MBBS1

MINERALS

ROB EVANS

After successfully studying the material covered in this lecture, students should be able to: State the dietary requirements for iron iron, zinc and copper, Describe Describe the clinical conditions associated with deficiencies of iron, zinc and copper, Explain E l i h how i iron is i transported t t d and d stored t d in i the body, Describe the causes and effects of iron and copper overload and outline the procedures for the treatment of iron and copper overload.

TRACE ELEMENTS IN MAN (70KG)

METAL Iron Zinc Copper Manganese y Molybdenum Chromium Cobalt TOTAL ( (mg) g) ______ ______ ______ 16 <9 6 1 [PLASMA] [ ] ( ) _______ _______ _______ 0.01 0.02 2 0.005 0 005

TRACE ELEMENTS IN MAN (70KG)

METAL Iron Zinc Copper Manganese y Molybdenum Chromium Cobalt TOTAL ( (mg) g) 3700 [PLASMA] [ ] ( ) 18

16 <9 6 1

0.01 0.02 2 0.005 0 005

But at pH 7, maximum solubility of Fe3+ is 10-18M

TRACE ELEMENTS IN MAN (70KG)

METAL Iron Zinc Copper Manganese y Molybdenum Chromium Cobalt TOTAL ( (mg) g) 3700 1800 16 <9 6 1 [PLASMA] [ ] ( ) 18 18 0.01 0.02 2 0.005 0 005

But at pH 7, maximum solubility of Fe3+ is 10-18M

TRACE ELEMENTS IN MAN (70KG)

METAL Iron Zinc Copper Manganese y Molybdenum Chromium Cobalt TOTAL ( (mg) g) 3700 1800 80 16 <9 6 1 [PLASMA] [ ] ( ) 18 18 17 0.01 0.02 2 0.005 0 005

But at pH 7, maximum solubility of Fe3+ is 10-18M

TRACE ELEMENTS: have an important STRUCTURAL and/or FUNCTIONAL role in many metalloproteins/enzymes The most important oxidases and O2 carrying proteins are Cu and/or Fe metalloproteins e.g. cytochrome oxidase (a Cu/Haem protein) y protect p aerobic organisms g Metalloenzymes against 2 toxic products of O2 reduction: SUPEROXIDE ION: O2PEROXIDE ION: O22-

SUPEROXIDE DISMUTASES (also known as SOD) (mainly Cu/Zn metalloenzymes, but also some contain Mn, Fe) 2O2 O2 + O22CATALASE HAEM-containing enzymes that act on or utilise H2O2 PEROXIDASE Catalase: 2H2O2 2H2O + O2 Peroxidase: H2O2 + SH2 2H2O + S (substrate in (oxidised form) reduced form) e.g. GLUTATHIONE PEROXIDASE: H2O2 + 2GSH 2H2O + GS-SG

GSH = Reduced Glutathione (a tripeptide containing Glu-Cys-Gly) GS-SG GS SG = Oxidised Glutathione (two tripeptides linked by a disulphide bond)

ZINC
Total body zinc: 2 to 3 g Found in all tissues but high in liver, kidney, bone, retina, muscle and prostate. Dietary intake of approx. 10 mg/day compensates for daily losses of approx. 2 to 3 mg. Vital constituent of > 600 enzymes, including deydrogenases, peptidases, phosphatases, carbonic anhydrase, enzymes involved in DNA and protein synthesis. synthesis Zinc deficiency growth retardation, hypogonadism, delayed wound healing.

