The uptake of iron by the enterocyte is an important regulatory step in body iron content. Iron can be absorbed into the enterocyte as heme iron or nonheme iron (both ferrous and ferric forms). Heme iron is soluble in the duodenum and is absorbed as an intact metalloproteinvia heme carrier protein 1 (HCP-1) (Fig. 2A). Ferrous iron is then released from heme via heme oxygenase. (5) Unbound iron is absorbed into the enterocyte in the ferrous or ferric form. In the duodenum, nonheme iron is converted to the ferrous (II) form by ascorbic acid and duodenal cytochrome B (DcytB) on the surface of the brush border (Fig. 2B). (6) Ferrous iron then binds to divalent metal transporter-1 (DMT1) and is transferred into the enterocyte.Iron available to gut is ferric form (Fe3+) and is absorbed as Ferrous form (Fe2+) by the enterocytes. This is facilitated by enzymatic reduction (ferrireductase) present in brush border epithelium.DMT is a non specific transporter of divalent ions. It has highest affinity with iron , after that which ever is in excess in diet. This forms the basis of one of the tests of iron deficiency which is known as Zn protoporphyrin. So if there is iron deficiency , it will lead to increase in ZnPP. Also if a child is iron deficient , he is at risk of lead poisoning.
Binds ferroportin, complex internalised and degraded.Resultant decrease in efflux of iron from cells to plasma
Binds ferroportin, complex internalised and degraded.Resultant decrease in efflux of iron from cells to plasma
In the balanced state, 1 to 2 mg of iron enters and leaves the body each day. Dietary iron is absorbed by duodenal enterocytes. It circulates in plasma bound to transferrin. Most of the iron in the body is incorporated into hemoglobin in erythroid precursors and mature red cells. Approximately 10 to 15 percent is presentin muscle fibers (in myoglobin) and other tissues (in enzymes and cytochromes). Iron is stored in parenchymal cells of the liver and reticuloendothelial macrophages. These macrophages provide most of the usable iron by degrading hemoglobin in senescent erythrocytes and reloading ferric iron onto transferrin for delivery to cells. Iron-laden transferrin (Fe2-Tf) binds to transferrin receptors (TfR) on the surface of erythroid precursors. These complexes localize toclathrin-coated pits, which invaginate to form specialized endosomes.2A proton pump decreases the pH within the endosomes,leading to conformational changes in proteins that result in the release of iron from transferrin. The iron transporter DMT1 moves ironacross the endosomal membrane, to enter the cytoplasm.3Meanwhile, transferrin (Apo-Tf) and transferrin receptor are recycled tothe cell surface, where each can be used for further cycles of iron binding and iron uptake. In erythroid cells, most iron moves intomitochondria, where it is incorporated into protoporphyrin to make heme. In nonerythroid cells, iron is stored as ferritin and hemosiderin.Tissue UptakeFor iron uptake in most tissues, transferrin binds totransferrin receptors on the surface of the cell, and thetransferrin receptor–transferrin complex is endocytosed.Protons are pumped into the endosome, lowering thepH and releasing iron from the transferrin. The free ironis released into the cell for use, and the transferrin isreleased back into the bloodstream. The number oftransferrin receptors expressed on the cell surface is regulatedby intracellular iron concentrations. In a low-ironstate, expression of the transferrin receptor is increasedand expression of ferritin is reduced. Conversely, whenthe intracellular iron concentration is high, expression ofthe transferrin receptor is reduced while expression offerritin is increased. (5)
2 identical bilobedstructrure which has an intracellular small portion and a large extracellular portion
Iron metabolism in neonates and role of hepcidin
Dr Kamal Arora MD, DM NeonataologyAll India Institute of medical sciences New Delhi India
OverviewIron – must needed micronutrient• Iron and developing brainPhysiology• Iron absorption• Iron transport and recyclingTests for iron measurement• Ferritin• Hepcidin• Zinc protoporphyrin ,sTFRIron dosing• AAP recommendations
Iron is an essential element for microbes, plants and higher animals. It plays a significant role in critical cellular functions in all organ systems in all species. It is required for early brain growth and function in humans since it supports neuronal and glial energy metabolism, neurotransmitter synthesis and myelination.
