The Importance of Zinc in Animal Health

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History

1509, recognized as element
Essentiality demonstrated
- Plants: 1869
- Animals: 1934
Deficiency
- Considered unlikely until 1955
  - swine parakeratosis shown to be caused by Zn deficiency
  - conditioned human deficiency demonstrated in 1956
  - 1961, hypogonadal dwarfism suggested to be zinc deficiency

Facts

30th element in the periodic table (IIB element)
- MW = 65.37, completely filled d orbitals
In aqueous solutions
- One oxidation state, namely Zn2+
- Prefers tetrahedral complex formation
Not a redox active metal
- readily complexes with amino acids, peptides, proteins and nucleotides
- affinity for thiols, hydroxy groups & ligands with electron-rich nitrogen donors

Distribution

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Sources

Relatively abundant mineral
- Good sources: shellfish, beef and other red meats
- Slightly less good: Whole-grains
  - most in bran and germ portions
  - 80% lost to milling
  - phytates, hexa & penta phosphates depress absorption
    - P/Zn ratios of 10 or more
- Relatively good sources: nuts and legumes
Eggs, milk, poultry & fish diets lower than pork, beef, lamb diets
- High meat diets enhance absorption
  - 280g or 10 oz fits right into food pyramid guide
  - cys & met form stable chelate complexes

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Effect of trace mineral source on animal performance

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Relative bioavailability of trace mineral sources

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Whole Body Fluxes

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Dietary Factors that Affect Zn Absorption

Feed/Food source
Phytate (calcium-phytate-zinc complex)
- Mainly hexa- and pentaphosphate derivatives
- Highly dependent on calcium
Amino Acids
- histidine, cysteine
Presence/Absence of other divalent cations
- Fe, Ca
Efficiency of absorption can vary from 15-60%
- Under normal conditions 1/3 of dietary Zn is absorbed
- Zn status alters efficiency of absorption
Uptake and retention is > in growing animals

Overview

Approximately 300 enzymes are associated with zinc
Biological functions of Zn are divided into three categories
- Catalytic, Structural, Regulatory
Role in metabolism
- Protein synthesis
- Nucleic acid metabolism
- Carbohydrate and energy metabolism
- Lipid
- Epithelial tissue integrity
- Cell repair and division
- Vitamin A and E transport and utilization
- Immune function
- Reproductive hormones

Absorption

Absorption takes place throughout the intestine
- Glycocalyx
  - Barrier? Storage site?
- Primarily in the jejunum
- Some absorption in the rumen
- No measurable amounts absorbed from stomach cecum or colon
In small intestine

Nonmediated (nonsaturable) process
- Not affected by dietary Zn intake
Mediated (saturable) process
- Stimulated by Zn depletion

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Transport in blood

Plasma contains approx .1% of the total zinc of the body
Albumin is major portal carrier
- Binds to albumin by tetrahedral ligation to sulfur atoms
- 70% of Zn is bound to albumin in plasma
- 20-30% bound to α-2 macroglobulin
- Other plasma proteins
  - Transferrin, histidine-rich glycoprotein, metallothionine
Plasma Zn concn's respond to external stimuli Intake fluctuations Fasting Acute stresses infection Plasma Zn levels do not influence absorption from mucosa
Most reductions in plasma levels reflect increased hepatic uptake
- Hormonal control

Transport

Rapidly cleared from plasma by liver
Fast component of 2 pool model (T1/2 = 12.3 da)
- Single dose of zinc is taken up with T1/2 = 20 s
Slow component, other tissues (T1/2 = 300 da)
Bone and CNS uptake slow
Pancreas, liver and kidney most rapid
RBC & muscle in between
Exchangeable pool & zinc status

Cellular Uptake

Hepatic uptake via a biphasic process
- Contribution to overall Zn flux
  - Sequesters newly absorbed Zn
  - Removes Zn from the circulation
- Saturable process – initial step
  - Temperature dependent
  - rapid
  - Stimulated by glucocorticoids
- Linear accumulation – subsequent step
  - slow
  - Not affected by dietary Zn intake
- Does not require energy
Erythrocytes
- Depends upon bicarbonate ions
Fibroblasts, proximal tubule, lymphocyte
- Biphasic uptake (same as liver)