ACRODERMATITIS ENTEROPATHICA SEVERE SKIN LESIONS DEATH DUE TO INFECTION

ACRODERMATITIS ENTEROPATHICA SEVERE SKIN LESIONS DEATH DUE TO INFECTION

COMPLETELY CURED BY ZINC THERAPY

IRON
4th most abundant element in the Earths crust 2nd most abundant metal in the Earths Earth s crust essential metal for almost all organisms (one exception is Lactobacillus family) low bioavailability toxic when present in excess

Metabolic functions of iron

Function Compound p
Cytochrome a,b,c Oxidative energy production Cytochrome P450 Catalase, peroxidase Hemoglobin Oxygen transport M Myoglobin l bi Mitochondrial respiration Inactivation of harmful O2 radicals DNA synthesis th i Succinate dehydrogenase Xanthine oxidase Rib Ribonucleotide l tid reductase d t

How much iron is there in our body? y

Normally

iron in body Transferrin-bound iron in plasma:

Iron overload: up to

iron in body

How much iron is there in our body? y

Normally 3 5 g iron in body

Transferrin-bound iron in plasma: 3 mg

Iron overload: up to 60 g iron in body

IRON
Used very efficiently in the body Small daily losses compensated for by absorption in duodenum Approx 1 mg iron lost per day (adult man) 0.1 mg urinary loss 0.1 mg sweating, desquamation of skin cells 0.3 0 3 mg desquamation of mucosal cells in intestine; biliary excretion 0.5 0 5 mg gastrointestinal bleeding Menstruation increases losses by approx 2-3 mg per day

Very important fact! NO O PHYSIOLOGICAL S O OG C MECHANISM TO REMOVE EXCESS IRON FROM THE BODY

Daily y Fe requirements q (mg) ( g) INFANTS: 1 CHILDREN: 05 0.5 YOUNG, NON-PREGNANT, WOMEN: 2 PREGNANT WOMEN: 3 MEN & POST-MENSTURAL WOMEN: 1
Approximately 10% dietary iron absorbed Minimal amount needed to be ingested g daily: y (Daily requirement) x 10

Bioavailability of iron (i.e. efficiency with which i iron i is used di in the h b body) d ) can b be quite i variable i bl
e.g. e g In Sudan, soil is very iron-rich and daily intake of iron can p to 300mg gp per day!!! y BUT DOES NOT LEAD TO be up IRON OVERLOAD. In Bantu tribes of South Africa there is a tendency to brew alcoholic drinks in iron pots. Daily intake can be up to 100mg per day. THIS GIVES RISE TO IRON OVERLOAD drinks contain chemicals (e.g. fructose, vitamin C) which promote uptake of dietary iron in the gut (there is probably also an as yet unidentified genetic component that also enhances dietary iron uptake) p )

Major Iron Compartments (Total body iron: 3 5 g)

COMPARTMENT 1 Haemoglobin 2,500 2 Storage iron (Ferritin & Haemosiderin) 3 M Myoglobin l bi 130 4 Uncharacterised iron-binding molecules 5 Other tissue iron (cytochromes,Fe-containing enzymes) 6 Transport iron (Transferrin) 80 3.5 2.2 67 IRON CONTENT(mg) % TOTAL BODY IRON

1,000

27

0.2

0.08

Body y iron distribution and storage g

Dietary iron Utilization Duodenum (average, 12 mg per day d ) Utilization

Muscle (myoglobin) (300 mg) Storage iron

Plasma transfe rrin (3 mg) Circulating erythrocytes (hemoglobin) (1 800 m g) (1,800 )

Bone marrow (300 mg)

Sloughed mucosal cells Desquamation/Menstruation Other blood loss (average, 12 mg per day) Liver (1,000 m g) Iron loss

Reticuloendothelial macrophages (600 mg) ( g)

Andre ws NC. N Engl J Med 1999;341:19 861995

Role of iron in the generation of a free radical (species with an unpaired electron) more damaging than the superoxide anion (O2-): SOD (= ( Superoxide id dismutase) i t ) 2O2- + 2H+ O2- + Fe3+ Fe2+ + H2O2

H2O2 + O2 Fe2+ + O2 Fe3+ + OH- + OH (hydroxyl radical)

Hydroxyl radical: highly reactive; capable of damaging all biological molecules (major product of radiolysis of water responsible for radiation-induced tissue damage)

Molecular mass 460,000 Da Hollow protein shell made up of 24 subunits ( l (polypeptide tid chains) h i ) th that t can bind up to 4,300 ferric (Fe3+ ) ions as an internal hydrated ferric oxide/phosphate c uste cluster Normally only about 2/3 full of iron