Iron deficiency during the fetal or postnatal periods ◦ Alter brain structure and cognitive functioning ◦ Lead to long-term cognitive and motor impairment ◦ Cannot be corrected by iron supplementation laterJ.L.Beard et al, Iron and neural functions , Annals review nutrition 2003 , 23:41–58
Iron: A Critical Nutrient for the Developing Brain • Controls oligodendrocyte production of myelin Delta 9- Iron Deficiency=> Hypomyelination desaturase 1. Delta 9-desaturase •Oxidative phosphorylation , determine neuronal and glial energy status 2. Cytochromes Iron Deficiency=> Impaired neuronal growth, differentiation, electrophysiology Cytochromes 3. Tyrosine Hydroxylase • Monamine neurotransmitter and receptor synthesis (dopamine, serotonin, norepinephrine) Tyrosine Iron Deficiency=> Altered neurotransmitter regulation Hydroxylase J.L.Beard et al, Iron and neural functions , Annals review nutrition 2003 , 23:41–58
Potent oxidant stressor ◦ Role in Fenton reaction to create reactive oxygen species Iron overload associated with neurodegenerative disorders in adults ◦ Hypoxic-ischemic reperfusion injury ◦ Parkinson’s, Alzheimer’s diseases Fetus/premature infant at high risk for iron toxicity ◦ Underdeveloped anti-oxidant systems ◦ Low Total Iron Binding Capacity
Fetuses have 75mg of elemental iron per kilogram body weight during 3rd trimester ◦ Term infant: 200 - 250mg ◦ 24 week (500g): 37.5 mg Majority is in the RBCs (55mg/kg) Liver storage pools are relatively large at term (12 mg/kg) Non-storage tissues, including brain, heart, skeletal muscle account for the rest (8 mg/kg) Preterm Small-for-gestational age1. Lozoff B, Georgieff M. Iron deficiency and brain development. Semin Pediatr Neurol. 2006;13:158–1652. Lozoff B, Beard J, Connor J, Felt B, Georgieff M, Schallert T.Long-lasting neural and behavioral effects of iron deficiency in infancy. Nutr Rev. 2006;64:S34–S43
Section II Iron – must needed micronutrient • Iron and developing brain√ Physiology • Iron absorption • Iron transport and recycling Tests for iron measurement • Ferritin • Hepcidin • Zinc protoporphyrin ,sTFR Iron dosing • AAP recommendations
Absorption of Iron DMT -1 with Ferroreductase Intestinal lumen Fe3+ --- Fe2+ DuodenumBrush border epitheliumApical membrane Divalent metal transporter (nonspecific) Ferritin Fe, Cu, Zn, Mn, Mg,Pb Heirarchy of binding (Fe is highest) Basolateral membrane Iron Deficiency => increases uptake of Ferroportin others (including Zn, Pb) channels PLASMA Apo-transferrin molecules Transferrin molecules
Action of Hepcidin- iron excess Intestinal lumen DMT -1 with Ferroreductase Apical membrane FerritinBasolateral membrane Ferroportin channels H PLASMA H H H
Action of Hepcidin- Iron deficiency Intestinal lumen DMT -1 with Ferroreductase Apical membrane FerritinBasolateral membrane Ferroportin channels PLASMA
Fate of iron in mitochondria+ Globin = Hemoglobin Fe Transferrin Fe 2+ Ferrous Protoporphyrin (Heme) 106 umol FC <5 umolProtoporphyrin Free Protoporphyrin 50 umolPorphobilinogen Absorbed through ALA DMT -1 channel Dehydrogenase ZnAminolevulinic acid Zinc Protoporphyrin (ZnPP)
Iron is efficiently recycled from senescent red blood cells. Erythrocytes are phagocytosed by macrophages in the spleen, where they are lysed and the protein is degraded. The released iron can either be stored in the macrophage or sent back into circulation bound to plasma transferrin
Section III Iron – must needed micronutrient • Iron and developing brain Physiology • Iron absorption • Iron transport and recycling Tests for iron measurement√ • Ferritin • Hepcidin • Zinc protoporphyrin • sTFR Iron dosing • AAP recommendations
Direct •Bone marrow aspiration and biopsy •Hemoglobin •Serum ferritin •Free erythrocyte protoporphyrin Indirect •Zinc protoporphyrin •Total iron binding capacity (TIBC) •Transferrin receptor concentration •Transferrin saturation •HepcidinEach test identifies iron availability at a different point in iron metabolism.
Bone marrow aspiration and biopsy ◦ Prussian blue staining of marrow hemosiderin to semi- quantitatively grade the amount of macrophage storage iron.Disadvantage Invasive Not possible in newborns
Indirect Measures Advantages Less invasive Lack of sensitivity or specificity or both. Easy to perform on peripheral blood. Affected by other factors such as: ◦ Concurrent infection ◦ Inflammation ◦ Maternal chorioamnionitis ◦ Liver disease
Most useful laboratory measure of iron status Universally available and well-standardized measurement that offers important advantages over bone-marrow examination for identifying iron deficiency A valuable feature of the measurement is that the concentration is directly proportional to body iron stores in healthy individuals; 1 mg/L serum ferritin corresponds to 8–10 mg or 120 ug storage iron/kg body weight
Numerous studies have demonstrated its superiority over other iron-related measurements for identifying IDA.
