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CB Chapter 13 Iron
CB Chapter 13 Iron
13
Heme metabolism disorders:
iron and porphyrine
CONTENT
13 Heme metabolism disorders: iron and porphyrine ............................................................. 1
13.1 Basic physiology ......................................................................................................... 2
13.2 Laboratory assessment of iron metabolism ............................................................... 4
13.3 Other biochemical aspects of anemia......................................................................... 9
13.4 Heme metabolism disorders ..................................................................................... 11
13.5 Laboratory diagnostics of porpyhrias ........................................................................ 13
Case studies and self-assessment questions ..................................................................... 14
KEY INFORMATION............................................................................................................. 16
Iron (Fe) is an essential element necessary not only for erythropoiesis, but also for cell
respiration, cell proliferation and differentiation, regulation of gene expression, cell signalling
and also for some immune functions. Iron deficiency, especially in the first two years of life,
affects the mental and physical development of children and can endanger cognitive and
motor functions. In adults, iron deficiency can result in poor physical performance and
undesirable complications in pregnancy. Excess iron can damage parenchymal organs,
especially the liver, heart and pancreas. Understanding Fe metabolism has changed
dramatically over the past two decades due to knowledge of its cellular and systemic
homeostasis and the identification of many key proteins that regulate Fe metabolism.
This chapter focuses on:
disorders of iron metabolism and heme with emphasis on laboratory tests used for their
diagnosis;
some biochemical aspects of anemia.
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Iron from food is absorbed mainly in the duodenum, in the brush border of enterocytes, which
regulate the intensity of absorption and storage of iron according to the needs of the organism
(Figure 13.1). From the total iron content in a mixed diet (10 – 20 mg/day), only about 5 – 10%
is absorbed, with heme Fe2+ being absorbed better than non-heme Fe3+ of plant origin, which
must be reduced (in the acidic environment of gastric juice) to Fe2+. In iron deficiency and
conditions with increased erythropoiesis, absorption by active transporters can increase up to
20 – 30%. On the contrary, the absorption of iron decreases if a diet is rich in calcium, phytates,
oxalates, polyphenols (tannins in tea, coffee, cocoa), or in case of increased iron body stores.
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enterocyte
FIGURE 13.1 Absorption of iron in enterocytes. DMT1 – divalent metal transporter 1, HCP1 – heme carrier
protein1, FPN – ferroportin, cytB – cytochrome M
The absorbed iron is again oxidized to Fe3+ by the activity of ceruloplasmin. The part of iron is
stored as ferritin in enterocytes, and the majority is incorporated into transferrin and
transported to cells that use it in different ways: erythroid precursor cells for hemoglobin
synthesis, muscle for myoglobin synthesis and other cells incorporate iron into storage
proteins.
Iron from disintegrated erythrocytes, stored as ferritin in macrophages of the bone marrow,
spleen, and liver, can also be reused for erythropoiesis. Physiological excretion of iron from the
body is possible only via stool (unabsorbed Fe from food) as well as exfoliated skin and
intestinal mucosa cells, which change relatively quickly (up to 1 mg/20 μmol per day). Women
also lose Fe via blood (menstruation, pregnancy).
Hepcidin, a peptide hormone (25 AK) produced by the liver, is a central regulator of iron
balance. Its physiological role is to inhibit the supply of iron from enterocytes and macrophages
to the circulation. This is ensured by reducing the number of transport channels called
ferroportin on the surface of these cells. Increasing iron stores and inflammation of any origin
stimulate synthesis of hepcidin. Hepcidin production during the inflammatory response is
induced by cytokines, especially IL-6. On contrary, in iron deficiency (e.g. in anemia, hypoxia
and increased erythropoiesis in the bone marrow) low concentration of hepcidin allows a
smooth iron supply to the blood (Figure 13.2).
a. liver b.
↓hepcidin liver
↑hepcidin
HFE hepcidín
HFE
TFR2 HFE
TFR2
HJV HJVTFR2
Fe HJV Fe
enterocyte enterocyte
macrophage
macrophage
At the systemic level, transferrin saturation with iron is a major factor regulating hepcidin
production. At the cellular level, several mechanisms control the synthesis of several regulatory
proteins in iron homeostasis (INFO 13.1).
binding sites on transferrin are saturated with iron. Transferrin can be measured directly as
a protein concentration, or indirectly through its ability to bind externally supplied Fe - as total
iron binding capacity (TIBC). The physiological TIBC for iron varies in range 45 – 72 μmol/L.
Because the half-life of transferrin is relatively long, changes in its concentration do not reflect
short-term fluctuations in S-Fe. The ratio between S-Fe and TIBC indicates the percentage of
transferrin saturation. There is an inverse relationship between TIBC and transferrin
saturation.
