ASSIGNMENT FOR BLOOD 1

1. Briefly describe the metabolism of mature red blood cells with regards to the following:

A. Major substrate

Glucose serves as the main energy source or fuel for the red blood cells. Mature red blood cells
lack the mitochondria that contain ATP synthase and the enzymes of the tricarboxylic acid cycle
(TCA), electron transport chain, and β-oxidation pathway. They are therefore incapable of
utilizing fatty acids or ketone bodies as metabolic fuel. Consequently, red blood cells are
completely reliant on glycolysis to generate ATP. Glucose enters red blood cells by facilitated
diffusion, a process mediated by glucose transporter 1 (GLUT1), also known as glucose
permease.

B. Pathways occurring in mature RBCs and their importances to normal RBC function.

● Embden-Meyerhof Pathway (anaerobic glycolysis)

○ The first phase of glycolysis employs glucose phosphory- lation, isomerization,
and diphosphorylation to yield fructose 1,6-bisphosphate (F1,6-BP). Fructose-
bisphosphate aldolase cleaves F1,6-BP to produce glyceraldehyde-3-phosphate
(G3P). Intermediate stages employ the enzymes hexokinase, glucose-6-
phosphate isomerase, and 6-phosphofructokinase. The initial hexokinase and 6-
phosphofructokinase steps consume a total of 2 ATP molecules and limit the rate
of glycolysis.
○ The second phase of glucose catabolism converts G3P to 3-phosphoglycerate
(3-PG). In the first step, G3P is oxidized to 1,3-bisphosphoglycerate (1,3-BPG)
through the action of glyceraldehyde-3-phosphate dehydrogenase (G3PD). 1,3-
BPG is dephosphorylated by phosphoglycerate kinase, which generates 2 ATP
molecules and 3-PG.
○ The third phase of glycolysis converts 3-PG to pyruvate and generates ATP. The
product 3-PG is isomerized by phosphoglycerate mutase to 2-phosphoglycerate
(2-PG). Enolase (phosphopyruvate hydratase) then converts 2-PG to
phosphoenolpyruvate (PEP). Pyruvate kinase (PK) splits off the phosphates,
forming 2 ATP molecules and pyruvate. PK activity is allosterically modulated by
increased concentrations of F1,6-BP, which enhances the affinity of PK for
PEP.5 Thus, when the F1,6-BP is plentiful, increased activity of PK favors
pyruvate production. Pyruvate may diffuse from the erythrocyte or may become a
substrate for lactate dehydrogenase with regeneration of the oxidized form of
nicotinamide adenine dinucleotide (NAD+). The ratio of NAD+ to the reduced
form (NADH) modulates the activity of this enzyme.
● Hexose Monophosphate Pathway/Pentose Phosphate Pathway
○ The pentose phosphate pathway of the RBC metabolizes about 5%-10% of the
total flux of glucose and produces NADPH.
 ○ HMP detoxifies peroxide (H2O2), which arises from O2 reduction in the cell’s
aqueous environment, where it oxidizes and destroys heme iron, pro- teins, and
lipids, especially lipids containing thiol groups.5 By detoxifying peroxide, the HMP
extends the functional lifespan of the RBC.
○ HMP diverts glucose-6-phosphate (G6P) to ribulose 5-phosphate by the action of
glucose-6-phosphate dehydrogenase (G6PD). In the process, oxidized
nicotinamide adenine dinu- cleotide phosphate (NADP) is converted to its
reduced form (NADPH). NADPH is then available to reduce oxidized gluta-
thione (GSSG) to reduced glutathione (GSH) in the presence of glutathione
reductase.
○ Reduced glutathione (GSH) is important in the metabolism of RBCs, in part to
counteract the action of potentially toxic peroxides. The RBC can synthesize
GSH and the NADPH required to return oxidized glutathione (G-S-S-G) to the
reduced state GSH.
● Rapoport-Luebering Pathway
○ The glycolytic pathway in red blood cells also possesses a unique branch, or
shunt, whose purpose is to isomerize 1,3-bisphosphoglycerate (1,3-BPG) to 2,3-
bisphosphoglycerate (2,3-BPG). 2,3-BPG binds to and stabilizes hemoglobin in
the T-state. Conversion of 1,3-BPG to 2,3-BPG is catalyzed by 2,3-BPG mutase,
a bifunctional enzyme that also catalyzes the hydrolysis of 2,3-BPG to the
glycolytic intermediate 3-phosphoglycerate.
○ Production of 2,3-bisphosphoglycerate is important in regulating the ability of Hb
to transport oxygen.
○ Increased 2,3-B/DPG in tissue increases the release of oxygen from hemoglobin
to the tissues, shifting the O2-Hgb dissociation curve to the right.
● Methemoglobin Reductase Pathway
○ The iron of Hb must be maintained in the ferrous state . Ferric iron is reduced to
the ferrous state by the action of an NADH- dependent methemoglobin reductase
system involving cytochrome b5 reductase and cytochrome b5.
○ Using H+ from NADH formed when G3P is converted to 1,3-BPG, cytochrome b5
reductase acts as an intermediate electron carrier, returning the oxidized ferric
iron to its ferrous, oxygen-carrying state. This enzyme accounts for more than
65% of the methemoglobin-reducing capacity within the RBC.
Figure 1. Glucose metabolism in RBCs [Keohane & Smith, 2016]
2. The red blood cell can pass and squeeze itself into the tiniest blood vessels without bursting.

A. Describe the unique RBC membrane proteins that makes this possible.

Red Blood Cell Membrane Lipids and Proteins

In order to maximize the efficiency of gas exchange, red blood cells must possess the
structural strength to maintain their biconcave shape, yet remain sufficiently flexible to squeeze
through peripheral capillaries and the sinusoids of the spleen. The red blood cell membrane’s
lipid bilayer is composed of about 50% lipid and 50% protein. Major lipids are cholesterol and
phospholipids. Choline-containing phospholipids (phosphatidylcholine and sphingomyelin) are
predominant in the outer leaflet while amino-containing phospholipids
(phosphatidylethanolamine and phosphatidylserine) are predominant in the inner leaflet.
Glycosphingolipids including the ABO blood groups constitute 5 to 10% of the total lipid of the
RBC membrane.

