Study Guide for Exam III
Clinical Chemistry I, CHM 651/751
A. Anatomy and Function of Liver
1. Know what the location and/or definition of the following terms are: superior, inferior,
anterior, posterior, visceral, peritoneum, ligament, liver lobe, liver lobule, central vein,
sinusoid, bile canaliculi, Kupffer cell, reticuloendothelial cell, hepatocyte, portal triad,
gallbladder, hepatic artery, portal vein, hepatic vein, lysosome, peroxisome
2. Know the following pathway from the bile canaliculi to the intestines:
canaliculi →intralobular ducts → interlobular bile duct → right (or left) hepatic duct →
common hepatic duct
↓
cystic duct
↓
gallbladder
common bile duct→ ampulla of vater →duodenum
↑
pancreatic duct
3. Know the sources of blood entering the liver:
a) portal vein (from digestive organs, 2/3 of blood entering the liver)
b) hepatic artery (from aorta, thus oxygenated, 1/3 of blood entering the liver)
4. Know that the blood exits the liver through the hepatic vein which enters the vena cava
which empties into the heart
5.
What are the four components of the liver lobule?
 central vein
 hepatocytes radiating from the central vein
 sinusoids
 bile canaliculi
6.
Know what the blood flow rate through the liver is (1/3 cardiac output) and the bile
output (600 to 800 mL/day)
7.
Know the following facts of the reticulo-endothelial (also called macrophage) system:
a) Development of reticulo-endothelial system

The precursor to the macrophage is called the monocyte

The development of the monocyte from stem cells goes through several
differentiation cell stages which occur in the bone marrow. The last stage
before the mature macrophage stage is the monocytes which are in the
bone marrow and blood.

The monocytes fully mature to macrophages in the bone marrow. In the
blood the macrophages settle into several tissues [such as the liver (the
largest proportion of RE cells in the body, 60%), spleen, lymph nodes,
others]
b) Degradation process
 Macrophages ingest antibody (or complement coated) antigens because
they have surface Fc receptors (which bind antibodies) and C3 receptors
(which binds C3b protein in the complement pathway)
1


These bound complexes are taken into the cell through a process called
endocytosis
Once inside the macrophage the vesicle containing component to be
degraded fuse with lysosomes (which contain many different hydrolytic
enzymes) and/or peroxisomes (which contain oxidizing enzymes) which
degrade the protein or other ingested component
c) Storage
Reticuloendothelial cells serve as a site to store chemical components. For
example it is the tissue store cell for iron
8. Know the two type of cells that line the sinusoids [endothelial cell and reticulo-endothelial
cell (Kupfffer Cell)]. Also know that the the space between the cells is large (intercellular
clefts) thus facilitating the liver’s secretion (such as plasma proteins) and chemical processing
role.
9. Liver is the principle organ of energy production for the body in the following way:
a) Carbohydrate metabolism – maintains blood glucose level
1’) Glycogen
 Storage of glucose by formation of glucose polymer (glycogen) when
amount of glucose is in excess of energy needs. Glycogen is in the
form 100-400 Ǻ diameter granules stored in hepatocytes
 When glucose levels in blood are low glycogen is broken down to
glucose-6-phophate and which is further hydrolysed to glucose to
restore blood glucose
 The two sites of glycogen storage are the liver and the muscles.
Glycogen in the muscle is used for the energy needs of the muscle
only (muscle cells lack the enzyme glucose-6-phosphatase thus
glucose -6posphate remains in the cell to be used for energy
production). Glycogen in the liver is used for the body’s energy needs
(12 hour supply of glucose under fasting conditions) as it maintains the
blood glucose level
2’) Gluconeogenesis
 Process occurs mostly in the liver (some occurs also in the kidney)
 This is the other process that maintains the blood glucose level. It is
the way that the body maintains blood glucose after the glycogen
stores in the liver are exhausted.
 It is the synthesis of glucose from non-carbohydrate sources.
Specifically, amino acids from protein breakdown, and glycerol from
triglycerides, are converted to oxaloacetate (which is in the citric acid
cycle) which is then used to synthesize glucose
 Oxaloacetate is synthesized from amino acids
 Certain amino acids are converted to pyruvate (of the
glycolytic pathway) which is then converted to
oxalolactate
 Other amino acids can be converted to various
intermediates of the citric acid cycle which are eventually
converted to oxaloacetate in the citric acid cycle
2

