 There is sustained release from enterocytes into portal
circulation for ~ 9h



Fractional Absorption

 Inversely proportional to the amount of zinc in the meal

 Does not depend on the ‘zinc status’ of the body

 Increased by breast milk; Decreased by phytates

 Protein hydrolysates and some amino acids, particularly
histidine and cysteine, increase fractional zinc absorption.



Transport and cellular uptake
 ZIP4 (SLC39A4), a member of the ZIP family .
 This transporter is expressed at the luminal side of enterocytes through
the small and large intestine.
 hoRF1, may contribute to zinc uptake from the colon.
 Genetic variants of this gen are associated with the rare familial
condition acrodermatitis enteropathica.
 DMT1,(SLCA11A2)
 The proton coupled divalant cation transporter 1 appears to provide a
minor uptake route. DMT1 also transports iron, copper, cadmium and
other divalent metal ions that may compete with zinc.
 PepT,(SLC15A1)
 Hydrogen ion/peptide cotransporter is a posible alternative route for zinc
uptake when complex to small peptides.uptake as a complex with
individual amino acids may explain why histidine and cysteine improve
intestinal zinc absorption.


 Metallothinein
 Regulates zinc transfer into portal blood through binding and
retaining it within the enterocyte until shedding into the
intestinal lumen.
 Zinc transporters
 Zinc is exported from enterocytes into portal blood by Zinc
transporters 1 (ZnT-1,SLC30A1) and 2
(ZnT-2,SLC30A2).ZnT-1 is upregulated when zinc intake is
high.
 Paracellular pathway
 With increasing intra luminal concentrations, net zinc
movement across the tight junctions of the epithelial layer
becomes more significant. Regulatory mechanisms involving
DMT1 or metallotionein thus are bypassed when high-dose
supplement are ingested.


Intestinal zinc absorption



Excretion
 Routes: intestine, kidneys, integument, and semen

 After a meal, maximum zinc secretion occurs through
pancreatobiliary secretions

 Maximum reabsorption occurs from mid-jejunum and ileum

 Total amount excreted = Amount secreted – Amount reabsorbed

 Excretion of endogenous zinc by the intestine depends on the
‘zinc status’ of the body.


Regulation
 Metallothionein expression
 The most important mechanism for maintaining zinc
adequacy.
 Increased expression in the small intestine decreases
intestinal absorption
 Increased expression in the liver expands stores.
 Metallothionein expression is induced by the metal
response element-binding transcription
factor-1(MTF-1)
 MTF-1 is bind to multiple metal response elements of the
metallothionein promoters when the free zinc ion
concentration is high.
 MTF-1 also induces expression of ZnT-1


Metallothioneins
 Major Zn2+ binding protein in mammalian
systems is metallothionein (MT)

 MT contain Zn2+ coordinated to Cys by
mercaptide bonds

 Binding of Zn2+ by MT depends on the
redox state of the cell – oxidization
releases Zn2+ from MT and vice versa.

 Zn2+ + Apothionein → Metallothionein



Cellular Zn2+ sensors
 The cellular Zn2+ sensor is
MTF-1 (Metal response
element binding transcription
factor-1)

 Intracellular Zn2+ binds to
MTF-1

 Zn-MTF-1 binds to MRE and
increases transcription of
Metallothionein and ZnT1
mRNA.

 MT binds intracellular Zn2+
and removes it from the free
pool.

 ZnT1 increases Zn2+ efflux
from cells



Compartments: Endogenous Zinc Pools (EZP)
 Plasma
 75% of Zn2+ is bound to albumin and 20% to α2-macroglobulin.
 Most of the remaining Zn2+ is complexed to His & Cys rich proteins.
 The free Zn2+ concentration of serum is in nM range.

 Rapidly Exchanging Pool/ Endogenous Zinc Pool
(EZP)
 Liver, spleen, kidneys, bone marrow, erythrocytes
 exchanges with plasma zinc within 3 days
 accounts for 10% of total body zinc

 Slow Exchanging Pools
 Nervous system, muscles, bones etc


Intracellular Distribution of Zn2+

 30-40% in nucleus
 50% in cytosol and cytosolic
organelles
 10-20% in membranes.

