A. Iron Functions and Absorption [5,8]

1. Functions of Iron
   1. Normal function of iron as complexes with porphyrin
   2. Hemoglobin - oxygen transport in blood
   3. Myoglobin - oxygen transport in muscle
   4. Cytochromes - participate in redux reactions including drug metabolism
2. Note humans cannot excrete iron actively, so concentrations maintained through absorption
3. Iron Absorption
   1. About 10% of the 10-20mg of daily iron intake is absorbed
   2. Absorption mainly in duodenum (near gastroduodenal junction) crypt cells
   3. Diets contain both heme and non-heme (inorganic) iron, each with specific transporters
   4. Most dietary iron exists as ferric (Fe3+) rather than ferrous (Fe2+) salts
   5. Fe3+ is reduced to Fe2+ by intestinal brush border ferric reductase (DCYTB)
   6. Fe3+ may also be reduced to Fe2+ by ascorbic acid
4. Heme Iron Absorption
   1. Heme iron is derived from from myoglobin (mainly in meats)
   2. Heme iron accounts for ~70% of daily iron intake
   3. Heme efficiently absorbed through putative heme-iron transporter HCP1
   4. HCP1 is upregulated by hypoxia and iron deficiency; may also transport folate
5. Non-Heme (Inorganic) Iron Absorption
   1. Transported from intestine into enterocytes mediated by DMT1
   2. DMT1 is divalent metal transporter 1, also called NRAMP2 or DCT1
   3. DMT1 transports only Fe2+ iron across apical enterocyte membrane
   4. Inorganic iron absorption is influenced by dietary chelating compounds
   5. Citrate and ascorbate increase iron absorption by forming soluble complexes
   6. Tannates (from tea), phytates and phosphates inhibit iron absorption
6. Transport Across Enterocytes
   1. Fe2+ crosses enterocyte and and is exported from basolateral surface by ferroportin 1
   2. Ferroportin 1 is also called IREG and MTP-1
   3. Hephaestin, an iron oxidase, forms a complex with ferroportin 1
   4. Hepcidin is also part of the complext
   5. In export process by ferroportin, Fe2+ is reoxidized to Fe3+ (via hephaestin activity)
7. Iron that is not directly transferred to circulation is stored as in ferritin in cells
8. Absorption of other heavy metals occurs through DMT1
   1. Manganese
   2. Cadmium
   3. Lead (lead intake competes with iron)
   4. Cobalt
   5. Copper
   6. Zinc

B. Iron Transport and Storage

1. Inorganic Iron is Highly Toxic Requiring Transport (Carrier) Proteins
   1. Absorption of iron by enterocyte is coupled to levels of transferrin-iron in blood
   2. Iron is transported and must be stored in complexes to prefent toxicity
   3. Once transported across epithelia, iron is complexed with transferrin
   4. Transferrin is an 80 kD serum glycoprotein used for iron transport through blood
   5. Each transferrin binds two iron atoms
   6. Aggregate binding sites of all transferrin in plasma is called TIBC
   7. TIBC is the total iron binding capacity
   8. Normal serum Iron levels are 14-43µmol/L (80-180µg/dL)
   9. Normal TIBC is 45-82µmol/L (250-460µg/dL)
   10. Transferrin saturation is normally 20-45% (% of TIBC utilized at a given time)
2. Cellular Uptake of Iron
   1. Specific receptors on plasma membranes of cells recognize transferrin
   2. This leads to internalization of protein complex with iron
   3. Iron is released intracellularly
   4. Excess iron is stored in the body as ferritin or hemosiderin
   5. Ferritin is a large protein which forms a circle around iron (Fe2+)
   6. Ferritin oxidizes Fe2+ to Fe3+ for storage
   7. Normally, there is a correlation between serum ferritin and body iron stores (see below)
3. Iron Stores
   1. Main store in red blood cells as hemoglobin (Hb): ~2500mg per adult
   2. There are about 1 billion iron atoms per RBC (red blood cell)
   3. Reticuloendothelial storage system - ferritin and hemosiderin
   4. Ferritin + Hemosiderin stored iron: 100-400mg in women, 1000mg in men
   5. Tissue iron (myoglobin, other enzymes): ~300mg
   6. Total body iron is ~3000mg in women, ~3800mg (35-45mg/kg) in men
   7. Each 1µg/L serum ferritin is equivalent to 10mg of storage iron
   8. Normal ferritin levels are 15-200µg/L
   9. Hemosiderin stored iron is usually minimal except in pathologic situations
   10. Hepatic iron stores accurately reflect total body iron (in thalassemia patients) [7]
4. Normal Iron Loss
   1. Humans have no specific physiological mechanisms for iron excretion
   2. Men lose about 1mg/day
   3. Menstruating women lose about 1.5-2mg/day
   4. Most daily loss is due to microerosions and microulcerations in GI tract
   5. Women lose a blood through menstruation
5. Regulation of Iron Levels [2,8]
   1. Iron stores in body are highly regulated and sensed by intestinal crypt cells
   2. Hemochromatosis protein (HFE) and hepcidin are sensors of iron levels
   3. HFE is expressed in crypt cells and hepcidin secreted by the liver
6. HFE
   1. HFE protein likely couples sensing mechanism of crypt cell to absorption by enterocyte
   2. As transferrin saturation rises, crypt cell accumulates iron and reduces iron absorption
   3. HFE either inhibits uptake or inhibits release of iron from cells
   4. HFE function depends on level of transferrin saturation
   5. HFE binds to transferrin receptor 1 and enhances uptake of iron or inhibits its release
   6. Mutations of HFE result in overabsorption of dietary iron
   7. C282Y mutation of HFE associated with elevated serum ferritin and transferrin saturation in whites [12]
   8. C282Y mutation not commonly found in non-whites regardless of iron status [12]
7. Hepcidin [5]
   1. Synthesized by hepatocytes in response to iron overload or inflammation
   2. Inhibits absorption and release of iron from macrophage sand other cell types
   3. Binds to ferroportin 1 at basolateral membrane of enterocyte causing internalization and degradation which decrease iron transfer to blood
   4. Thus, hepcidin has key role in down-regulating intestinal iron absorption
   5. Macrophages are important because they ingest senescent RBC
   6. The heme-iron from RBC is recycled through macrophages to transferrin for transport
   7. Hemojuvelin modulates hepcidin expression (little is known about it)
8. Other Genes Involved in Hemochromatosis [2]
   1. TFR2 has 66% homology to TFR1 and can mediate uptake of transferrin-bound iron
   2. TFR2 has high level expression in hepatocytes