Most of the red blood cell membrane proteins are glycoproteins in nature. As shown in
Figure 2, several of these proteins span the membrane bilayer (integral membrane proteins)
while others associate with its surface, generally via protein-protein interactions (peripheral
membrane proteins). Integral membrane proteins, also known as transmembrane proteins,
function as channels or carriers and sites of attachment for peripheral proteins. Peripheral
proteins, on the other hand, are the proteins that determine the shape and flexibility of the
RBCs. The principal proteins are enumerated in Table 1 below.

Figure 2. Interactions of cytoskeletal proteins with each other and with certain integral proteins
of the membrane of the red blood cell.
 Table 1. Principal Proteins of the Red Cell Membrane

Integral Proteins of the RBC Membrane

As shown in Table 1, the principal transmembrane proteins of the RBC membrane

include Anion Exchange Proteins and the Glycophorins. Band 3 protein is a transmembrane
glycoprotein oriented with its carboxyl terminal end projecting from the external surface of the
erythrocyte membrane and its amino terminal end from the cytosolic face. Thought to exist as a
dimer, band 3 protein is a multipass membrane protein whose polypeptide chain crosses the
bilayer 14 times. The principal function of this anion exchange protein is to provide a channel
within the membrane through which chloride and bicarbonate anions can be exchanged. At the
tissues, bicarbonate generated from the hydration of CO2 is exchanged for chloride. At the
lungs, where carbon dioxide is exhaled, this process is reversed. The amino terminal end
serves as an anchoring point for several red blood cell proteins, including band 4.1 and 4.2
proteins, ankyrin, hemoglobin, and several glycolytic enzymes.

Glycophorins A, B, and C (Band 4) are single-pass transmembrane proteins (the

polypeptide chain crosses the membrane only once). The 23 amino acid transmembrane
segment is α-helical in configuration. Glycophorin A, the predominant form, is heavily
glycosylated. The amino terminal end of this 131-amino acid polypeptide is modified by 16
oligosaccharide chains, 15 of them O-linked, that account for roughly 60% of its mass. The
oligosaccharide chains of glycophorin A account for nearly 90% of the sialic acid residues
bound to the red cell membrane. The carboxyl terminal end extends into the cytosol and binds
to band 4.1 protein, which in turn binds to spectrin. Polymorphism of glycophorin A provides
the basis of the MN blood group system (see below). Some viral and bacterial pathogens, such
as influenza virus and Plasmodium falciparum, target erythrocytes by recognizing and binding to
glycophorin A. Intriguingly, individuals whose red cells lack glycophorin A exhibit no adverse
effects.
Proteins of the RBC Membrane

The lipid bilayer of RBCs is inherently fluid, contributing significantly to the deformability
of the erythrocyte membrane. As discussed above, it is flexible as it is pulled into the biconcave
shape by a strong but flexible network of cytoskeletal proteins.

Spectrin is the most abundant protein of the erythrocyte cytoskeleton. It is composed of two
polypeptides more than 2100 residues in length: spectrin 1 (α chain) and spectrin 2 (b chain). The α and b
chains of each spectrin dimer intertwine in an antiparallel orientation to form a highly extended structural
unit ≈ 100 nm in length. Normally, two spectrin dimers self-associate head-to-head to form an
approximately 200 nm long tetramer that is linked to the inner surface of the plasma membrane (and is
bridged to other spectrin tetramers) via ankyrin, actin, and band 4.1 protein. The result is an internal
mesh, the cytoskeleton, that is strong enough to maintain cell shape and resist swelling due to osmotic
pressure, yet flexible enough to allow the erythrocyte to fold when needed.

Ankyrin is a pyramid-shaped protein that binds spectrin. In turn, ankyrin binds tightly to
band 3, securing attachment of spectrin to the membrane. Ankyrin is sensitive to proteolysis,
accounting for the appearance of bands 2.2, 2.3, and 2.6, all of which are derived from band
2.1.

Actin (band 5) exists in red blood cells as short, doublehelical filaments of F-actin. The
tail end of spectrin dimers binds to actin. Actin also binds to protein 4.1.

Protein 4.1, a globular protein, binds tightly to the tail end of spectrin, near the actin-
binding site of the latter, and thus is part of a protein 4.1-spectrin-actin ternary complex. Protein
4.1 also binds to the integral proteins glycophorin A and glycophorin C, thereby attaching the
ternary complex to the membrane. In addition, protein 4.1 may interact with certain membrane
phospholipids, thus connecting the lipid bilayer to the cytoskeleton.

Certain other less quantitatively prominent proteins, such as band 4.9, adducin, and
tropomyosin, also participate in cytoskeletal assembly.

B. Describe what happens if a person has Hereditary Spherocytosis and why their RBCs are
prone to hemolysis under certain conditions.

Hereditary Spherocytosis is an abnormality wherein there are mutations in the genes encoding
spectrin or other structural proteins in the red cell membrane. Its major cause is deficiencies in
the amount or in the structure of α- or β-spectrin, ankyrin, band 3, or band 4.1. Basically,
deficiency of spectrin results in hereditary spherocytosis and hereditary elliptocytosis which are
both causes of hemolytic anemia. Hemolytic anemias can be caused by extrinsic, intrinsic, or
membrane-specific factors. For this guide question, membrane-specific factors that render red
blood cells vulnerable to lysis include mutations that affect the cytoskeletal proteins responsible
for maintaining their biconcave shape and resistance to osmotic pressure. The most important
of these defects include hereditary spherocytosis and hereditary elliptocytosis, which arise from
abnormalities in the amount or structure of the cytoskeletal protein spectrin. The figure below
shows a schematic diagram of some causes of hemolytic anemias.

Figure 3. Schematic diagram of some causes of hemolytic anemias.

Going back to the guide question, hereditary spherocytosis is a genetic disease transmitted as
an autosomal dominant that affects about 1:5000 persons of Northern European ancestry. It is
characterized by the presence of spherocytes (spherical red blood cells, with a low surface-to-
volume ratio) in the peripheral blood, by a hemolytic anemia, and by splenomegaly.
Spherocytes are more vulnerable to lysis when exposed to lower than normal osmotic pressure,
since their spherical shape offers little capacity to accommodate additional water. Their
abnormal shape also renders them less deformable and more prone to destruction in the
spleen, thus greatly shortening their life in the circulation. The figures below show visualization
of the spherocytes.
 Figure 4. Spherocytes in comparison to normal RBC.