Typical physiological and pathological changes in serum transferrin are listed in Table 13.1.
Examination of transferrin saturation has a diagnostic value especially in screennig of latent
hemosiderosis, which is indicated by values > 60%. Examination of TIBC in patients with chronic
kidney disease treated with erythropoietin provides better information on the availability of Fe
than ferritin, which is elevated due to a decrease in GFR.
Ferritin
The concentration of ferritin in the serum is low; its source is apoptotic cells. The amount of
iron bound in serum ferritin is minimal, so it does not influence the iron balance. Serum ferritin
levels reflect the intracellular iron stores available for erythropoiesis.
Decreased serum ferritin is an early marker of iron deficiency. In interpretation of results, it is
necessary to take into account the fact that it is a positive acute-phase reactant. Therefore,
ferritin may not be reduced in sideropenic patients with concomitant inflammatory or
malignant disease. Ferritin concentrations > 100 μg/L reliably exclude iron deficiency.
Elevated serum ferritin is a sign of iron excess in the body, regardless of its origin. Other causes
of the increased serum ferritin are listed in the Table 13.2. Patients with CKD treated by
hemodialysis have elevated serum ferritin levels, but this does not reflect iron available for
hematopoiesis. Even with elevated ferritin levels, they suffer from multifactorial anemia and
other hematological parameters inform better about iron deficiency (e.g. low hemoglobin
content in erythrocytes and reticulocytes).
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Fe3+
TrfR
mitochondria
endosome
Hb synthesis
Iron deficiency
Iron deficiency (sideropenia) is the most widespread form of nutritional deficiency worldwide
and causes 50% of all anemias. Anemia is microcytic, hypochromic with MCV < 80 fL.
Sideropenia occurs as a result of:
insufficient dietary intake of iron, including its low bioavailability with high fiber, and
phytate content in food;
absorption disorders (malabsorption syndromes and conditions after bowel surgery);
increased iron losses.
Several laboratory parameters inform about the iron deficit (Table 13.3). The best diagnostic
test to assess iron deficiency is low ferritin. In patients with CKD, alternative tests (e.g. %
hypochromic erythrocytes, and reticulocytes) have better informational value.
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Iron excess
Excess iron is not as frequent as iron deficiency. The most common causes are chronically
increased peroral or parenteral intake and increased absorption in enterocytes in genetically
determined hemochromatosis (Table 13.4).
Laboratory signs of excess iron in the body are:
increased S-Fe concentration;
increased transferrin saturation;
increased serum ferritin.
If transferrin saturation rises above 35 – 40%, a part of the circulating iron exists in potentially
toxic form - in complexes with citrate or albumin. Iron easily leaves that bond and enters the
cells, where due to its high reactivity, it can cause damage or even organ failure.
Mechanism Examples
High intake Excessive content in diet, repeated transfusions
High intestinal absorption Hereditary hemochromatosis
Increased but ineffective thalassemia, congenital sideroblastic anemia, myelodysplastic
erythropoiesis syndrome
Increased RBC destruction congenital hemolytic anemias, sickle-cell anemia
Hemochromatosis is confirmed by genetic testing for a mutation in the HFE gene. A liver
biopsy is not necessary to confirm the diagnosis, but can be used to evaluate fibrosis and rule
out other liver diseases. The content of iron in the bone marrow is usually normal in hereditary
hemochromatosis, rather the accumulation of iron in parenchymal organs predominates.
Increased iron content in the monocyte-macrophage system occurs in ineffective or defective
erythropoiesis (haematological disorders, malignancies and chronic inflammatory diseases).
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Sideropenic anemia is only one of the possible forms of anemia - the condition with a decrease
in the concentration of hemoglobin in the blood below the reference interval. Regardless of
the cause, all types of anemia negatively affect the supply of oxygen to the tissues. Anemias
are classified according to erythrocyte size into microcytic, normocytic, and macrocytic, or
based on etiopathogenetic criteria (Table 13.6). Possible causes of anemia are:
1. Insufficient RBC production:
a. for nutritional deficiency of substances necessary for hematopoiesis (Fe, folic acid,
vitamin B12, protein malnutrition),
b. from congenital or acquired causes with sufficient essential factors (chronic disease
anemia, aplastic and dysplastic anemia malignant bone marrow infiltration);
2. Increased RBC loss: due to bleeding or hemolysis.
In addition to the complete blood count and the morphological examination of the peripheral
blood or bone marrow smear, other, especially immunological and biochemical tests are used
in the laboratory diagnosis of anemia. Biochemical tests are particularly important in
macrocytic anemias due to folic acid or vitamin B12 deficiency (Figure 13.4) as well as in
hemolytic anemias.