C. Iron Assays in Humans

1. Direct assays for evaluation of iron require tissue biopsies
2. Normal Levels
   1. Iron: 14-43µmol/L (80-180µg/dL)
   2. TIBC: 45-82µmol/L (250-460µg/dL)
   3. Transferrin saturation is normally 20-45%
   4. Ferritin: 15-200µg/L (an acute phase reactant)
3. Increase in Total Body Iron
   1. Serum iron normal or slightly elevated
   2. Ferritin levels elevated (>500µg/dL)
   3. TIBC normal
   4. Transferrin saturation increased (>80%)
4. Decrease in Total Body Iron
   1. Serum iron normal or decreased
   2. Ferritin levels depressed (<15µg/dL)
   3. TIBC normal
   4. Transferrin saturation reduced (<10%)

D. Iron Deficiency

1. Iron deficiency only occurs with loss of >5mL blood per day
   1. About 10% of women in USA have iron deficiency
   2. About 3% of men in USA have iron deficiency
   3. Menstruating women require greater iron intake to replenish for blood loss
2. Anemia is late manifestation of iron loss
3. Iron Deficiency Anemia (IDA) [5]
   1. IDA occurs in 5% of women and 2% of men in USA
   2. Most common cause in women is chronic iron loss due to menstruation
   3. Earliest manifestation of IDA is increased free erythrocyte protoprophyrin (FEP)
   4. Frank microcytosis with anisocytosis will occur if IDA persists
   5. Laboratory changes as above (Transferrin saturation is most sensitive)

E. Iron Overload [2]

1. Causes of Iron Overload
   1. Transfusion related - usually ß°-thalassemia or sickle cell anemia
   2. Hereditary hemochromatosis - genetic disease
2. Hereditary Hemochromatosis
   1. Abnormal Iron metabolism most often due to mutation in HFE gene (Type I)
   2. HFE mutations lead to malregulation of intestinal iron absorption
   3. Iron (Fe) deposition in abnormal sites
   4. Various other types of hemochromatosis have been defined, due to other mutations
   5. Types 2-4 hemochromatosis due to mutations in other iron regulatory genes
3. Damage Due to Hemochromatosis
   1. Organ dysfunction occurs due to iron deposition and toxicity
   2. Liver disease (cirrhosis), diabetes, hypogonadism, cardiomyopathy, arthritis
   3. Presents earlier in men than in women due to menstrual iron (blood) losses
   4. Diagnosis of hemochromatosis should be made on genotype, not on hepatic iron stores
   5. Hepatic iron stores can, however, be estimated by T2 weighted gradient echo MRI [11]
4. Treatment of Iron Overload
   1. Phlebotomy is mainstay
   2. Usually 500cc removed every 1-2 weeks
   3. Follow transferrin saturation and ferritin levels, which should fall to normal range
   4. Once in normal range, phlebotomy is done every 2-4 months
   5. Goal is to maintain low normal ferritin and transferrin saturation levels
   6. Iron binding agents may be used if phlebotomy is contraindicated
   7. Deferoxamine (Desferal®) - given intraperitoneally (also for aluminum toxicity in CAPD)
   8. Deferiprone - oral chelation therapy, questionable longer term efficacy [3,4]
   9. Deferiprone more effective than deferoxamine in removing cardiac iron in ß-thalassemia patients [9]
   10. Oral deferasirox (Exjade®) now available for age over 2 years for iron overload [13]
   11. Initial dose deferasirox is 20mg/kg oral initially, adjusted every 3-6 months; discontinue temporarily if ferritin remains <500µg/L [13]
   12. ICL670 is another oral iron chelator with very promising early clinical data [10]

References

1. Rockey DC. 1999. NEJM. 341(1):38
2. Pietrangelo A. 2004. NEJM. 350(23):2383
3. Olivieri NF, Brittenham GM, Matsui D, et al. 1996. NEJM. 332:918
4. Olivieri NF, Brittenham GM, McLaren CE, et al. 1998. NEJM. 339(7):417
5. Zimmermann MB and Hurrell RF. 2007. Lancet. 370(9586):511
6. Provan D and Weatherall D. 2000. Lancet. 355(9211):1260
7. Angelucci E, Brittenham GM, McLaren CE, et al. 2000. NEJM. 343(5):327
8. Townsend A and Drakesmith H. 2002. Lancet. 359(9308):786
9. Anderson LJ, Wonke B, Prescott E, et al. 2002. Lancet. 360(9332):516
10. Nisbet-Brown E, Olivieri NF, Giardina PJ, et al. 2003. Lancet. 361(9369):1597
11. Gandon Y, Olivie D, Guyader D, et al. 2004. Lancet. 363(9406):357
12. Adams PC, Reboussin DM, Barton JC, et al. 2005. NEJM. 352(17):1769
13. Deferasirox. 2006. Med Let. 48(1233):35