 The cytosolic free [Zn2+] is 1-2
nM

 Zinquin fluorescence shows
 Fluorescent cytosol
 Non-fluorescent nucleus
 Zincosomes (vesicles)



Intracellular Distribution of Zn2+

 Confocal microphotographs of
mouse fibroblasts stained with
probes for DNA (blue),
microtubules (green). Next
generation zinc probes reveal
vesicular Zn(II) pools (purple).
Note the change in vesicular
distribution as the cell in the
upper right hand corner
undergoing mitosis.



Zinc Transporters



Zinc deficiency
Deficiency Manifestation

Severe dermatitis, alopecia, diarrhea,
 Causes: emotional disorder, weight loss,
infections, hypogonadism in males

 Malnutrition
 Alcoholism Moderate growth retardation and delayed puberty
 Malabsorption in adolescents, hypogonadism in males,
rough skin, poor appetite, mental
 Burns lethargy, delayed wound healing, taste
 Chronic renal disease abnormalities and abnormal dark
 Acrodermatitis adaptation
enteropathica

Mild oligospermia, slight weight loss and
hyperammonaemia



(Left) This boy has a zinc deficiency, and his hair is very thin and sparse; (right) after
treatment his hair is growing more strongly



Acrodermatitis Enteropathica
 hZIP4 gene mutation

 Erythematous, dry, scaly,
eczematous skin.

 Periorificial and acral pattern on
the face, the scalp, the hands, the
feet, and the anogenital areas.

 Infants may also experience
withdrawal, photophobia, and
loss of appetite.



Zinc deficiency as a cause of anorexia nervosa
 Zinc deficiency causes a decrease in appetite -- which could
degenerate in anorexia nervosa (AN). Appetite disorders, in turn,
cause malnutrition and, notably, inadequate zinc nutriture. The
use of zinc in the treatment of anorexia nervosa has been
advocated since 1979 by Bakan. At least 5 trials showed that
zinc improved weight gain in anorexia. A 1994 randomized,
double-blind, placebo-controlled trial showed that zinc (14 mg
per day) doubled the rate of body mass increase in the treatment
of anorexia nervosa (AN). Deficiency of other nutrients such as
tyrosine and tryptophan (precursors of the monoamine
neurotransmitters norepinephrine and serotonin, respectively), as
well as vitamin B1 (thiamine) could contribute to this
phenomenon of malnutrition-induced malnutrition.


Zinc toxicity
 Even though zinc is an essential requirement for a healthy body,
too much zinc can be harmful. Excessive absorption of zinc can
also suppress copper and iron absorption. The free zinc ion in
solution is highly toxic to plants, invertebrates, and even
vertebrate fish. The Free Ion Activity Model (FIAM) is well-
established in the literature, and shows that just micromolar
amounts of the free ion kills some organisms. A recent example
showed 6 micromolar killing 93% of all daphnia in water.
Swallowing an American one cent piece (98% zinc) can also
cause damage to the stomach lining due to the high solubility of
the zinc ion in the acidic stomach.



Zinc toxicity
 Zinc toxicity, mostly in the form of the ingestion of US pennies
minted after 1982, is commonly fatal in dogs where it causes a
severe hemolytic anemia. In pet parrots zinc is highly toxic and
poisoning can often be fatal.

 There is evidence of induced copper deficiency at low intakes of
100-300 mg Zn/d. The USDA RDA is 15 mg Zn/d. Even lower
levels, closer to the RDA, may interfere with the utilization of
copper and iron or to adversely affect cholesterol.



Physiological functions of zinc
 Biochemical functions :
 Cofactor for enzymes
 Activity of zinc finger proteins

 Cellular functions :
 Growth & cell development
 Cell membrane integrity
 Tissue growth & repair
 Wound healing

 Endocrinological functions:
 Reproduction: spermatogenesis & oogenesis
 Thyroid function
 Pancreatic function
 Prolactin secretion
 Thymopoetin synthesis

 Immunological functions : function of neutrophils, T cells, B cells and NK cells
 Neurological function: Cognition, memory, taste acuity, vision
 Hematological function : coagulation factors
 Skeletal function : Bone mineralization


Z in c

B io c h e m ic a l
f u n c t io n



Zn2+ containing enzymes

 Zn2+ is an essential cofactor for ~ 300 enzymes from all 6
classes.