2+ and Iron is taken up as Fe (each of the 24 subunits is the displayed in a different colour to then oxidised within 3+ protein shell to Fe convey the complexity of the protein)

STRUCTURE OF FERRITIN

Soluble form of storage iron

FERRITIN (continued)
Mobilisation requires either reduction of Fe3+ to Fe2+ or proteolytic degradation of protein shell Provides a readily usable form of storage iron Presence of iron stimulates the biosynthesis of apo ferritin (= iron-free ferritin shells) Mucosal cells have low [ferritin] but [ferritin] increases with conditions of iron overload electron-dense aggregates (can be visualised using electron microscopy) Present P t at t low l levels l l in i plasma l (normally ( ll 15-300 15 300 g/litre) but in patients with iron overload can increase up to 10,000 10 000 g/litre

HAEMOSIDERIN
Heterogeneous W t i Water-insoluble, l bl forms f large l aggregates t Derived from ferritin Iron is only slowly mobilised from haemosiderin Found mainly in spleen, bone marrow, Kupfer cells in liver In conditions of iron overload, haemosiderin is deposited in most cells

TRANSFERRIN (Th C (The Central lP Protein i of fM Mammalian li Iron Metabolism) )

Domain I

N-LOBE
Domain II

N C

Domain I Domain II

C-LOBE

Dr Schellenberg D S h ll b f found d that th t the protein transferrin, transferrin which is found in the liver and the spleen and carries iron to the rest of the body, undergoes specific changes in heavy drinkers and could be used as a marker for alcoholism.

TRANSFERRIN NS N
Domain I

N-LOBE
Domain II

N C

Domain I

Molecular mass 77,000 Da Bilobal protein with one Fe3+ binding site per lobe Approx. pp 3 mg/ml g in plasma Normally only partially iron saturated (25- 40% of the ironbinding sites occupied)

Domain o II

C-LOBE

TRANSFERRIN S (continued) (co t ued)

Transports iron in the body from sites of absorption (intestinal mucosal cells) to sites of iron utilisation (e.g. bone marrow) and storage (e.g. liver) and from sites of haemoglobin breakdown (reticuloendothelial system) Affinity for iron is high at physiological pH but affinity is reduced at acidic pH Iron deficiency leads to increase in [transferrin] Iron overload leads to decrease in [transferrin]

UPTAKE OF IRON INTO CELLS (by receptor-mediated endocytosis) Iron-loaded transferrin binds to transferrin receptors on plasma membrane Receptor/transferrin complexes migrate to clathrinp coated pits Clathrin-coated vesicles, carrying transferrin/receptor complexes, move away from membrane Loss of clathrin coat; reduction of intra-vesicular pH to approx. pH 5.5 Dissociation of iron from transferrin; transport of iron, as Fe2+ via DMT1, into cytoplasm for storage in ferritin &/or utilisation in mitochondria Vesicle returns to plasma membrane, with receptorbound iron-free iron free transferrin, transferrin where iron iron-free free transferrin dissociates from receptor

Delivery of Cellular Iron through Receptormediated Endocytosis of Transferrin

[N.B. DMT1 = Divalent Metal Transporter 1]

UPTAKE OF IRON INTO CELLS (by receptor-mediated endocytosis) Iron-loaded transferrin binds to transferrin receptors on plasma membrane Receptor/transferrin complexes migrate to clathrinp coated pits Clathrin-coated vesicles, carrying transferrin/receptor complexes, move away from membrane Loss of clathrin coat; reduction of intra-vesicular pH to approx. pH 5.5 Dissociation of iron from transferrin; transport of iron, as Fe2+ via DMT1, into cytoplasm for storage in ferritin &/or utilisation in mitochondria Vesicle returns to plasma membrane, with receptorbound iron-free iron free transferrin, transferrin where iron iron-free free transferrin dissociates from receptor

Delivery of Cellular Iron through Receptor-mediated Endocytosis of Transferrin

[N.B. [N B DMT1 = Divalent Metal Transporter 1]

