zinc

Biological Function of ZincIsfahan University of Medical Science, School of Pharmacy

Department of Clinical Biochemistry

April 8, 2023 Total slides: 78 2

Biological Function of Zinc

Zinc

Biology

By: A.N. Emami Razavi

(An overview)



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)



Introduction

Zinc



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

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)

1746



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 .



Sources, Requierment & Hemostasis

Zinc



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 .



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

Causes:

Malnutrition Alcoholism Malabsorption Burns Chronic renal disease Acrodermatitis

enteropathica

Deficiency Manifestation

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

Moderate growth retardation and delayed puberty in adolescents, hypogonadism in males, rough skin, poor appetite, mental lethargy, delayed wound healing, taste abnormalities and abnormal dark adaptation

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.



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

Physiological functions of zinc



Biochemical function

Zinc



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

Displacement of water → Lewis acid catalysis

Carbonic anhydraseAlcohol dehydrogenaseCarboxypeptidaseMatrix metalloproteinaseThermolysinβ lactamase



Carbonic anhydrase



Zn 2+Polarizes H2O, making it a better nucleophile

His –Zn2+

His

His

O

H

CO

O

H2O

His –Zn2+

His

His

O

H

..+ C

O

O

His –Zn2+

His

His

O

H

..+ H+ + H O C

O

O

Bicarbonate

Displaces HCO3-



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.



C2H2 motif

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

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

Classes of zinc finger



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




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

Zinc Finger Motifs: Protein-Protein Interaction



Molecular biology

Zinc



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 Zn2+ uptake



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.



Immunology

Endocrinology

function

Zinc



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



Case Report

Zinc



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.



Questions?

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