Iron metabolism in neonates is a difficult topic to understand, have made it simple by simple animations and diagrams if any queries mail at dockamalpaeds@yahoo.com

1. Dr Kamal Arora
MD, DM
Neonataology
All India Institute of medical sciences
New Delhi
India

2. Overview
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

3.  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.

4. 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 later
J.L.Beard et al, Iron and neural functions , Annals review nutrition 2003 , 23:41–58

5. 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

6.  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

7. Deficiency Adequacy Overload
Y
Risk
X
Dose

8.  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 age
1. Lozoff B, Georgieff M. Iron deficiency and brain development. Semin Pediatr Neurol. 2006;13:158–165
2. 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

9. 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

11. Absorption of Iron
DMT -1 with
Ferroreductase Intestinal lumen
Fe3+ --- Fe2+
Duodenum
Brush border epithelium
Apical 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

12. Action of Hepcidin- iron excess
Intestinal lumen
DMT -1 with
Ferroreductase
Apical membrane
Ferritin
Basolateral membrane
Ferroportin
channels
H
PLASMA
H
H H

13. Action of Hepcidin- Iron deficiency
Intestinal lumen
DMT -1 with
Ferroreductase
Apical membrane
Ferritin
Basolateral membrane
Ferroportin
channels
PLASMA

14. Apo-transferrin molecules
Transferrin
receptors
Transferrin
receptors Clathrin pit
Clathrin pit
Cytoplasm of Erythroid
precursor cell
Ferritin
Proton pump
H+ H+
Endosome

15. Fate of iron in mitochondria+ Globin = Hemoglobin
Fe
Transferrin Fe 2+ Ferrous Protoporphyrin
(Heme)
106 umol
FC
<5 umol
Protoporphyrin Free Protoporphyrin
50 umol
Porphobilinogen
Absorbed through
ALA DMT -1 channel
Dehydrogenase Zn
Aminolevulinic acid
Zinc Protoporphyrin (ZnPP)

16.  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

17. 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

19. 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
•Hepcidin
Each test identifies iron availability at a different point in iron metabolism.

20.  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

21. 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

23.  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

24.  Numerous studies have demonstrated its
superiority over other iron-related measurements
for identifying IDA.

25.  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.

26. S. Author, Study Study group(s) Outcome
No. year population
1. 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 indices
2. 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

27. 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 study
4 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 infants
5. 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 group
6. 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

29. 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

30.  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

31.  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

32.  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

33.  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

35. Hepcidin
Urinary Antimicrobial Peptide Synthesized in the Liver
•25 aminoacid peptide (from clevage
of a 84 aminoacid propeptide)
•Defensin-like (family of natural
antimicrobial peptides involved in
innate immunity)
HEP (atic) CIDIN (antimicrobial)
Park CH, J Biol Chem 2001; 276:7806-10

36. Human
Pig
Rat
Mice
dog
conserved cysteines
Ganz T, Am J Physiol 2006

38.  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).

39. HEPCIDIN REGULATES ALL MAJOR
IRON FLOWS INTO PLASMA
STORAGE RECYCLING
DIET

40. Rivera S, Blood 2005; 105:1797-1802
50 mg HEPCIDIN I.P. in mouse (1 hour)

42. 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

44. Hepcidin studies in newborns
S. Author, Study Intervention Outcome
No. year population
1. 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)

45. Ferritin and hepcidin in various conditions
Disease Serum iron Hepcidin Ferritin
1. Iron deficiency Low Low Low
2. Transfusional iron High High High
Overload
3. Anaemia of Low (?) High/normal High
Inflammation
4. Hereditary High Low or absent High
Hemochromatosis

47. 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.

48.  ZnPP is found in blood in healthy individuals at a
ratio of nearly 50 ZnPP molecules per 1 x 106
heme molecules .

49.  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.

50. 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

51.  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

53.  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)

54.  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

55.  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

56.  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