Hereditary spherocytosis is caused by a deficiency in the amount of spectrin or abnormalities of

its structure that undermine its capacity to associate with other cytoskeletal components. The
consequent weakening of the links that anchor the erythrocyte membrane to the cytoskeleton
leads to the adoption of the spherocytic shape. Hereditary spherocytosis also can result from
mutations that produce abnormalities in ankyrin or in bands 3, 4.1, or 4.2. The anemia
associated with hereditary spherocytosis is generally relieved by surgical removal of the
patient’s spleen (splenectomy).

Hereditary elliptocytosis also results from genetic disorders that generate abnormalities in
spectrin or, less frequently, in band 4.1 protein or in glycophorin C. It can readily be
distinguished from hereditary spherocytosis by virtue of the fact that the affected red blood cells
assume an elliptic, disk-like shape.

In both hereditary spherocytosis and hereditary elliptocytosis, symptoms and signs are usually
mild. In hereditary spherocytosis, the anemia may be so well compensated that it is not
recognized until an intercurrent viral illness, such as parvovirus infection, transiently decreases
RBC production, causing an aplastic crisis. Moderate jaundice and symptoms of anemia are
present in severe cases. Hepatomegaly may be present. Cholelithiasis (pigment stones) is
common and may be the presenting symptom. In hereditary elliptocytosis, splenomegaly may
be present.

The treatment for these conditions is sometimes splenectomy. It is rarely needed and is
indicated in patients with symptomatic hemolysis or complications such as biliary colic or
persistent aplastic crisis. If the gallbladder has stones or other evidence of cholestasis, it should
be removed. Although spherocytosis persists after splenectomy, the cells survive longer in the
circulation. Usually, symptoms resolve and anemia and reticulocytosis decrease. However, RBC
fragility remains high.

3. What is erythroblastosis fetalis and what is the usual set-up for this to occur? Explain how this
can be prevented.

Hemolytic disease of the fetus and newborn (HDFN) is also known as alloimmune HDFN or
erythroblastosis fetalis. It is caused by the destruction of neonatal red blood cells by
maternal immunoglobulin G (IgG) antibodies. The formation of maternal antibodies in response
to a fetal antigen is called isoimmunization. These antibodies form when fetal erythrocytes
expressing certain red blood cell antigens that are not expressed in the mother cross the
placenta and gain access to maternal blood. This antibody response may be sufficient enough
to destroy fetal red cells leading to hemolysis, the release of bilirubin, and anemia.
The severity of the illness in the fetus depends on various factors, including the amount and
strength of antibody produced by the mother, the gestational age of the fetus, and the ability of
the fetus to replenish the destroyed RBCs and clear bilirubin.
Numerous blood group systems have been implicated in HDFN including, ABO, Rhesus (Rh),
Kell, Duffy, Kidd, MNS, Diego. P, Lutheran, and Xg. Rhesus and ABO are by far the most
common. ABO incompatibility generally occurs in a group O mother with a group A or B baby,
but ABO incompatibility causes less severe hemolytic disease of the newborn than does Rh(D)
incompatibility. Affected infants are usually asymptomatic at birth with absent or mild anemia
and develop neonatal jaundice, which is usually successfully treated with phototherapy.
Because Rhesus factor incompatibility is more severe, it will be the focus of the rest of this
discussion.

Pathophysiology
Following maternal exposure to RhD-positive blood, B-lymphocyte clones that recognize the
RBC antigen are established. The primary maternal immune response is the production of
IgM isotype. It is important to note that this primary maternal immune response is dose-
dependent. It occurs in about 15% of pregnancies with 1 mL of Rh-positive cells and 70% after
250 mL of Rh-positive cell exposure. Maternal IgG response occurs later in subsequent
pregnancies. The secondary immune response follows repeat exposure to as little as 0.03 mL
of Rh-positive cells. Maternal anti-D (IgG) antibodies cross the placenta and attach to Rh
antigens on fetal RBCs. RBC destruction occurs by lysis of antibody-coated RBCs by
macrophage lysosomal enzymes. The fetus initially responds to the subsequent anemia and
tissue hypoxia through reticulocytosis, and a rise in umbilical artery lactate indicates severe
fetal anemia. Erythroblastosis fetalis results when RBC destruction exceeds production.

Evaluation for Prevention

For patients who are Rh-negative and also have a negative antibody screen, it is important to try
and prevent them from becoming sensitized during the pregnancy course.
Possible reasons a patient may become exposed to fetal blood and thus sensitized include:
-miscarriage,
-amniocentesis,
-vaginal bleeding,
-placental abruption, and
abdominal trauma.
If any of these instances occur, RhoGAM (anti-D immunoglobulin or Rhesus factor IgG)
should be administered.

If the antibody screen for Rh comes back positive during the initial prenatal visit, the titer is
checked as well. Antibody titers of 1:16 and greater have been associated with fetal hydrops. If
paternity is not in question, blood type can be performed on the father of the baby to determine
whether the fetus is at risk. However, because approximately 5% of all pregnancies have
unknown or incorrect paternity, the safest course is to treat all pregnancies as if the fetus is
at risk.

Throughout pregnancy, the antibody titer is followed approximately every 4 weeks. As long as it
remains less than 1:16, the pregnancy can be managed expectantly. However, if it exceeds
1:16, serial amniocentesis should be started as early as 16 to 20 weeks. At the first
amniocentesis, fetal cells can be collected and analyzed for the Rh antigen to determine fetal
Rh status. If negative, the pregnancy can be followed expectantly. However, if the fetus is Rh-
positive, fetal anemia is screened for, using fetal middle cerebral artery (MCA) Doppler
measurements. It was demonstrated more than a decade ago that in anemic fetuses, there is
greater blood flow to the brain; thus, the MCA Doppler measures peak systolic velocity
(PSV). In fetuses with greater PSV measurements, concern for fetal anemia merits more
invasive testing and potential treatment.

Historically, prior to the use of MCA Doppler, the evaluation of Rh-positive fetus in an Rh-
negative woman with positive titers 1:16 or greater was done with serial amniocenteses to
assess the amniotic fluid by a spectrophotometer.

Treatment/ Management
Rho(D) immune globulin is a preparation of human IgG containing antibodies against the
Rho(D) antigen of the red cell. Rho(D) immune globulin is used for the prevention of Rh
hemolytic disease of the newborn. Administration of Rho(D) immune globulin to Rho(D)
negative mothers at the time of antigen exposure, such as the birth of a Rho(D) positive child,
blocks the primary immune response to the foreign cells. Therefore, maternal antibodies to
Rh-positive cells are not produced in subsequent pregnancies, and hemolytic disease of the
neonate is averted.