Intracellular Transport

Zinc transporters regulate Zn ion concentrations through import, export or sequestering Zn into vesicles
- Storage, toxicity
2 families exist:
- ZnT- mainly exports Zn ions from cells
- ZIP – important for Zn influx
Number of transporters
- ZnT-1: all organs, small intestine (basolateral membrane), kidney (tubular cells), placenta
  - Efflux
- ZnT-2: intestine, kidney, testis
  - Efflux & (?) intracellular vesicles
- ZnT-3: brain (synaptic vesicles) & testis
  - Influx, intracellular retention
- ZnT-4: mammary gland & brain
  - Efflux (into milk) Lethal mouse transgenic
ZIP family transporters:
- Consist of:
  - hZIP1
  - hZIP2
  - hZIP3
- Responsible for influx of Zn as well as Mn2+, Cd2+, and other divalent cations into cells
Number of transporters
- DCT1: duodenum, jejunum, kidney, bone marrow, others
  - Non-specific: Zn, Cd, Mn & Cu actually have slightly higher affinity than Fe, the mineral for which the transport actions of this protein was first identified.
  - Competition between Fe & Zn & Cu

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Storage

Storage sites
- No specfic storage sites are recognized
  - Within cells, amounts sequestered within metallothionine could be considered as stores
  - Anorexia, muscle catabolism, tissue zinc release
    - Metalloenzymes cling tenaciously to zinc
- Serum/plasma zinc drops rapidly (~1 week) with zinc deficient diet
  - Zinc turnover is extensive and rapid
    - Two-components of turnover, fast ~12.3 days, and slow, ~300 days
  - Fast pool is also called the "exchangeable" pool
    - Usually amounts to 157-183 mg Zn

Excretion

Lost via hair, sweat, desquamation, bile pancreatic secretions, seminal fluid, urine, feces
Main endogenous loss
- Secretions into gut
  - Bile and pancreas
- Mucosal cells
Urinary and integumental losses
- < 20% under normal conditions
  - Losses increase with trauma, muscle catabolism, and administration of chelating agents (EDTA)
Primarily in fecal material
- Unabsorbed Zn
- Secreted Zn (endogenous sources)
  - From pancreatic and intestinal sources

Regulation

Metallothionein
- Concentrated in liver, kidney, pancreas, intestine
- Acts as a Zn2+ buffer
  - Controls free Zn2+ level
  - Control intracellular Zn pool responsive to both hormones and diet
Zn-binding protein, metallothionein (MT), is involved in the regulation of Zn metabolism
MT is inducible by dietary Zn via the metal response element (MRE) and MTF-1 mechanism of transcriptional regulation
- ↑ in cellular MT ↑ Zn binding within cells
Acute infections associated with proinflammatory cytokines increses Zn uptake into liver, bone marrow and thymus and reduces the amount going to bone, skin and intestine

Metabolic Interactions

Interactions of other divalent cations in the intestinal lumen ?
- ↑Fe, ? ↑Sn, ↑? Cd → ↓ Zn
- ↑ Zn → ↓ Cu

Interactions

Copper
- High Zn diets reduce Cu absorption
  - electronic configuration competition
- Metallothionine synthesis induced
  - sequesters Cu in mucosal cell preventing serosal transfer
    - Happens with 150mg Zn for two years
    - Can be used with Wilson's disease patients
    - High copper diets do not interfere with Zinc absorption
Iron
- Supplements inhibit zinc absorption
  - Ferrous > Ferric, heme no effect
  - Pregnant and taking >60mg Fe/day should also take Zn
Calcium
- High Ca diets reduce Zn absorption
  - effect enhanced in phytate rich diets
  - not sure how much of a problem in humans
    - post menopausal women yes, adolescent girls, no
Other
- Tin (Sb), not usually high in diet, but diets high in Tin can increase fecal Zn excretion
- Cadmium (Cd), alter Zn distribution in body rather than altering absorption
- Folic acid, conjugase requires Zn
  - High doses sometimes impair Zn status further in low Zn situation - mechanism currently unclear

