Iron, TIBC, and Copper

source and occurrence

n Iron (Fe2+, Ferrous) or (Fe3+, Ferric)
n Vital part of erythroid maturation
n Less than 5 grams total in body
n Found in compounds such as
– Hemoglobin: (2500 mg)
– Myoglobin: (300 mg)
– Enzymes
• Catalase: H2O2 decomposition (300 mg)
• Peroxidase: Oxidation (300 mg)
• Cytochromes: Electron Transfer (300 mg)
• Iron-sulfur: Electron Transfer (300 mg)

free vs bound vs stored

free iron can lead to tissue damage

• generation of free radicals by reacting with peroxide

transported in the plasma bound to transferrin

stored as ferritin or hemosiderin

intake requirements

RDA for male is 10 mg/day

for females 15mg/day

~1 mg of iron is lost per day (adults)

due to loss of RBCs and intestinal shedding

in females, the menstrual cycle can drain 30 mg of iron

iron metabolism source

– Majority of circulating iron derived form
destroyed RBCs (recycled)
– Intestinal absorption
– Occurs mainly in the duodenum
– Accounts for only 5-10% of daily iron intake
– Dietary, ferric Fe3+ form
• reduced to the ferrous Fe2+ form to be absorbed
– Diet is usually 10-20 mg/day

iron metabolism transport

n Transferrin (4 mg)
– A singled chained polypeptide formed in the liver
– Transports Iron to normoblasts
n Binds iron in the ferric, Fe3+ state
n Delivers iron to cells with transferrin surface
receptors
n 40 mg of iron is transported daily

storage of iron

n Over 65% of the body iron is in hemoglobin
n Stored in the rest of the body as
– Ferritin and Hemosiderin

ferritin (apoferritin)

n A soluble multi subunit protein shell which
surrounds iron atoms
n Takes up iron in the ferrous, Fe2+ state and
oxidizes it to the ferric state
n 1/3 in the liver
n 1/3 in the bone marrow
n 1/3 in the spleen
n Assesses iron storage
n Assays: Immunoassays
n ↓: Sensitive for the detection of iron deficiency
n ↑: In chronic diseases, infection, inflammation

hemosiderin

ferritin which has lost surface tension

is soluble

stains with prussian blue stain

measurement of serum iron

n Specimen Requirements
– SERUM
• do not use EDTA, Potassium oxalate, citrate
– Bind the iron in the specimen
– Avoid hemolyzed serum
n
n Reference Methods:
– AAS

colorimetric methods

n Colorimetric
– Splitting off of Fe3+ from Transferrin by acid;
• HCL, H2SO4, Tricholoroacetic acid (TCA)
– Separation of Fe3+ and protein
– Reduction of Fe3+  Fe2+
• Ascorbic Acid
• Hydrazine
• Thioglycollic acid, Hydroxylamine
– Reaction of Fe2+ with Chromogen

chromogens

– Bathophenanthroline
– Diphenylphenanthroline
– Ferrozine (reference method)
• Method used by Student Lab Instruments
– Tripyridyl triazine (TPZ) pyridylazo dye

measuring iron

the specimen is acidified to release iron from transferrin and reduce Fe3+ to Fe2+ (ferrous iron)

instrumentation methods

n Vitros (Johnson & Johnson)
n Reflectance colorimetry
– 1st layer
• -remove iron from the transferrin by acid
– 2nd layer
• ascorbic acid - Fe3+  Fe2+
– 3rd layer:
• chelate out iron
– 4th layer
• Reagent layer
– Two readings taken-one at 54 seconds and one at
297 seconds

instrument methods: Roche

reference ranges

males - 59-158 ug/dl

female - 37-145 ug/dl

clinical significance of increased serum iron

– Hemolytic anemia
– Sideroblastic anemia
– Primary Hereditary Hemochromatosis
– Iron intoxication
– Iron overload (transfusion)
– Increased Red Cell Destruction
– Ineffective Erythropoiesis
• Following thalassemia
• Lead poisoning

clinical significance of decreased serum iron

n Iron deficiency anemia
– (↓ Hb, Hct, MCV, MCH,
MCHC)
n Inadequate intake
n Chronic Diarrhea
n Malabsorption
n Increased Requirements
n Growth
n Premenopausal Women
n Pregnancy
n Chronic Blood Loss
n Excessive menstruation
n Peptic Ulcer
– Gastritis
n Anemia of Chronic
Diseases
– Infections
– Inflammatory
diseases: SLE, RA
– Malignancies
n Thalassemia

total iron binding capacity (TIBC)

n Reflects Transferrin levels
n Not bound with serum Fe
n Capability of transferrin to binding Fe
n Helpful in the determining cause of iron
deficiency anemia