RhoGAM should be administered at 28 weeks since it has a half-life of about 12 weeks and
covers the mother until term or 40 weeks, and postpartum if the neonate is Rh-positive. A
standard dose of RhoGAM (0.3 mg) will eradicate 15 mL of fetal RBCs. This dose is adequate
for a routine pregnancy. In cases of antepartum bleeding, abdominal trauma, amniocentesis, or
placental abruption where more blood is transferred from the fetus to the mother than normal,
the standard 0.3 mg dose of RhoGAM may not be sufficient. A Kleihauer-Betke test that
determines the amount of fetal RBCs in the maternal circulation should be performed. If the
amount of fetal RBCs is more than can be eliminated by the single RhoGAM dose, additional
dosages must be given.

Cordocentesis and measurement of fetal hemoglobin are used to assess the severity of anemia
when MCA dopplers are elevated. Fetal hemoglobin is two standard deviations below the mean
value for gestational age. A hemoglobin level of more than 7 g/dL below the normal mean for
gestational age or hydrops (actual hemoglobin level of less than 5 g/dL). A hematocrit of less
than 30% also can be used as the threshold for fetal transfusion.

4. Explain why patients with advanced liver disease and children suffering from Nephrotic
syndrome develop edema. Discuss the role of plasma proteins in the maintenance of normal
fluid compartments.

A person who has liver disease can't make plasma proteins. Because about 70% to 80%
of all blood proteins are made in the liver (except the immunoglobulins and von willebrand
factor, which is synthesized by the plasma cells and in the vascular endothelium respectively).
Albumin is the main protein in the blood (60 percent in total). Oncotic pressure in the blood is a
very important factor in this (75-80 percent of oncotic pressure). Plasma oncotic pressure is
what keeps fluid inside the vascular space. With liver disease or not enough food, there will be
less plasma oncotic pressure, which leads to edema. Proteins in the blood are made by
membrane-bound ribosomes, which means they have signal peptides and are mostly made of
sugars. Plasma proteins control how much fluid moves between inside and outside cells through
starling forces. Each has a certain half-life that can be changed by certain things, like the
albumin, which has a half-life of about 20 days.

People who have advanced liver disease or cirrhosis have a hard time controlling how
much extracellular fluid they have, which causes fluid to build up as ascites or edema. As portal
hypertension gets worse, splanchnic arterial vasodilation also happens, mostly because of the
production of nitric oxide (NO). Splanchnic arterial vasodilation reduces effective arterial blood
volume, which leads to fluid accumulation and kidney function problems. This is because the
homeostatic activation of vasoconstrictor and antinatriuretic factors causes vasoconstrictor and
antinatriuretic factors to be released. Sodium and water are kept in the body and the renal
vasoconstriction is also caused. The pressure in the splanchnic capillaries rises because of the
portal hypertension and the splanchnic hyperdynamic circulation. This leads to the accumulation
of retained fluid as ascites.

Plasma proteins are mostly made in the liver. Immunoglobulins are made by
lymphocytes and plasma cells. Albumin, globulins, and fibrinogen make up the total amount of
protein (in plasma only). Proteins help control oncotic pressure, transport substances like
hemoglobin, lipids, and calcium, and promote inflammation and the complement cascade,
among other things. There is a big difference in the amount of albumin in the total protein in the
body.

There is too much fluid in the tissues, either inside cells (cellular edema) or in the
collagen-mucopolysaccharide matrix in the interstitial spaces (interstitial edema). It can happen
when there are changes in the pressures (hydrostatic and oncotic) that act on the microvascular
walls, changes in the molecular structures that make up the barrier to fluid and solute flow in the
endothelial wall, or changes in the lymphatic outflow. The extracellular matrix or interstitial
edema is what we're looking at.

Hydrostatic edema is when there is too much interstitial fluid because of high capillary
hydrostatic pressure and low permeability. Edema is caused by a change in the physical
structure of the pores in the microvascular membrane. This makes the barrier less effective at
stopping macromolecules from moving from the blood to the interstitium. In liver disease and
severe malnutrition, edema is caused by a drop in the amount of plasma proteins, especially
albumin, that circulate in the body.

Another reason for edema that is caused by intravascular factors is when there are big
changes in the amount of proteins in the blood, especially albumin. Hypoproteinemia can
happen if your kidneys aren't working well, your liver isn't making enough plasma proteins, you
don't get enough food, or you get intravenous fluids that don't have any macromolecules in
them, which makes it hard for your body to make proteins. If you have low protein levels, this
can cause a big transcapillary flow of protein-poor fluid into the interstitial spaces. This is
because low protein levels make it easier to reabsorb fluid in the non-steady state and make it
harder to filter it out in the steady state. Like capillary hypertension, this effect is countered by
rises in tissue hydrostatic pressure, which boosts lymph flow, both of which help to keep tissue
fluid from building up in the body. Also, more capillary filtration helps to lower the concentration
of proteins in the extracellular spaces. This effect is even more pronounced when the
extracellular matrix gel has more space for proteins to move through. The resulting drop in
interstitial colloid osmotic pressure reduces net filtration pressure, which reduces the risk of
edema. People who have low levels of protein don't have the same response to high blood
pressure as people who have high blood pressure, so the amount of space available for edema
to form after a stressor like this one is smaller. Tissues can't make up for changes in plasma
colloid osmotic pressure that are the same as a rise in capillary hydrostatic pressure.

In nephrotic syndrome, a lot of protein comes out of the kidneys and is sent to the urine.
The swelling of body tissues and the risk of getting infections can happen as a result of this, and
it can be hard to get better. Blood that doesn't have a lot of protein makes it hard for water to
move from the body to the blood vessels, which causes swelling (edema). First, the eyes and
lower legs get swollen. Then the rest of the body gets swollen, too.

When a lot of protein is flushed out of the body into the urine, it can make it foamy. They
may also pass less urine than usual when they have relapses. It's possible for children who
have nephrotic syndrome to pass out important proteins that help keep blood from clotting in
their urine, which can be dangerous. This can make them more likely to get blood clots that
could be very bad. A relapse also makes the blood more concentrated, which can cause it to
clot.

5. Discuss briefly how iron is metabolized in the body and the important proteins required for its
metabolism, transport and storage. Why is excess iron considered toxic to the body? What
mechanism reduces the toxicity of free iron ?