Function

Zinc-containing enzymes
- More than 70 enzymes
  - Secondary & tertiary protein structures
    - Metal stabilized active sites
- Examples of general types
  - dehydrogenases
  - phosphatases
  - peptidases
  - kinases
  - deaminases
Insulin
Cu/Zn Superoxide Dismutase
- General class of enzymes that protect against oxidative damage in the body.
Insulin
- Zn important structurally
- Zn needed for insulin "stored" in pancreas
  - Functionality drops rapidly so more of a "working store" than a static store
Nuclear transcription factors (>130)
- Same protein structural role forms "zinc-fingers"
- "Zn-fingers" bind DNA
  - allow different nuclear hormones to interact with DNA via different DNA binding proteins
    - up to 37 "fingers" have been found on a single transcription factor
    - Vit. A, Vit. D, steroid hormones, insulin-like growth factor-1, growth hormone, and others bind to zinc-finger proteins to modulate gene expression
  - Zn is responsible for thymidine incorporation
Cell Differentiation
- Thymidine kinase activity
- Creatine kinase activity

Transcription Factors

Transcription factors
- Regulate gene expression
- Involved in virtually all biological processes:
  - Development, differentiation, cell proliferation, response to external stimuli
- Consists of 2 domains
  - DNA Binding Domain (DBD) – recognizes and binds to specific DNA sequence elements in the promoter of target genes
  - Protein-interacting Transactivation Domain (TAD) – influences the rate of transcription

Zinc Finger Proteins

Zinc finger proteins are characterized by their utilization of zinc ions as structural components
C2H2 zinc finger binding motif
- Predominant motif in eukaryotic transcription
- Involved in skeletal differentiation
- Zinc binding motif is determined by the presence of 2 cysteine and 2 histidine residues that engage in a four coordinate bond with a singe Zn ion
- Bind to response elements in the upstream promoters of genes transcribed by RNA poly 2
- Binds to 5S ribosomal RNA gene, and 5S RNA, and activates transcription by RNA polymerase 3.

Mech of Transcription

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Function

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Zinc Fingers
- Mutation c/ablation of binding
  - in case of Zif268, loss in sequence-specific DNA binding that allowed viral infection
- Iron can replace Zn in "fingers"
  - Low Zn and high Fe
  - Fe gives rise to ROS more readily
    - DNA damage & carcinogenesis?
- Cadmium can replace Zn in "fingers"
  - Non-functional, cytotoxic

Transcription Factors

Revelation
- Gene expression is controlled by specific proteins call transcription factors
  - Zinc containing transcription factors account for 1% of genome
- Zinc plays key structural role in transcription factor proteins
- Ligands for transcription factors include:
  - Vitamin A
  - Vitamin D
  - Bile acids
  - Thyroid hormones

Membrane Stability

Membrane fractions contain high concentrations of Zn
- Increases rigidity of cell
Protection from oxidative damage
- Competition for binding sites with redox metals

Membrane Function

In deficient animals:
- Failure of platelet aggregation
  - Due to impaired Calcium uptake
- Peripheral neuropathy
  - Brain synaptic vesicles exhibit impaired calcium uptake
- Increased osmotic fragility in RBCs
  - Decreased plasma membrane sulfhydryl concentration

Immune Function

After Zinc depletion
- All functions within monocytes were impaired
- Cytotoxicity decreased in Natural Killer Cells
- Phagocytosis is reduced in neutrophils
- Normal function of T-cells are impaired
- B cells undergo apoptosis
High Zn supplementation shows alterations in cells similar to Zn depletion