UPTAKE OF IRON INTO CELLS (by receptor-mediated endocytosis)

Iron-loaded transferrin binds to transferrin receptors on plasma membrane Receptor/transferrin complexes migrate to clathrin-coated pits Clathrin-coated vesicles, , carrying y g transferrin/receptor p complexes, p , move away y from membrane Loss of clathrin coat; reduction of intra-vesicular pH to approx. pH 5.5 Dissociation of iron from transferrin; transport of iron, as Fe2+ via DMT1, into cytoplasm for storage in ferritin &/or utilisation in mitochondria Vesicle returns to plasma membrane, with receptor-bound iron-free transferrin, where iron-free transferrin dissociates from receptor

Delivery of Cellular Iron through Receptormediated Endocytosis of Transferrin

[N.B. DMT1 = Divalent Metal Transporter 1]

DISORDERS OF IRON METABOLISM

Iron deficiency

Lack of sufficient iron in the diet Excessive bleeding Pregnancy P ANAEMIA T t by Treat b supplementation l t ti of f di diet t with ith iron i tablets

DISORDERS OF IRON METABOLISM

Iron overload
G Genetic ti (hereditary) (h dit )h haemochromatosis h t i (defect in proteins of iron uptake, iron transport or regulation l ti of fi iron metabolism) t b li ) Transfusional (secondary) iron overload Dietary iron overload Atransferrinaemia (absence of transferrin) or hypotransferrinaemia (very low levels of transferrin very rare disorders, can be treated by i j ti injections of ft transferrin) f i ) Aceruloplasminaemia (absence of ceruloplasmin, l l i enzyme i in plasma l th that t oxidises idi Fe2+ to Fe3+)

DISORDERS OF IRON METABOLISM

Iron overload
G Genetic ti (hereditary) (h dit )h haemochromatosis h t i (defect in proteins of iron uptake, iron transport or regulation l ti of fi iron metabolism) t b li ) Transfusional (secondary) iron overload Dietary iron overload Atransferrinaemia (absence of transferrin) or hypotransferrinaemia (very low levels of transferrin very rare disorders, can be treated by i j ti injections of ft transferrin) f i ) Aceruloplasminaemia (absence of ceruloplasmin, l l i enzyme i in plasma l th that t oxidises idi Fe2+ to Fe3+)

Classification of genetic haemochromatosis

Type 1
Mutations M t ti in i gene for f protein t i called ll d HFE (High (Hi h Fe) One of the most common autosomal recessive conditions found in populations of Northern Europe Most common mutation is C282Y (i.e. cysteine 282 mutated to tyrosine). In UK about 1 in 7 people are carriers of C282Y mutation and about 1 in 150 are homozygous for the mutation

Type yp 1 genetic g haemochromatosis

In homozygotes an extra 3-4 mg of iron is absorbed from the diet every day Large g accumulation of iron ( (as ferritin and haemosiderin) ) over a number of years, especially in liver, heart & pancreas Liver failure, diabetes mellitus, cardiac failure & pigmentation of the skin (ash-grey colour) due to increased production of melanin Cell destruction can result in iron-catalysed lipid peroxidation reactions (particularly lysosomal membranes) N.B. the actual role of HFE in iron metabolism is still unclear, but mutations in the HFE gene can lead to iron overload

Tests for Type 1 genetic haemochromatosis (GH)

1. Measure transferrin saturation (i.e. % occupation of iron-binding sites on t transferrin) f i ) after ft an overnight i ht fast. f t If on 2 occasions the transferrin saturation is >55% in men or >50% in women then: Measure the concentration of serum ferritin (is an indication of the amount of iron stored in the body). body) Levels >300 g/litre in men or >200 g/litre in women in indicative of Type 1 GH Genetic Ge et c test for o t the e co common o mutations utat o s in t the e HFE gene Liver biopsy tissue sample removed and examined for tissue damage

2.

3. 3 4.