Iron is a key constituent of many human proteins, including hemoglobin, myoglobin, cytochrome
P450 group of enzymes, numerous components of the electron transport chain, and
ribonucleotide reductase, which catalyzes the conversion of ribonucleotides into
deoxyribonucleotides. Body iron, which is distributed as shown in Table 2, is highly conserved.
A healthy adult loses only about 1.0 to 1.5 mg (<0.05%) of their 3 to 4 g of body iron each day.
However, an adult premenopausal female can experience iron deficiency due to blood loss
during menstruation.
 Table 2. Distribution of Iron in a 70-kg Adult Male

Absorption of nonheme iron by enterocytes of the proximal duodenum is a highly regulated

process. Inorganic dietary iron in the ferric state (Fe3+) is reduced to its ferrous form (Fe2+) by
a brush border membrane-bound ferrireductase, duodenal cytochrome b (Dcytb). Vitamin C,
gastric acid, and a number of other reducing agents present in food may also favor reduction of
ferric to ferrous iron. The transfer of iron across the apical membrane of the enterocytes is
accomplished via the divalent metal transporter 1 (DMT1 or SLC11A2). DMT1 is relatively
nonspecific, and may also be involved in the transport of other divalent cations, such as Mn2+,
Co2+, Zn2+, Cu2+, and Pb2+. Once inside the enterocytes, iron can either be stored bound to
the iron storage protein ferritin or transferred across the basolateral membrane into the
circulation by the iron exporter protein, ferroportin or iron-regulated protein 1 (IREG1 or
SLC40A1). Hephaestin, a copper-containing ferroxidase homologous to ceruloplasmin, oxidizes
Fe2+ to Fe3+ prior to export. Iron is transported in plasma in the Fe3+ form by the transport
protein, transferrin. Any excess ferritin-bound iron retained by the enterocytes is disposed of
when the enterocytes are sloughed off into the gut lumen.

Dietary iron that is ingested as heme is taken up by a distinct mechanism. Following absorption
by the enterocytes, iron is released from heme by the enzymatic action of heme oxygenase.
Once released, the iron is either stored in association with ferritin or transported into circulation
by ferroportin.

For nonheme iron transport in enterocytes in the intestinal lumen, referring to Figure 5, Ferric
iron (Fe+3) is reduced to the ferrous form (or Fe+2) by a luminal ferrireductase, duodenal
cytochrome b (Dcytb). Ferrous iron is then transported into the enterocyte via divalent metal
transporter-1 (DMT-1). Within the enterocyte, iron is either stored as ferritin, or
transported out of the cell by ferroportin (Fp). Ferrous iron can also be converted back to
its ferric form by hephaestin. The ferric iron will then be bound by transferrin for transport
by the blood to various sites in the body.

Figure 5. Nonheme iron transport in enterocytes

The extreme toxicity of free iron is largely a consequence of its ability to catalyze the
formation of damaging reactive oxygen species (Figure 2xx). Iron in the free state can react
with hydrogen peroxide which will result in a temporary product – HYDROXY RADICAL.
Hydroxy radical is the most reactive among the ROS and can cause extensive tissue damage.

Figure 6. The Fenton Reaction

Biological organisms minimize the potential toxicity of iron by employing specialized

storage and transport proteins (refer to Figure 6). In humans, iron is transported through
the body tightly bound to the plasma protein transferrin (Tf). This β1-globulin has a
molecular mass of approximately 76 kDa and contains two high-affinity binding sites for Fe3+.
The form of the protein in which both sites are occupied is called holotransferrin (Tf- Fe).
Transferrin is a glycoprotein that is synthesized in the liver. The concentration of Tf in plasma is
approximately 300 mg/dL, sufficient to carry a total of approximately 300 μg of iron per deciliter
of plasma. This figure represents the total iron-binding capacity (TIBC) of plasma (Figure 2xx).
The binding sites in transferrin are not normally fully occupied, or saturated. Typically, about
30% of the iron binding sites in transferrin are occupied. Saturation can decrease to less than
16% during severe iron deficiency and may increase to more than 45% in iron overload
conditions. Thus, due to transferrin’s ability to bind with free iron, free iron can now travel
by the blood and be distributed to the different parts of the body. This reduces free iron
accumulation and limits its ability to catalyze ROS, which is the reason for its toxicity.

Glycosylation of transferrin is impaired in congenital disorders of glycosylation as well as in

chronic alcoholism. The presence of carbohydrate-deficient transferrin (CDT), which can be
measured by isoelectric focussing (IEF), is used as a biomarker of chronic alcoholism.

6. How do neutrophils which are formed elements in the blood reach the site of infections that
are extravascular? Explain the 4 cardinal signs of inflammation : heat, pain, redness and loss of
function. Briefly describe the different mechanisms how neutrophils kill invading
microorganisms.

Leukocytes Migrate in response to Chemical Signals

Leukocytes can be found throughout the body, migrating from the blood to sites of infection in
response to chemical signals, a process referred to as chemotaxis. This takes place via a
stepwise, amoeboid mechanism. Impelled by the proteins of the cytoskeleton, the white blood
cell extends a projection, called a pseudopod. Once the pseudopod anchors itself, the
cytoskeletal proteins associated with the main body of the cell contract, squeezing the cell’s
contents toward and into the pseudopod. The pseudopod fills with cytoplasm and organelles,
forming a new, translocated cell body. The deflated remains of the old cell body are absorbed
and a new pseudopod extends to initiate the next step.

White blood cells begin their migration from the bloodstream into the surrounding tissues by
squeezing through capillary walls. Called diapedesis, this process mirrors amoeboid motion in
its reliance on the ability of cytoskeletal proteins to dramatically contort the shape of the
leukocyte. The process begins with the extension of a thin, pseudopod-like projection between
the cells that comprise the capillary epithelium. As with amoeboid motion, the contents of the
cell then are squeezed through the narrow passage formed by the projection into the distal end,
which fills to form a new cell body on the opposite side of the capillary wall.
 Figure 7. Diapedesis

Chemotaxis Is Mediated By G-protein Coupled receptors

Leukocytes are attracted into tissues by chemotactic factors that include chemokines,
complement fragment C5a, small peptides derived from bacteria (eg, N-formyl-methionyl-
leucyl-phenylalanine), and a number of leukotrienes. The binding of these factors to specific
cell-surface receptors activates a signal transduction cascade similar to that which mediates
activation of platelets. Both cascades are initiated by ligand binding to receptors containing
seven membrane-spanning domains that are closely coupled with heterotrimeric guanosine
nucleotide binding proteins (G proteins). The G proteins activate phospholipase C, which
hydrolyses phosphatidylinositol 4,5-bisphosphate to produce diacylglycerols and the water sol-
uble second messenger inositol 1,4,5-triphosphate (IP3). The appearance of IP3 triggers the
release of Ca2+, leading to a tran- sient increase in the level of cytoplasmic Ca2+. In
neutrophils, the appearance of cytoplasmic Ca2+ activates the components of the actin–myosin
cytoskeleton responsible for effecting cell migration and granule secretion. Diacylglycerol,
together with Ca2+, stimulates protein kinase C and induces its translocation from the cytosol to
the plasma membrane, where it catalyzes the phosphorylation of various proteins, including
some involved in triggering the respiratory burst.