Vitamin A & Zinc

Zn influences Vitamin A metabolism
- Absorption, transport, and utilization
  - Vitamin A transport is mediated through protein synthesis
    - Zn deficiency can depress synthesis of retinol-binding protein in liver
  - Oxidative conversion of retinol to retinal requires Zn-dependent retinol dehydrogenase enzyme
    - Retinol to retinaldehyde (retinal), for visual processes
Night Blindness
- Hallmark deficiency sign for Vitamin A
  - Seen with Zn deficiency as well, why?
Stojanovic, Stitham and Hwa: Critical Rose of Transmembrane segment Zn binding I the structure and function of rhodopsin JBC 279(34):35932-35941, 2004
- Rhodopsin proteins

Vitamin A

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Zn and Vitamin A Interaction

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Mechanisms of Toxicity

Excess accumulation within cells may disrupt functions of biological molecules
- Protein, enzymes, DNA
  - Leads to toxic consequences
Anemia
- Impaired copper availability
Acute excessive intakes
- Local irritant to tissues and membranes
  - GI distress, nausea, vomiting, abdominal cramps, diarrhea
Relatively non-toxic
- Sources of exposure – drinking water, feed, polluted air

Deficiency

Signs
- Growth retardation
- Delayed sexual maturation & impotence
  - Impaired testicular development
- Hypogonadism & hypospermia
- Alopecia
- Acroorifical skin lesions
  - Other, glossitis, alopecia & nail dystrophy
- Immune deficiencies
- Behavioral changes
More signs
- Night blindness
- Impaired taste (hypoguesia)
- Delayed healing of wounds, burns, decubitus ulcers
- Impaired appetite & food intake
- Eye lesions including photophobia & lack of dark adaptation
Monogastric more susceptible
- Chickens & pigs used to become deficient with high corn diets
  - Old enemy phytate
- Ruminants resistant due to ability to break down phytates
Diabetes
- Increases urinary zinc excretion
  - Can cause deficiency
Elderly
- Poor intakes & altered physiology

Deficiency During Pregnancy

Zn deficient rats failed to conceive
Abnormalities of blastocyst development
Offspring had high incidence of abnormalities
- Deformities of brain, skull, limbs, eyes, heart, lungs
Low Zn intake during the third trimester may not have such profound effects
- Main stages of differentiation are already complete
- Can result in low birth weight, and prolonged and difficult parturition

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Malformations in Zn deficiency

Cleft lip
Cleft palate
Brain (Hydrocephalus, anencephalus or exencephalus)
Micro- or agnathia
Micro- or anopthalmia
Clubbed feet
A- or syndactyly
Curly or stubby tail
Dorsal herniation
Heart (abnormal position)
Lung (missing lobes)
Urogentital (Hydronephrosis, missing kidney, or abnormal positions)

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Stress Response

Factors that decrease plasma Zn concentration
- Infection
- Bacterial endotoxins
- Surgery
- Burns
- Pregnancy
IL-1 causes increased Zn uptake by liver thymus and bone marrow
Severe trauma or death can result from Zn supplementation to stressed animals

2002 DRI's

Infants UL=(x)
- 0-6 mo: 2 mg/d AI (4)
Children & adolescents
- 7mos-1 yr: 3 mg/d (5)
- 1-3 yrs: 3 mg/d (7)
- 4-8 yrs: 5 mg/d (12)
- 9-13 yrs: 8 mg/d (23)
- 14-18 yrs: (34)
  - Males 11 mg/da
  - Females 9 mg/da

Adults: 19 yrs & older (40)
Men: 11 mg/da
Women: 8 mg/da

Pregnancy:
11-18 yrs: 12 mg/da (34)
19-50 yrs: 11 mg/day (40)

Lactation:
11-18 yrs: 13mg/da (34)
19-50 yrs: 12 mg/day (40)

Footnote
Males need more than females due to high Zn content of seminal fluids & relatively low Zn loss through menstruation

The Importance of Zinc in Animal Health

Chelated Minerals

Transitioning to Zero ZnO: The changes in diet formulation