Treatment of Type 1 genetic haemochromatosis

Treatment of Type 1 genetic haemochromatosis

Phlebotomy (removal of 500ml of blood, once or twice t i per week) 500 ml of blood contains about 250 mg iron

Effect of phlebotomy on transferrin saturation and serum ferritin levels

(taken from website of the Haemochromatosis Society: www.ghsoc.org)

Classification of genetic haemochromatosis (continued) ( )

Type 2a
Mutations in g gene for p protein called HEMOJUVELIN (newly-identified, rare disorder) Mutation in gene for HEPCIDIN (also known as HAMP Human Anti-Microbial Peptide) [newly-identified, rare disorder] Rare di R disorder d f found di in newly-identified l id tifi d gene f for Transferrin T f i Receptor 2 (TfR2) Newly-identified, rare disorders, arising from mutations in the iron transporter called FERROPORTIN 1 (FP1) [also known as IReg1]

Type 2b

Type 3

Type 4

TRANSFUSIONAL (SECONDARY) IRON OVERLOAD Repeated blood transfusions (e.g. in cases of thalassaemia) Accumulation of iron in the body Ability of the reticuloendothelial (RE) cells (in spleen, bone marrow, , lymph y p nodes) ) to store iron is exceeded Release of iron to transferrin Deposition of iron at other sites in body, especially the heart Cardiac failure, , also damage g to endocrine organs g and liver

TREATMENT OF SECONDARY IRON OVERLOAD:

R Remove i iron b by CHELATION THERAPY

(i.e. treat patients with drugs that are able to bind iron the iron drug complex is then excreted from the body) iron-drug IDEAL REQUIREMENTS FOR A USEFUL CHELATOR (CHELATOR ( CHELATOR from the Greek word CHELE CHELE for CLAW) CLAW ) 1. High association constant for iron 2. Is specific p for iron 3. Non-toxic 4. Highly efficient at promoting iron removal 5. Orally active One widely used chelator is DEFEROXAMINE (DESFERAL=DFO) administered by subcutaneous infusion (1 to 2g over period of 12-22 hour, 5 or 6 days per week) Iron balance or net excretion of iron [BUT DFO IS NOT ORALLY ACTIVE]

New orally active chelators There is a pressing need for orally active chelators that either replace DFO or can be used in combination with DFO There is a worldwide search for orally y active chelators by y both academic researchers (e.g Professor Robert Hider and his group at Kings College London) and Pharmaceutical Industries (e.g ( Novartis) ) Family of iron-binding chelators, based on the hydroxypyridinones developed by Professor Hiders hydroxypyridinones, Hider s group DEFERIPRONE (also known as L1) is orally active and approved pp for clinical use in some countries ( (Mol Wt 139) )

Deferiprone (L1)

3:1 complex of Deferiprone (L1) and Fe3+

Gut Lumen
Heme Fe HCP1 Heme oxygenase

Portal Circulation

Non Heme Fe Non-Heme

Fe3+ F Dcytb Fe2+ DMT1 Ferritin

Labile iron pool

FP1

Fe2+ Fe3+ Hp IRP Tf

Mitochondria

Molecular pathways of dietary iron p in the duodenum absorption

Dcytb (Duodenal cytochrome b1): reduces Fe3+ to Fe2+ DMT1 (Divalent (Di l t metal t lt transporter t 1) 1): t transports t F Fe2+ into i t cell ll IRP (Iron Regulatory Protein): regulate the synthesis of ferritin, transferrin receptor and other proteins) FP1 (Ferroprotin 1) (also known as Ireg1, (Iron Regulated Protein 1): exports Fe2+ out of cell Hp (Hephaestin): membrane-bound ferroxidase (oxidises Fe2+ Fe3+) C (Ceruloplasmin): Cp (C l l i ) soluble l bl f ferroxidase id i in plasma l (oxidises Fe2+ Fe3+) Tf (Transferrin): iron tranport protein in plasma Ferritin: iron storage protein, primarily intracellular but present at low concentrations in plasma) Haem oxygenase: catalyses the breakdown of haem and release of iron Haem receptor (also kno known n as HCP1, (haem carrier protein 1) 1): membrane-bound protein that transports haem into cell