Chemokines are Stabilized by Disulfide Bonds

Chemokines are small, generally 6 to 10 kDa, proteins secreted by activated white blood cells
that attract additional leukocytes to a site of infection or injury. Chemokines can be divided into
four subclasses based on the number and spacing of the cysteine residues that participate in
the formation of the disulfide bonds that stabilize the protein’s conformation. Type C
chemokines are characterized by the presence of a pair of conserved cysteine residues that
form an intrachain disulfide bond. In addition to the conserved disulfide bond present in type C,
the other three recognized chemokine groups possess a second disulfide bond. In type CC
chemokines, one of the additional cysteine residues lies adjacent to the first of the first pair of
universally conserved residues. In types CXC and CX3C, these cysteines are separated by one
and three intervening amino acid residues, respectively. CX3C chemokines, the largest of the
four types of cytokines, have a longer C-terminus that includes sites of covalent modification by
glycosylation.

Integrins Facilitate Diapedesis

The adhesion of leukocytes to vascular endothelial cells is mediated by transmembrane

glycoproteins of the integrin and selectin families. Integrins consist of an α and a β subunit
linked noncovalently. Each subunit contains extracellular, transmembrane, and intracellular
segments. The extracellular segments bind to various cell surface proteins that possess Arg-
Gly-Asp sequences (eg, several components of the extracellular matrix). The intracellular
domains bind to various proteins of the cytoskeleton, such as actin and vinculin. Integrins help
to integrate leukocyte responses (eg, movement and phagocytosis) to changes in the
environment by virtue of their ability to link the outsides of cells to their insides via their dual
binding domains. Some inte- grins of specific interest with regard to neutrophils are listed in
Table 3. Type 1 leukocyte adhesion deficiency is caused by a lack of the β2 subunit (also
designated CD18) of LFA-1 and of two related integrins found in neutrophils and macrophages,
Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18). The loss of these proteins impairs the ability
of the affected leukocytes to adhere to endothelial cells, the first step in diapedesis. Since fewer
white blood cells enter their infected tissues, affected individuals tend to suffer from recurrent
bacterial and fungal infections.

Table 3. Principal Integrin of White Blood Cells and of Platelets

Acute Inflammatory Response and the 4 Cardinal Signs

Acute inflammation is the early (almost immediate) response of a tissue to injury. It is

nonspecific and may be evoked by any injury short of one that is immediately lethal. Acute
inflammation may be regarded as the first line of defense against injury and is characterized by
changes in the microcirculation: exudation of fluid and emigration of leukocytes from blood
vessels to the area of injury. Acute inflammation is typically of short duration, occurring before
the immune response becomes established, and it is aimed primarily at removing the injurious
agent.

Until the late 18th century, acute inflammation was regarded as a disease. John Hunter (1728–
1793, London surgeon and anatomist) was the first to realize that acute inflammation was a
response to injury that was generally beneficial to the host: “But if inflammation develops,
regardless of the cause, still it is an effort whose purpose is to restore the parts to their natural
functions.”

Clinically, acute inflammation is characterized by 5 cardinal signs: rubor (redness), calor

(increased heat), tumor (swelling), dolor (pain), and functio laesa (loss of function). The first four
were described by Celsus; the fifth was a later addition by Virchow in the nineteenth century.
Redness and heat are due to increased blood flow to the inflamed area; swelling is due to
accumulation of fluid; pain is due to release of chemicals that stimulate nerve endings; and loss
of function is due to a combination of factors. These signs are manifested when acute
inflammation occurs on the surface of the body, but not all of them will be apparent in acute
inflammation of internal organs. Pain occurs only when there are appropriate sensory nerve
endings in the inflamed site—for example, acute inflammation of the lung (pneumonia) does not
cause pain unless the inflammation involves the parietal pleura, where there are pain-sensitive
nerve endings. The increased heat of inflamed skin is due to the entry of a large amount of
blood at body core temperature into the normally cooler skin. When inflammation occurs
internally—where tissue is normally at body core temperature—no increase in heat is apparent.

Microcirculatory Response with Regards the Cardinal Signs

Vasodilation and Stasis. The first change in the microcirculation is a transient and insignificant
vasoconstriction, which is then followed by marked, active dilation of arterioles, capillaries, and
venules. This vasodilation causes an initial marked increase in blood in the area (hyperemia).
Subsequently, as fluid is lost into the exudate, stasis may supervene, with very sluggish blood
flow.

Increased Permeability. The permeability of capillaries and venules is a function of the

intercellular junctions between vascular endothelial cells. These pores normally permit the
passage of small molecules (MW < 40,000). Pinocytosis permits selective transfer of larger
molecules across the capillary into the interstitium. In normal capillaries, fluid passes out of the
microcirculation and into tissues under the influence of capillary hydrostatic pressure—and
returns because of plasma colloid osmotic pressure. Normally, fluid that passes out of the
microcirculation is an ultrafiltrate of plasma. In acute inflammation, there is an immediate (but
reversible) marked increase in the permeability of venules and capillaries due to active
contraction of actin filaments in endothelial cells. The effect is separation of intercellular
junctions from one another (widening of the pores). Direct damage to the endothelial cells by the
noxious agent may also contribute. Increased amounts of fluid and high-molecular-weight
proteins are able to pass through these abnormally permeable vessels.

Increase in permeability in acute inflammation occurs in several phases, principally an

immediate phase and a sustained or delayed phase. These permeability changes are effected
by various chemical mediators.