Macrocytic anemia
B12, folate
Reticulocytes
vit B12 + folate >100 109/L
vit B12 deficiency folate deficiency
deficiency
YES NO
Vitamin B12 (cobalamin) is a compound similar to porphyrin with centrally bound cobalt
instead of iron. Its only source for humans is food of animal origin. The vitamin stores in an
adult's body last for 3 – 4 years until a deficit develops. The resorption of cobalamin in the
terminal ileum requires an intrinsic factor (IF) formed in the gastric mucosa to protect it from
degradation during transport through the ileum. After reabsorption, it binds to a transport
protein (transcobalamin) in the bloodstream. This form is called holotranscobalamin, or an
active vitamin B12, because it releases cobalamin into the cells with active hematopoiesis or
cells storing cobalamin (liver and other internal organs). The end products of intracellular
metabolism of cobalamin - methylcobalamin and adenosylcobalamin - are involved in the
metabolism of homocysteine and methyl malonyl-coenzyme A. As both molecules are essential
for the proper metabolism and function of neurons, cobalamin deficiency manifests by a variety
of neurological symptoms.
The causes of vitamin B12 deficiency are in particular:
insufficient intake in food (vegetarians, vegans);
impaired absorption - gastric factors (atrophic gastritis, antibodies against IF or parietal
cells of the gastric mucosa, gastrectomy, proton pump inhibitors);
impaired absorption - intestinal causes: resection of the ileum, celiac disease, chronic
inflammatory diseases (Crohn), rare genetic defects of the cubilin receptor in enterocytes.
Heme is an iron-containing pigment essential for life, synthesized in the liver and maturing
erythroid cells in the bone marrow. Disorders of heme synthesis due to enzyme defects lead to
diseases such as porphyria and some types of anemia (e.g., X chromosome-linked
sideroblastic anemia). Degradation of heme is a strictly controlled process that takes place in
the spleen and liver. By opening the cyclic heme molecule, linear tetrapyrrole biliverdin is
formed, subsequently reduced to bilirubin (see Chapter 5). Porphyrias (gr. porphyros = purple)
are rare metabolic diseases caused by a congenital or acquired defect of enzymes necessary
for heme biosynthesis. Each enzymopathy leads to the accumulation of typical precursor
tetrapyrroles - porphyrins, which are intermediates in heme biosynthesis (Figure 13.4).
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CUTANEUS
porphobilinogen (PBG)
PBG deaminase Acute intermitent porphyria
hydroxymetylbilan
uroporphyrinogen III cosyntase Congenital erythropetic porphyria
uroporphyrinogen III
uroporphyrinogen decarboxylase Porphyria cutanea tarda
coproporphyrinogen III
coproporphyrinogen decarboxylase Congenital coproporphyria
protoporphyrinogen IX
protoporphyrinogen decarboxylase Porphyria variegata
protoporphyrin IX
mitochondria ferrochelatase Erythropetic protoporphyria
HEM
Questions:
a. What is the expected diagnosis based on biochemical findings?
b. What examination is needed to confirm the diagnosis?
c. What may be the cause of elevated serum ferritin?
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Questions:
a. What is the expected diagnosis based on biochemical findings?
b. What examination is needed to confirm the diagnosis?
c. What is the most frequent cause of iron deficiency?
Self-assessing questions
1. Identify the main regulator of the iron balance and explain the mechanism of its effect?
2. List the tests used to diagnose iron deficiency.
3. What factors may affect serum iron level?
4. What biochemical tests would you indicate in a patient with macrocytic anemia?
5. Name the biochemical signs of hemolysis.
6. Explain the causes of porphyria?
7. When is the highest probability of positive laboratory results in an examination of
porphyria?
16 13 HEME METABOLISM DISORDERS
KEY INFORMATION
Absorption, transport, and iron stocks are strictly controlled. Most of the Fe in the
body is in the form of heme.
Daily absorption of iron rom food is about 1 mg, which is transported in the blood
bound to transferrin, stored intracellularly in the form of ferritin and hemosiderin.
Serum iron as an isolated test is of little diagnostic value, with the exception of iron
intoxication.
The best indicator of iron stores in the body is serum ferritin.
Anemia from iron deficiency can occur due to insufficient food intake, malabsorption,
or excessive blood loss.
Iron excess in the body is less common iron deficiency. It arises on a genetic basis
with increased intestinal absorption of iron (hemochromatosis) or for non-genetic
reasons with excessive intake of iron (transfusion, chronic alcoholism).
Porphyria’s are disorders of heme biosynthesis that are clinically manifested by neuro-
visceral attacks or cutaneous manifestations due to photosensitization.
Acute porphyrias are life-threatening situations, there is evidence of increased urinary
excretion of heme biosynthesis intermediates, especially porphobilinogen.
Beside clinical presentation, the differential diagnosis of porphyrias is based on the
detection of a different spectrum of produced heme precursors in body fluids.