 There are 3 primary types of Zn2+ sites: catalytic, structural & co-
catalytic

 His, Glu, Asp and Cys are the main amino acids that supply
ligands to these sites.



Catalytic sites

3 protein ligands, bound
water
 Conformational change during
catalysis activates Zn2+ bound
water

 Ionization or polarization of water
→ acid base catalysis Carbonic anhydrase
Alcohol dehydrogenase
Carboxypeptidase
 Displacement of water Matrix metalloproteinase
→ Lewis acid catalysis Thermolysin
β lactamase



Carbonic anhydrase



Zn 2+Polarizes H2O, making it a better nucleophile

His His
O O
..
His –Zn2+ O + C His –Zn2+ O C
O
H O H
His His
H2O
His Displaces HCO3-
O
..
His –Zn 2+
O + H+ + H O C
O
H
His Bicarbonate



Structural sites

4 protein ligands, no bound
water

 Responsible for chemical
properties

 Alcohol dehydrogenase
 Protein kinase family
 DNA, RNA polymerase
 Matrix metalloproteinase
 tRNA synthase family



R-alcohol Dehydrogenase from Lactobacillus brevis

 Alcohol dehydrogenase
uses two molecular
tools to convert ethanol
to acetaldehyde. The
first is a zinc atom
which is used to hold
and position the alcohol
group on ethanol. The
second is a large NAD
cofactor, which actually
performs the reaction.



Co-catalytic sites

bridging of two metal sites by
an AA usually Asp

 Responsible for the overall
fold of the protein as well as
catalysis

 Superoxide dismutase
 Phosphatase
 Aminopeptidase
 β-lactamase



Zinc fingers
 A zinc finger is a protein domain
that can bind to DNA. A zinc finger
consists of two antiparallel β sheets,
and an α helix. The zinc ion is
crucial for the stability of this
domain type -in absence of the
metal ion the domain unfolds as it is
too small to have a hydrophobic
core
 Zinc fingers are important in
regulation because when interacted
with DNA and zinc ion, they
provide a unique structural motif for
DNA-binding proteins.



Classes of zinc finger

 C2H2 motif

 2 Cys and 2 His residues bind
to one Zn2+ .

 E.g. TFIIA, developmental/cell
cycle regulators, metabolic
regulators




 C4 motif

 Nuclear hormone
receptors

 E.g. Estrogen Receptor
(ER). ER forms a dimer.
Each ER binds to 2 Zn2+ .
All steroid receptors
have the C4 motif




 C6 motif

 Gal4. Forms a dimer. 6
Cys residues bind to 2
Zn2+

 E.g. metabolic
regulators



Zinc Finger Motifs: Protein-Protein Interaction

 RING-finger

 specialized Zn-finger involved
in mediating protein-protein
interactions

 a series of His and Cys residues
with a characteristic spacing
that allows the coordination of
two zinc ions

 Ubiquitin Ligase complex



Zinc Finger Motifs: Protein-Protein Interaction

 LIM domain

 2 tandemly repeating Zn-fingers

 Homeobox proteins

 Transcription regulatory proteins



Z in c

M o le c u la r b io lo g y



Zn2+ and cell signaling

 Interaction with signal transduction pathways

 Protein phosphorylation and dephosphorylation

 Second messenger metabolism

 Enzyme regulatory activity

 Activity of transcription factors



Interaction with signal transduction pathways &
Protein phosphorylation / dephosphorylation



Interaction with signal transduction pathways: Ca2+
signaling pathways

 Electrical stimulation of cardiac cells can cause Zn2+ entry through voltage
gated Ca2+ channels

 Elevation of extracellular Zn2+ can act via HHS-R to mobilize Ca2+ from
hormone sensitive Ca2+ stores → increases intracellular [Ca2+]

 Ca2+ -Calmodulin dependent PK activity:
 Low [Zn2+] increase calmodulin independent activity
 High [Zn2+] inhibit the binding of Ca2+ -Calmodulin