A well-known limitation of the serum ferritin is the elevation in values that occurs independently of iron status in patients with acute or chronic inflammation, malignancy, or liver disease.
S. Author, Study Study group(s) OutcomeNo. year population1. Mukhopadhy Mother Group 1: Cord ferritin –low in SGA group. ay K et al infant pair : Term AGA (n=50) 68 vs141(p=0.0007) 2010 ≥37 weeks Group 2: Proportion of infants with low Birth Term SGA (n=50) cord ferritin more in SGA weight≥ Primary outcome-cord ferritin (p=0.05) 1500 gm Secondary outcome –infants with No correlation in maternal and (n=126) 1.low cord ferritin (< 40ug/l) neonatal cord iron parameters 2. Serum iron and TIBC Serum ferritin levels were same 3. Serum ferritin at 28 days in both groups (p=0.16) 4. Correlation b/w maternal and neonatal iron indices2. Olivares et Birth weight: Group 1: At birth, preterm SGA infants al,1992 1500 to 2500 Preterm AGA (n=29) have low iron stores as compared grams; Group 2: to preterm AGA and term SGA gestation: 33 Preterm SGA (n=17) infants: 55% preterm SGA group - 40 weeks Group-3: had abnormally low cord serum Term SGA (n=38) ferritin <60mcg/l as compared to (SGA was defined as per the 20% and 9% in the preterm AGA curves by Thomson ) and term SGA groups (a sub-group of the study were respectively. given iron supplements from 2 Preterm SGA<Preterm months of age) AGA<Term SGA
3 Haga P et al, Birth weight: Group 1: At birth, preterm SGA 1980 600-2000 Preterm AGA (n=24) infants have low iron grams Group 2: stores as compared to Preterm SGA (n=8) preterm AGA and term Group 3: AGA infants Term AGA (n=22) Term SGA infants were not included in the study4 Karaduman Group 1: Iron stores (as measured D et al, 2001 Term SGA (n=21) by serum ferritin) are low in term SGA infants Group 2: as compared to term Term AGA (n=19) AGA infants5. Scott PH et Total no. Group 1 At birth, plasma levels of al,1975 infants-106 PT SGA/AGA transferrin and iron in Group 2 the SGA infants were T-SGA/AGA similar to those in the AGA group6. Dr Bijan 34 SGA Late preterm and term No difference in SGA Saha 30 AGA Group 1 :SGA and AGA group (unpublished) Group 2: AGA
Structure of cellular transferrin receptor C terminal 671 AA residues Disulphide bond 61 AA residues N terminal 2 identical subunits Molecular mass – 95000 daltons (each)Erythrocyte precursor cell, placental cell
A soluble form of the transferrin receptor was first identified in serum in 1986 by Japanese Controls flow of transferrin iron inside the cell Serum levels represent the total mass of tissue receptor Serum receptor levels rises significantly with tissue iron deficiency. Quantitative measure of iron deficiency and distinguishes from the iron deficiency of chronic disease
Highest no. of these receptors - ◦ Rapidly dividing cells ◦ Haemoglobin synthesis tissues ◦ Placenta ◦ Total absence in patients with aplastic anaemia Iron replete cells – less no of receptors- protects from excess iron
The only determinant of the sTfR other than the erythroid precursor mass is tissue iron deficiency which increases the sTfR in proportion to the severity of the iron deficit
Several commercial assays are now available, Wider application of sTfR measurements has been limited to date by the marked differences in normal values reported with different assays
Hepcidin Urinary Antimicrobial Peptide Synthesized in the Liver•25 aminoacid peptide (from clevageof a 84 aminoacid propeptide)•Defensin-like (family of naturalantimicrobial peptides involved ininnate immunity)HEP (atic) CIDIN (antimicrobial) Park CH, J Biol Chem 2001; 276:7806-10
HumanPigRatMicedog conserved cysteines Ganz T, Am J Physiol 2006
Production stimulated by increased plasma iron and tissue stores. Negative feedback - hepcidin decreases release of iron into plasma (from macrophages and enterocytes). Fe-Tf increases hepcidin mRNA production (dose dependent relationship).