methodology

n Saturation of transferrin with excess Fe3+
– Ferric ammonium citrate
– Ferric chloride
n Removal of unbound Fe3+
– Precipitation with
• MgCO3
n Centrifuge
n Analyze supernatant for iron
– Measures iron bound to transferrin
n Reaction Sequence for TIBC
– Apotransferrin + free Fe+3  Fe-transferrin + free Fe+3
– Fe-transferrin + Fe+3 + alumina Fe-transferrin +alumina-Fe
n Reference Range 250-450 ug/dl

clinical significance for TIBC increased and decreased levels

n Increased
– Iron deficiency anemia
n Decreased in
– chronic disease
– Infections, inflammation, and malignancy
– Thalassemia
– Hemochromatosis
• Iron storage disease
n Normal in Sideroblastic anemia

percent saturation of transferrin

n Serum iron x 100 = % Saturation
TIBC
n Unsaturated Iron Binding Capacity (UIBC)
n TIBC = UIBC + Serum Iron
n UIBC = TIBC - Serum Iron
n This is latent iron binding capacity
n Only 1/3 of the transferrin binding sites are
normally bound to Fe

plasma ferritin

n Ferritin
– the most sensitive indicator of iron deficiency
n Ferritin levels decline early in anemia and increase
early in chronic diseases
n Methodology
– RIA - Radioimmune Assay
– IRMA - Immunoradiometric assay
– EIA - Enzyme marker
– Flurometric enzyme immunoassay (FPIA-Abbott IMx)
– CHEMILUMINESCENT
n Reference Values for Ferritin
– Male: 15-200 ug/L
– Females: 12-150 ug/L

significance of plasma ferritin levels

n Decreased
– in iron deficiency anemia
n Normal - Increased
– in anemia of chronic disorders and Thalassemia
n Increased
– in Sideroblastic anemia
– Familial Hemochromatosis

laboratory findings in microcytic/hypochromic anemia

laboratory markers of iron status in several disease states

free erythrocyte protoporphyrin

n Measured by the Zinc Protoporphyrin: Heme ratio or
ZPP/Heme
n Useful in screening for iron deficiency demonstrated
in lead poisoning
n Methodology
– Hematoflurometry
– Hemoglobin is converted to cyanmethemoglobin, measure
light absorbance at 420nm
– Then measure the zinc protoporphyrin at 595 nm
– Reference Values: <80 umol ZPP/mole of heme
– Significance:
• Increased in iron deficiency

copper (functions)

n Function of Copper
• Oxidation reduction reactions
• Involved with the synthesis of hemoglobin
• Formation of collagen
• Maintenance of the myelin sheath skeletal development
• Function of the immune system
• Formation of melanin pigmentation
• Component of
– enzyme superoxide dismutase
– Cytochrome C oxidase
– Tyrosinase
– Ascorbate oxidase
n RDA = 1.5 - 3.0 mg/day
n Transported primarily by ceruloplasmin

measurement of copper

AAS, (flame or electro thermal)

n Reference Values
– Males: 70-140 ug/dl
– Females: 80-155 ug/dl
– Pregnancy: 120-300 ug/dl
– Erythrocytes: 90-150 ug/dl
– Wilsons disease:
• Serum 40-60 ug/dl
• Urine >100 ug/dl

clinical significance of copper

n Deficiency (Rare)
– Wilson’s Disease (Heptolenticular disease)
• Autosomal recessive, ATP-7B gene mutation
• Copper transport is abnormal
• Copper accumulates in the liver, brain, cornea, and
kidney
• Kayser Fleisher rings in the cornea by copper deposition
• Decreased Ceruloplasmin, Increased Urinary copper
– Menke's Steely Hair Syndrome:
• Extreme form of Cu deficiency
• X linked defect in Cu transport and storage
• Decreased ceruloplasmin
– Severe copper deficiency
– Transient deficiencies
• Sprue, nephrosis

increases in copper levels clinical significance

n Addison’s disease
n Hypopituitarism
n Infection
n Lymphoma
n Liver disease
n Pregnancy
n Bronze baby syndrome
– Complication of UV treatment of infants
with jaundice
– Copper-porphyrin conjugates

ceruloplasmin

n Indicator of Copper status
n Function:
– Transport of Copper
– Synthesized in the liver
– An acute phase reactant and involved in the
formation of heme
– Considered an anti-oxidant

measurement of ceruloplasmin

n Colorimetric
– oxidizes a substrate to color change
n Immunoassay
– Immune-Nephelometry
– RID - Radial-Immunodiffusion
– Chemiluminescence
– FLPIA
n Reference Values:
– 15-60 mg/dl
– (excretion is 2-37 g/day)

ceruloplasmin levels

n Increased values
– Rheumatoid arthritis (gives blood a greenish cast)
– Biliary cirrhosis
– Pregnancy
– Oral contraception
– Some infections
– Cancer
n Decreased Values:
– Wilson’s Disease
– Menke’s Steely Hair Syndrome

menkes syndrome vs wilson’s disease