Second messenger metabolism: Cyclic Nucleotides

 cGMP PDE is activated by
Zn2+ upto 1µM; at > 1µM
cGMP PDE is inhibited

 Increase in intracellular Zn2+ >
1µM increases cGMP

 Increase in cGMP in turn
downregulates Zn2+ uptake

 NO, an activator of guanylyl
cyclase enhances the
downregulation of Zn uptake
2+



Enzyme regulatory activity : Protein Kinase C function

 PKC contain 2 Zn2+ binding motifs in the regulatory region

 Zn2+ in nM concentrations can activate PKC and increase its
translocation to the cell membrane and cytoskeleton

 The cellular redox state can affect PKC activity: Oxidation
causes release of Zn2+ from Zn2+ binding motifs of PKC →
 increases autonomous activity of PKC,
 decreases sensitivity to regulating cofactors.



Activity of transcription factors

 Regulation through zinc finger domains:
 MTF-1 (metal transcription factor-1)
 CREB (cAMP response element binding protein)
 Steroid receptors
 Gal-4

 Direct regulation:
 Activation of NF-κB



Neuronal Zn2+ signals

 There are three types of neuronal Zn2+ signals:

 Zn2+ -SYN

 Zn2+ -TRANS

 Zn2+ -INT



Zn2+ -SYN

 Synaptic vesicular Zn2+ in the presynaptic boutons is released on
axonal depolarization.

 Zn2+ can reach 10-30μM in the extracellular fluid

 Zn2+ -SYN reaches multiple Zn2+ modulated postsynaptic sites
 Amino acid receptors: glutamate- and GABA- R
 Tonic defacilitation of glutamate receptors: absence of synaptic Zn2+
increases seizures



Zn2+ -TRANS

 Transmembrane flux of Zn2+. Zn2+-TRANS is analogous to
transmembrane Ca2+ flux.

 The Zn2+ channels are Ca-AK channels and NMDA channels



Zn2+ -INT

 Analogous to intracellular Ca2+ signal, but no organelle
analogous to SR/ER has been identified for Zn2+

 Likely source of Zn2+ -INT is MT

 NO is one of the main inducers of Zn2+ -INT signal



Role of Zn2+ in cell proliferation

 Zn2+ increases IGF-1. IGF-1
causes G1→S transition.

 Zn2+ stimulates GF-R which
act via MAPK pathway

 Zn2+ increases phosphorylation
of Jun & ATF-2



Role of metallothioneins in Zn2+ mediated cell proliferation
& differentiation
 Cellular MT levels oscillate during the cell cycle reaching a
maximum at G1→S transition.

 MT translocates to nucleus during S phase in rapidly
proliferating cells.

 The nuclear translocation of MT is a vehicle for achieving high
nuclear zinc level in the S-phase of the cell cycle.

 Similarly MT translocates into the nucleus of differentiating
cells. E.g. preadipocytes, myoblasts. After differentiation, MT
translocates back to the cytoplasm.



Zn2+ inhibits apoptosis
 Mitochondria releases ROS which causes
lipid peroxidation, DNA damage, Protein
SH oxidation

 Mitochondria channel input signal
pathways to the central pathway of
Bcl-2(anti-apoptotic) or Bax (pro-
apoptotic) genes.

 Bcl2/Bax ratio determines whether cells
are apoptosed or not.

 When cells pass the Bcl2/Bax
checkpoint, Cytochrome C is released

 Cyt C activates the caspase cascade.


 Zn2+ inactivates the
caspase cascade

 Zn2+ binds to Cys163 and
prevents disulfide bond
formation

 This prevents dimerization
of Caspase-3 and inhibits
its activity.



Z in c

Im m u n o lo g y
E n d o c r in o lo g y
f u n c t io n



Immunological function of Zn2+
 Effect of Zn2+ deficiency on neutrophils:
 Decreases bone marrow production
 Decreases chemotaxis and adhesion
 Impairs phagocytosis and oxidative burst



 Decreases NK cell lytic activity and IFNα production

Decreases monocyte/macrophage activation, phagocytosis



 In B cells Zn2+ deficiency produces apoptosis

 In T cells Zn2+ deficiency produces thymic atrophy → impaired
T cell development and decreased counts.