Exudation of Fluid. The passage of a large amount of fluid from the circulation into the
interstitial tissue produces swelling, one of the major features of acute inflammation. Increased
passage of fluid out of the microcirculation because of increased vascular permeability is termed
exudation. The composition of an exudate approaches that of plasma; it is rich in plasma
proteins, including immunoglobulins, complement, and fibrinogen, because the abnormally
permeable endothelium no longer prevents passage of these large molecules. Fibrinogen in an
acute inflammatory exudate is rapidly converted to fibrin by tissue thromboplastins. Fibrin can
be recognized microscopically in an exudate as pink strands or clumps. Grossly, fibrin is most
easily seen on an acutely inflamed serosal surface that changes from its normal shiny
appearance to a rough, yellowish bread and butter-like surface, covered by fibrin and
coagulated proteins. Exudation should be distinguished from transudation. Transudation
denotes increased passage of fluid into tissues through vessels of normal permeability. The
force that causes outward passage of fluid from the microcirculation into the tissues is either
increased hydrostatic pressure or decreased plasma colloid osmotic pressure. A transudate has
a composition similar to that of an ultrafiltrate of plasma. In clinical practice, identification of
edema fluid as a transudate or an exudate is of considerable diagnostic value because it
provides clues to the cause of the disorder, eg, examination of peritoneal (ascites) fluid.
Exudation helps combat the offending agent (1) by diluting it; (2) by causing increased lymphatic
flow; and (3) by flooding the area with plasma, which contains numerous defensive proteins
such as immunoglobulins and complement. The increased lymphatic drainage conveys noxious
agents to the draining lymph nodes, thereby facilitating a protective immune response.
Occasionally, with virulent organisms, the lymphatics may inadvertently promote spread and
may actually themselves become inflamed (lymphangitis), together with the lymph nodes.

Respiratory Burst and Enzymes involved

Respiratory burst (RB) is a rapid increase in the production of reactive oxygen species (ROS)
like superoxide anion and hydrogen peroxide during the phagocytosis of microbes. Usually it
denotes the release of these chemicals from immune cells, such as neutrophils and
macrophages because they are infected by different bacteria or fungi. On the one hand, RB is
an essential component of innate immunity enabling phagocytic cells to eliminate microbes.
However, excessive production of ROS may result in the induction of oxidative stress in cells.
Both pathogenic bacteria and viruses can induce oxidative stress in host cells during infection.

There are also enzymes involved in infection response which give the neutrophils its
microorganism-killing capability. A phagocyte can kill a microorganism in different ways such as
through the production of ROS, antimicrobial peptides (defensin, cathepsin), and hydrolytic
enzymes like proteases, MPO, and lysozyme.

ROS production

There is an increase in oxygen consumption by phagocytic cells upon engulfment of bacteria.

The purpose of this oxygen consumption is to produce large amounts of microbicidal ROS
through NADPH oxidase. Superoxide anion, Hydrogen peroxide, Hydroxide, and Hypochlorite
are the most common products of microbicidal ROS. Production of this species happens via the
Electron Transport Chain system wherein NADPH oxidase system (cyt b558), a flavoprotein,
generates superoxide (ETC via reduction of oxygen by one electron) and H2O2.

Superoxide dismutase protects the cell from superoxide that may escape from phagosomes by
catalyzing the transformation of 2 superoxide radical anions into H2O2 and O. NADPH oxidase
will utilize O2 to produce superoxide. NADPH needed in this reaction comes from the pentose
phosphate pathway. The superoxide dismutase reaction converts superoxide to H2O2 which is
already bactericidal. H2O2 that escapes from the phagolysosome is potentially harmful but is
usually taken care of by catalase and glutathione peroxidase. H2O2 reacts with Cl- through the
reaction catalyzed by myeloperoxidase producing Hypochlorous acid (HOCl), the chief
microbicidal agent in phagolysosomes. Hypochlorites are mixed with amines to produce
chloramines which are less irritating but still microbicidal. With the help of cathepsins, defensins,
and increased pH, these will all ensure the death of the bacteria that results in arresting
inflammation.

Myeloperoxidase (MPO)

Myeloperoxidase (MPO) is present in neutrophil granules. It acts on H2O2 with chloride to

produce HYPOHALOUS ACIDS (HOCl) which can be determined as the reason for greenish
coloration of the pus. Hypochlorite (HOCL) is an oxidizing agent, and microbicidal that
stimulates proteinases. In tissues, HOCl reacts with the primary or secondary amine to produce
Chloramines. Chloramines are oxidizing agents and microbicidal without causing tissue
damage.

Proteinases

Proteinases are lysosomal enzymes in neutrophils that is capable of hydrolyzing elastin by

elastase, collagen by collagenase, and proteins of the ECM. It is activated by HOCl and affects
the equilibrium between proteinases and anti-proteinases. Normally there is a balance between
them. Usually inactive in the absence of inflammation but become active when an infection is
present. If left unchecked, they can cause much tissue damage even as they try to destroy the
microbial protein. Anti-proteinases can neutralize and prevent excessive tissue damage, and it
is inactivated by HOCl. Α deficiency of Α1-antitrypsin, an anti-proteinases, results in unopposed
elastase activity which digests pulmonary tissue resulting in emphysema. Α2-Mactoglobulin is a
plasma protein that defends against excessive action of proteases by combining and
neutralizing them. A deficiency or any damage of these sample anti-proteinases can imbalance
proteinases which can result in tissue damage or destruction.

7. Briefly describe prehepatic, hepatic and posthepatic hyperbilirubinemia and the type of
bilirubin you expect to be elevated in each of these conditions. When do you develop choluric
and acholuric jaundice? What is kernicterus and what causes it?

Hyperbilirubinemia is a blood level of bilirubin that exceeds 1 mg of bilirubin per dL (17 μmol/L)
which may result from:

1. production of more bilirubin than the normal liver can excrete, also known as
overproduction of bilirubin (retention hyperbilirubinemia)
2. failure of a damaged liver to excrete normal amounts of bilirubin
3. obstruction of the excretory ducts of the liver in the absence of hepatic damage which
may cause reflux to bloodstream (regurgitation hyperbilirubinemia)

In prehepatic hyperbilirubinemia serum bilirubin are elevated due to overproduction

of bilirubin in the bloodstream. Massive lysis of red blood cells (for example, in patients with
sickle cell anemia, pyruvate kinase or glucose 6-phosphate dehydrogenase deficiency) may
produce bilirubin faster than it can be conjugated. Unconjugated bilirubin (indirect
bilirubin) levels in the blood become elevated, causing jaundice. More conjugated bilirubin is
excreted into the bile, the amount of urobilinogen entering the enterohepatic circulation is
increased, and urinary urobilinogen is increased.

