Iron transport and homeostasis

• Describe the function of iron in biological molecules

  Iron is a metal with redox potential that favours its use in a number of protein complexes, especially those involved in electron transfer. Proteins using iron can be divided into:

  • Haemoproteins- acts as a prosthetic group oxygen binding, e.g., oxidases
  • Non-heme iron containing proteins- mitochondria aconitase, Fe-S proteins in the electron transport chain and ribonucleotide reductase in DNA synthesis
• Describe iron distribution in adults

  • The duodenum absorbs 1-2mg of iron per day
  • Plasma transferrin- 3mg
  • Storage in liver- 1000mg
  • Muscle myoglobin- 300mg
  • Bone marrow- 300mg
  • Circulating erythrocytes- 1800mg
  • Reticulo-endothelial macrophages- 600mg
• Describe Iron excretion in adults

  Adults excrete around 1-2mg per day, via sloughed mucosal cells, skin loss (desquamation), menstruation and other forms of blood loss.
• What is the function of transferrin?

  Transferrin transports iron, in Fe2+ state, between sites of absorption, storage and utilisation.
• Describe the affinity and saturation of transferrin

  Iron plasma concentration is significantly smaller (around 38 umol/litre less) than the maximum binding capacity of transferrin, therefore transferrin, under normal conditions, is around 20-30% saturated. Affinity for iron is dependent on:

  • pH 7.4- transferrin binds iron very strongly
  • pH <4.5- transferrin exhibits around zero binding

  Most of the iron bound to transferrin comes from haemoglobin catabolism by macrophages and binding of iron requires an anion, usually carbonate.
• Describe the catabolism of erythrocytes and haemoglobin

  Senescent erythrocytes are internalised by macrophages of the reticuloendotheLila system and they liberate iron from the protoporphyrin ring of haem via the action of haem oxygenase, which leads to the release of iron to transferrin.
• Where is transferrin synthesised?

  Major site of synthesis is the liver, but it is also synthesised in lymph nodes, macrophages and the bone marrow.
• Describe the absorption of iron in the gut

  Vitamin C/ L-absorbic acid and low pH support the conversion of Fe III to Fe II, which is catalysed by duodenal cytochrome B. Sodium hydrogen antiporter maintains the low pH in the gut.
• Describe iron absorption in the gut-molecular methods

  Iron deficiency in rats results in induction of vitamin C transporter, SVCT1, and increased enterocyte ascorbate levels in mice. Mice and rats can synthesis vitamin C in the liver.
• Describe the action of duodenal cytochrome B

  This is a ferric reductase expressed in the intestinal mucosa that promotes reduction of dietary ferric ion to ferrous iron so it can be transported across the apical enterocyte membrane. Ascorbate (vitamin C) acts as a co-factor. DcytB is strongly upregulated when dietary iron restricted, during anaemia, and in response to hypoxia.
• Describe identification of DcytB

  Isolated by sequencing clones from hypotransferrinaemic mouse model. RACE PCR was used to isolate the full length cDNA fragment. It was also found that DcytB is weakly expressed in control duodenum and expression is upregulated in iron deficient duodenum.
• Describe the action of DMT1- molecular methods

  1. Removal of intestine-specific DMT1 produces a severe iron deficiency anaemia in mice
  2. Absorption of iron from an oral dose was abolished in the DMT1 deficient mice, compared to the absorption of copper and manganese, which shows that DMT1 is critical for the absorption of iron but not copper or manganese.
  3. This is supported by normal tissues levels of copper, manganese and zine in DMT1 deficient mice compared depleted iron levels.
• Measuring selectivity of DMT1

  Oocytes were injected with DMT1 RNA and voltage clamped at -70mV. The selectivity of DMT1 was expressed as a ratio IM max/KM 0.5 and measured over a series of concentrations.
• Describe the characteristics of DMT1

  Consists of 12 membrane spanning domains with a glycosylated extracellular loop. The N and C termini reside in the cytosol and there is a consensus transport motif within the fourth intracellular loop. There is an IRE in the 3’ UTR, which is sensitive to iron.
• DMT1 distribution- molecular methods

  IRE+ increases DMT1 in the kidney outer medulla

  IRE- less DMT1 expression in the kidney outer medulla

  Northern blot
• Why is DMT1 in the kidney?

  DMT1 is found on the apical membrane on the early DCT and may act as an reuptake transporter.
• What happens to iron when it enters the cell?

  PRCB2 is a metallo chaperone that mediates the transfer of iron to ferritin.

  Poly (RC) Binding protein
• Describe the identification of basolateral membrane iron transporter

  cDNA isolated independently by three groups:

  • Ferroportin
  • Ireg1
  • Metal transporter protein 1

  All the same thing lol
• Describe the action of ferroportin

  It has 10 transmembrane domains and mediates temperature-dependent iron effluent. It also mediates the efflux of other metal ions. Ferroportin is localised within the basolateral enterocyte membrane.

  Hepcidin treatment of oocytes expressing FPN1 inhibited efflux of iron, zinc and cobalt.
• Describe ferroportin regulation-molecular methods

  Two splice variants: FPN1A and FPN1B

  • FPN1A: has a 5’ IRE and is repressed in iron deficient conditions
  • FPN1B: lacks the IRE and is not repressed in iron deficient conditions

  NORTHER BLOT
• What is Hephaestin?

  This is a multi copper oxidase with feroxidase activity that promoters the movement of ions from intestinal enterocytes into the circulation by facilitating binding to transferrin. It expressed the most within the small intestine and limited to the enterocytes of the villi and almost absent within the cyrpt. It also converts Fe II to Fe III and works in cooperation with ferroportin 1.
• Describe the identification of Hephaestin

  Positional cloning approach was used in combination with sequence miming to identify the gene on the X chromosome responsible for sex linked anaemia in mice.