Endocrinological function of Zn2+
 Insulin is stored in pancreatic islets as osmotically stable zinc-
insulin complex (2 Zn2+ : 6 insulin)

 The hexamers are formed in the trans-Golgi complex.

 Stimulating the islets by glucose and other stimulators result in
the release of the hexamer which immediately dissociates into
insulin and Zn2+.

 Zn2+ ions released from β cells stimulate the secretion of
glucagon from α cells by a paracrine mechanism.



Zn2+ and Diabetes Mellitus

 Plasma Zn2+ concentration is decreased

 Glucose-mediated hyperzincuria and decreased gastrointestinal absorption of
zinc are responsible. Hyperzincuria responds partly to insulin treatment.

 Hyperglycemia interferes with the active transport of Zn back into the renal
tubular cells.

 Zinc supplementation reduces blood glucose level in type 1 diabetics.

 At the molecular level:
 Zn2+ inhibits post insulin receptor intracellular events which results in a decreased
glucose tolerance, and a relative decrease in insulin secretion.



Zn2+ and Type 1 Diabetes Mellitus
 Type I DM is an autoimmune destruction of islets of pancreas
mediated by free radicals.

 Zn2+ is required for the function of Superoxide dismutase,
catalase and peroxidase.

 Zn-metallothionein complex in the islet cell provides protection
against free radicals.

 Zn2+ deficiency impairs the function of these enzymes and
increases autoimmune destruction.



Zn2+ and Type 2 Diabetes Mellitus
 In Type 2 DM there is insulin resistance
at the receptor level.

 In the initial stages this leads to
increased insulin secretion

 Excessive Zn2+ ions are co-secreted
with insulin during hyperglycemia

 Zn2+ induces islet cell death

 Endogenous Zn2+ translocates to other
islet cells, probably leading to their
death

 Chelation of released Zn2+ inhibits islet
cell death in vitro and diabetes in vivo


Summary
 Zn2+ is an essential micronutrient that is maintained in the physiological range
by various homeostatic mechanisms

 Zn2+ is essential for the function of enzymes and zinc finger proteins

 Metallothioneins, MTF-1 & ZnT1 regulate the intracellular level of Zn2+

 Zn2+ plays an important role in various signaling pathways

 Zn2+ is essential for cell proliferation, differentiation and apoptosis

 Zn2+ deficiency can decrease the function of the immune system

 Zn2+ is essential for the normal function of the pancreas



Z in c

C a s e R e port



ACRODERMATITIS ENTEROPATHICA TYPE OF
DERMATOSIS
ZINC DEFICIENCY PRESENT

 Bilateral, erythematous areas,
some of which are weeping, plus
vesicles, pustules and ulcers of the
lower extremities



 Note the weeping nature of
some of the lesions on the
dorsum of the foot. Yellowish
foci probably correspond to
pustules histologically. One of
the small blisters it at the tip of
the arrow. Erosions are present.



 Low power view, biopsy #1. The keratinocytes in the upper 1/3
of the epidermis are slightly pale when compared to those in the
lower epidermis. All of the keratinocytes are much larger than
normal.



 High power view of above (biopsy #1). Dyskeratosis of the type
that has been described as being the result of condensation of
tonofilaments in other diseases is apparently present here
(arrows). Spongiosis and edema of the papillary dermis are also
present.



 High power view of the superficial part of biopsy #1 showing
polymorphonuclear leukocytes within the upper levels of the
epidermis. More of these would have resulted in a pustule. The
pallor of the keratinocytes in this area may be the result of
increased cytoplasmic volume. The parakeratotic cap would
account for a scaly lesion.



 Low power view of biopsy #2. The pallor of the superficial part
of the epidermis is striking. Intraepidermal vesicles may result
from severe ballooning of keratinocytes (intracellular edema in
this case) or the coalescence of ballooned keratinocytes.
Spongiotic vesicles also are forming.



 High power view of biopsy #2. The keratinocytes, even those in
the basal layer, are huge, and some seem about to explode. There
are small defects at the dermoepidermal junction in this area.



 Very high power view of the dermoepidermal junction in biopsy
#2.



Q u e s t io n s ?


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