Hepatic hyperbilirubinemia is primarily due to decreased conjugation of bilirubin

resulting from damage to liver cells. Urobilinogen is increased in the urine because hepatic
damage decreases the enterohepatic circulation of this compound, allowing more to enter the
blood, from which it is filtered into the urine. The urine thus darkens, whereas stools may be a
pale, clay color. Plasma levels of AST and ALT are elevated. If conjugated bilirubin is not
efficiently secreted from the liver into bile (intrahepatic cholestasis), it can diffuse (“leak”) into
the blood, causing a conjugated hyperbilirubinemia. In this condition conjugated (direct) and
unconjugated (indirect) bilirubin can both be elevated.

Post hepatic bilirubinemia refers to events in the biliary tree, for which the major
causes are obstruction of the common bile duct by a gallstone (biliary calculus) or by cancer
of the head of the pancreas. Patients with obstructive jaundice experience gastrointestinal pain
and nausea, and produce stools that are a pale, clay color, and urine that darkens upon
standing. The liver “regurgitates'' conjugated bilirubin (direct) into the blood increasing its
serum level. The compound is eventually excreted in the urine. Urinary urobilinogen is absent.

To differentiate the types of hyperbilirubinemia based on affected part of heme

catabolism here is a table with their corresponding laboratory results:

Table 4. Laboratory results in normal patients and patients with three different causes of
jaundice [Rodwell, 2018]

Condition Serum Urine Urine Bilirubin Fecal

Bilirubin urobilinogen Urobilinogen

Normal Direct: 0.1–0.4 0-4 mg/24 hr Absent 40-280 mg/24 hr

mg/dL
Indirect: 0.2-0.7
mg/dL

Hemolytic ↑ Indirect Increased Absent Increased

Anemia
(e.g of prehepatic
hyperbilirubinemi
a)

Hepatitis ↑ Direct and Decreased if Present if micro Decreased

(e.g of hepatic Indirect micro- obstruction is
hyperbilirubinemi obstruction is present
a) present

Obstructive ↑ Direct Absent Present Trace to absent

Jaundice (e.g of
post hepatic
hyperbilirubinemi
 a)

Direct bilirubin is also conjugated bilirubin. In aqueous solution, the water soluble,
conjugated bilirubin reacts rapidly with the reagent (within one minute), and is said to be “direct-
reacting”.

Indirect bilirubin is also unconjugated bilirubin which is much less soluble in aqueous
solution, & reacts more slowly. However, when the reaction is carried out in methanol, both
conjugated and unconjugated bilirubin are soluble and react with the reagent, providing the total
bilirubin value. The “indirect-reacting” bilirubin is obtained by subtracting the direct-reacting
bilirubin from the total bilirubin.

Since there are two forms of bilirubin in the body, it should be noted that bilirubin should
be conjugated since its hydrophobic unconjugated form can cross the brain-blood barrier.
In retention hyperbilirubinemia unconjugated bilirubin is elevated and encephalopathy due to
hyperbilirubinemia (kernicterus) and acholuric jaundice can occur. It is acholuric since
unconjugated bilirubin is not soluble in urine. Note that in retention hyperbilirubinemia the
problem is in the bloodstream with overproduction of bilirubin.

Alternatively, in regurgitation bilirubinemia wherein hydrophilic conjugated bilirubin is

elevated, choluric jaundice occurs. Conjugated bilirubin is the only form of bilirubin that can be
present in urine because it is polar. Choluria is the presence of bile pigments in urine. Note that
in regurgitation bilirubinemia, the problem is in the obstructed bile duct which impedes excretion
of conjugated bilirubin from the liver.

Heme catabolism

The types of hyperbilirubinemia can be explained by understanding how the normal

steps in heme catabolism can be interrupted precipitating elevated bilirubin levels.The first step
in the degradation of heme is catalyzed by the microsomal heme oxygenase system of the
reticuloendothelial cells. The iron of the heme that reaches heme oxygenase has usually been
oxidized to its ferric form (hemin). Conversion of one mole of heme-Fe3+ to biliverdin, carbon
monoxide, and Fe3+ consumes three moles of O 2, plus seven electrons provided by NADH
and NADPH–cytochrome P450 reductase. In humans, biliverdin reductase reduces the
central methylene bridge of biliverdin to a methyl group, producing the yellow-pigment bilirubin.
Unlike bilirubin, which is only sparingly water soluble, bilirubin bound to serum albumin is
readily transported to the liver.
 Figure 8. Formation of bilirubin from heme [Harvey, 2011]

Hepatic catabolism of bilirubin takes place in three stages: uptake by the liver,
conjugation with glucuronic acid, and secretion in the bile. Bilirubin dissociates from the
carrier albumin molecule, enters a hepatocyte via facilitated diffusion, and binds to intracellular
proteins, particularly the protein ligandin (glutathione S-transferase). Bilirubin is nonpolar,
and would persist in cells (eg, bound to lipids) if not converted to a more water-soluble form thus
it is converted to a more polar molecule by conjugation with glucuronic acid. A bilirubin-specific
UDP-glucuronosyltransferase of the endoplasmic reticulum catalyzes stepwise transfer to
bilirubin of two glucosyl moieties from UDP-glucuronate. Secretion of conjugated bilirubin
into the bile occurs by an active transport mechanism, which probably is rate-limiting for the
entire process of hepatic bilirubin metabolism. The protein involved is a multispecific organic
anion transporter (MOAT) located in the plasma membrane of the bile canaliculi. Note that
unconjugated bilirubin is normally not secreted. Lastly, bilirubin diglucuronide is hydrolyzed and
reduced by bacteria in the gut to yield urobilinogen, a colorless compound. Most of the
urobilinogen is oxidized by intestinal bacteria to stercobilin, which gives feces the characteristic
brown color. However, some of the urobilinogen is reabsorbed from the gut and enters the
portal blood. A portion of this urobilinogen participates in the enterohepatic urobilinogen cycle
in which it is taken up by the liver, and then re-secreted into the bile. The remainder of the
urobilinogen is transported by the blood to the kidney, where it is converted to yellow urobilin
and excreted, giving urine its characteristic color.
 Figure 9. Summary of heme catabolism [Harvey, 2011]

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