Gut Lumen
Heme Fe HCP1 Heme oxygenase

Portal Circulation

Non-Heme Fe

Fe3+ Dcytb Fe2+ DMT1 Ferritin

Labile i iron pool

FP1

Fe2+ Fe3+ Hp IRP Tf

Mitochondria

Dcytb (Duodenal cytochrome b1): reduces to DMT1 ( (Divalent metal transporter p 1): ) transports p Fe2+ into cell; IRP (Iron Regulatory Protein): regulates the synthesis of ferritin, transferrin receptor and other proteins; FP1 (Ferroportin 1) (also known as Ireg1,Iron Regulated Protein 1): exports Fe2+ out of cell; Hp (Hephaestin): membrane membrane-bound bound ferroxidase (oxidises Fe2+ Fe3+); Cp (Ceruloplasmin): soluble ferroxidase in plasma (oxidises Fe2+ Fe3+); Tf (Transferrin): iron transport protein in plasma; Ferritin: iron storage g p protein, primarily p y intracellular but p present at low concentrations in p plasma; Haem oxygenase: catalyses the breakdown of haem and release of iron; Haem receptor (also known as HCP1, haem carrier protein 1):membrane-bound protein that transports haem into cell.

Fe3+

Fe2+;

COPPER
Total body copper: 75 to 150 mg High levels of copper in liver, brain, h t kid heart, kidneys. Dietary intake of approx. 1 to 2 mg per day to o co compensate pe sa e for o da daily y losses osses (primarily by biliary excretion). Approx 30% of dietary copper absorbed b b dt to compensate t f for l losses.

Copper is required for synthesis of a large number of copper-containing enzymes:

e.g.
Enzyme Functional role Known/expected effect of deficiency Ceruloplasmin Ferroxidase (Fe2+ Fe3+) ( Lysyl oxidase Cross-linking of collagen & elastin Melanin production

Anaemia

Vascular rupture Failure of pigmentation

Tyrosinase Dopamine -hydoxylase

Catecholamine production

Neurological effects

DISORDERS OF COPPER METABOLISM Copper deficiency MENKES DISEASE Defective D f ti absorption b ti of fC Cu f from intestine, X-linked disease (1 in 50,000 to 100,000), ( steely Results in depigmentation of hair (steely hair),

DISORDERS OF COPPER METABOLISM Copper deficiency MENKES DISEASE Defective D f ti absorption b ti of fC Cu f from intestine, X-linked disease (1 in 50,000 to 100,000), ( steely Results in depigmentation of hair (steely hair), Life expectancy is less than 2 years years. COPPER THERAPY HAS NO SIGNIFICANT EFFECT

DISORDERS OF COPPER METABOLISM C Copper overload l d

WILSONS DISEASE (hepatolenticular degeneration) Autosomal recessive disease (1 in 100,000) Impaired biliary excretion of Cu and incorporation p of Cu into ceruloplasmin, p , Build-up of Cu in liver liver damage Overflow of Cu into brain mental deterioration & loss of coordination, Yellow-brown ring on cornea (Kayser-Fleischer rings).

DISORDERS OF COPPER METABOLISM C Copper overload l d

WILSONS DISEASE (hepatolenticular degeneration) Autosomal recessive disease (1 in 100,000) Impaired biliary excretion of Cu and incorporation p of Cu into ceruloplasmin, p , Build-up of Cu in liver liver damage Overflow of Cu into brain mental deterioration & loss of coordination, Yellow-brown ring on cornea (Kayser-Fleischer rings). CHELATION THERAPY (using D-penicillamine) D penicillamine) IS EFFECTIVE

Happy Easter Holidays!! Rob Evans

robert.evans@brunel.ac.uk robertevans49@yahoo.co.uk b t 49@ h k

BUT BEFORE YOU GO FOR YOUR LUNCH 2 VOLUNTEERS O S ARE NEEDED TO HELP WITH A QUICK EXPERIMENT!!!
Rob Evans robert.evans@brunel.ac.uk @ robertevans49@yahoo.co.uk