HEPCIDIN REGULATES ALL MAJOR IRON FLOWS INTO PLASMASTORAGE RECYCLING DIET
GENETICALLY DETERMINED IRON OVERLOAD SYNDROMES (HEMOCHROMATOSIS)OMIM classification Gene chr. Remarks Type 1: “classical HFE 6p21.3 90%, only Caucasians Type 2: ”juvenile” 2a. HJV 1q21 = penetrance M and F 2b. Hepcidin 19q13.1 Type 3: TfR2 7q22 similar to “classical” Type 4: Ferroportin 2q32 dominant
Hepcidin studies in newborns S. Author, Study Intervention OutcomeNo. year population1. Ervasti Mari Pregnant mothers Mothers sample and newborn Maternal prohepcidin > cord et al,2009(25) and newborns cord blood. (325ug/L vs. 235 ug/L not Gestation: 37 – Main outcome : maternal and cord significant) 42 weeks serum prohepcidin , transferrin (n =193 pairs) receptors, serum ferritin Correlation b/w maternal and cord prohepcidin –very significant spearmans coefficient=0.600 Prohepcidin levels did not correlate with iron status in mothers or newborns.2. Amarilyo G et Gestation >35 Group 1: AGA (n=20) Hemoglobin and al, 2010(26) weeks Group 2: SGA (n=20) prohepcidin – same (All neonates- apgar >7 at 1 min Cord pH->7.25) EPO and Erythrocyte Measured progenitors –higher in SGA 1. Hemoglobin infants 2. Prohepcidin, 3. EPO, 4. Erythrocyte Progenitors (CD71/CD45)
Ferritin and hepcidin in various conditionsDisease Serum iron Hepcidin Ferritin1. Iron deficiency Low Low Low2. Transfusional iron High High High Overload3. Anaemia of Low (?) High/normal High Inflammation4. Hereditary High Low or absent High Hemochromatosis
Zn Zinc protoporphyrin (ZnPP) - normal metabolite that is formed in trace amounts during heme biosynthesis Final reaction in the biosynthetic pathway of heme is the chelation of iron with protoporphyrin During periods of iron insufficiency or impaired iron utilization, zinc becomes an alternative metal substrate for ferrochelatase, leading to increased ZnPP formation.
ZnPP is found in blood in healthy individuals at a ratio of nearly 50 ZnPP molecules per 1 x 106 heme molecules .
Simple and reliable measurement of IDA. Advantage of this well established assay is the ability to measure the ratio ZPP/haem directly on a drop of blood using a dedicated portable instrument called a haematofluorimeter. The ZPP is ideally suited to screening for IDA in field surveys of iron status or in paediatric and obstetrical clinics where uncomplicated iron deficiency is the major cause of IDA.
1. The ZPP is not widely used in large clinical laboratories, partly because of the difficulty in automating the assay.2. Zinc protoporphyrin levels can be elevated : Lead poisoning Sickle cell anemia Sideroblastic anemia Anemia of chronic disease
The sensitivity and specificity of ZnPP/H in preterm and term infants, have not been clearly determined. A normal range for ZnPP/H of preterm infants has been proposed, but the sample size was small.Juul SE et al ; Zinc protoporphyrin/heme as an indicator of iron status in NICU patients. J Pediatr. 2003;142:273–278
Current AAP dosing recommendations appear appropriate for preterms in NICU ◦ 2-4 mg/kg/day enteral iron 4mg/kg if <30 weeks 2-3 mg/kg if >30 weeks ◦ 6 mg/kg/day if on rhEpo Post-discharge recommendations (2.25 mg/kg/d) appear low and should be increased to 3.3 mg/kg/d Consider monitoring ferritin at birth, at discharge and at follow-up (along with hemoglobin & indices)
Term AGA 1 mg/kg daily Term SGA 2 mg/kg daily Preterm >30 w 2 mg/kg daily Preterm <30 w 4 mg/kg daily Preterm on rhEpo 6 mg/kg daily Preterm; ferritin <35 +2 mg/kg daily
AAP recommends hemoglobin screening at 12 months of age ◦ Earlier screening for premies, SGAs ◦ sTfR, ZnPP, MCV might screen pre-anemia sTfR, ZnPP not available everywhere, lacking standards for < 12 month olds Pre-anemic screening ◦ Ferritin is a good pre-anemic screen But, infant cannot have acute illness (acute phase reactant) ◦ NHANES and CDC testing sTfR/Heme ratio ◦ Hepcidin
Hepcidin is an iron-regulatory hormone that maintains plasma iron levels and iron stores within normal range Hepcidin regulates the entry of iron into plasma from duodenal enterocytes, from macrophages (and from hepatocytes) Hepcidin acts by binding the receptor/iron channel ferroportin and causing its degradation Hepcidin is regulated by iron, erythropoiesis and inflammation Excess hepcidin causes the hypoferremia and anemia of inflammation Hepcidin deficiency, or resistance to hepcidin, cause hemochromatosis