Oxaloacetate can also be synthesized from glycerol released from the
hydrolysis of triglyceride. The glycerol enters the glycolytic pathway,
which then is converted to pyruvate which is then converted to
oxaloacetate
 Note: you need to know only the information above on
gluconeogensis, you DO NOT need to know the steps of glycolysis
nor the steps of the citric acid cycle, nor the chemical structures of the
intermediates in these pathways, nor what particular amino acids enter
the pathways at what point
3’) Other comments
 The brain and red blood cells use glucose as its exclusive energy
source (although the brain does use ketone bodies as an energy source
in conditions of starvation) (Fatty acids cannot pass the blood brain
barrier for processing for energy
b) Lipid metabolism
1’) General comments about lipid metabolism not related to the liver. Note: the
specifics about the general comments given below for point b.1’ you DO NOT
need to know, except for 1) how bile aids in the digestion of fat 2) what the
general formula for a triglyceride, glycerol and fatty acid are, and 3) that the
breakdown product of fatty acid oxidation is acetyl-CoA which enter the
citric acid cycle for energy generation.. This is provided for context
understanding of the place of the liver in lipid metabolism, which you will
need to know
 83% of the stored energy of the body is stored as lipids
 The chemical form of stored lipids is triglyceride [know the general
chemical formula of a triglyceride, which consists of a glycerol group
(know this chemical structure) and three fatty acid groups (know the
general chemical formula for a fatty acid group)]
 Triglycerides are stored in adipose tissue [consisting of adipocytes (or fat
cells)]
 Lipases act on triglycerides to hydrolyze triglycerides to its component
parts glycerol and fatty acids. It is the fatty acid component which
contains the most energy, which is broken down to acetyl-CoA, which is
then utilized in the citric acid cycle and electron transport to generate
energy This process occurs in all cells except the brain.
 Energy utilization by muscles: glucose is primary source with rapid
contraction of muscle, but with sustained exercise the glucose supply is
exhausted and fatty acids and ketone bodies take over as the principal
source
 The process of lipid transport and storage after digestion of a meal
 Lipases released into the duodenum from the pancreas to
hydrolyze triglycerides. Bile is important in the process because it
emulsifies the triglycerides so that the hydrolysis reaction can
occur more efficiently as large globules of triglycerides are broken
up into small droplets.
3
 Bile salts are also important for forming miscelles in the intestinal
lumen promoting the absorption of fatty acids by epithelial cells of
the duodenum
 Fatty acids are then reesterfied with glycerol to triglycerides in the
epithelial cell and then are packaged with proteins, cholesterol,
phospholipids as chylomicrons
 Chylomicrons go into the lymphatics and then into the blood
 From the blood the fatty acids are taken up principally by adipose
cells and the liver (for further packaging as other lipoproteins, for
further transport through the body). Other organs also take fatty
acids into the cells from chylomicrons (liberated from TG by
lipases present on the tissue cell’s membrane)
 Fatty acid metabolism to acetyl-CoA is an oxidation process (βoxidation). It requires oxygen.
 Acetyl-CoA is also synthesized from pyruvate meaning that it is
synthesized from the breakdown of glucose as well as from amino
acids that can be converted to pyruvate
 The exception to the above points in lipid transport is small chain
fatty acids (2 to 10 carbons) are directly absorbed through the
epithelia cells into the blood system where it is bound by albumin
to be transported to the liver
2’) Lipid processes involving the liver
a’) A large percentage of the initial metabolism of fatty acids occurs in the
liver (60%)
b’) The liver is the principal site of synthesis of cholesterol, triglyceride,
phospholipids, and lipoproteins
c’) The synthesis of ketone bodies from acetyl-CoA occurs exclusively in
the liver. Know the following:

Know the identity of ketone bodies: acetoacetate, acetone, βhydroxybutyrate and the reaction pathway

Know that ketone bodies are a normally utilized by muscle cells
(along with metabolism of fatty acids) when glucose is low

In cases of starvation or untreated diabetes the brain utilizes ketone
bodies for energy since it is hydrophilic enough to not require a
transport protein

Acetoacetate can be directly used as an energy source.
Acetoacetate and β-hydroxybutyrate are also converted back to
acetyl-CoA for utilization in the citric acid and electron transport
pathways for energy generation

In untreated type I diabetes and starvation increased fatty acid
breakdown occurs because of diminished glucose availability
leading to excess acetyl-CoA synthesis. This excess is shifted to
ketone body synthesis because gluconeogensis consumes all the
oxaloacetate meaning that the acetyl-CoA cannot be utilized for
energy production in the citric acid cycle. This process leads to the
pathologic condition called ketoacidosis, with a lowering of blood
4
pH due to the acidic ketone bodies and acetone breath. Βhydroxybutyrate is a marker for uncontrolled diabetes.
10. Other processes which occur predominantly in the liver
a) Ammonia from the degradation of amino acids is converted to urea (urea less
toxic than ammonia)
b) Conjugation of bilirubin
c) Synthesis of bile acid from cholesterol (80% of the cholesterol synthesized is used
for this purpose)
d) The majority of the main serum proteins are synthesized by the liver, except for
immunoglobulins
e) The liver is the principal place of biotransformation of exogenous (drugs and
toxins) and endogenous compounds (such as steroid hormones). These reactions
take place to allow the body to be able to excrete the compound by diminishing
their intestinal and renal tubular reabsorption The process normally inactivates the
compound (or makes the compound less toxic) However in some instances it can
make the compound more toxic. Many different types of reactions can take place
promoted by specific enzymes. Cytochrome P-450 is the most important oxidases
(promoted oxidation). The conjuagation reaction (with glucoronic acid, sulfate)
makes the compound soluble so that it can be excreted.
f) 33% of the iron of the body is stored in the Kupffer cells
g) 90% of the Vitamin A of the body is stored in the Kupffer cells
11. In severe long standing liver disease:
a) Decreased serum proteins are noted (except for IgG which is increased because
IgG is not synthesized by the liver and the liver is not degrading antigens because
of its decreased function leading to increase in IgG)
b) Prealbumin is a better short-term marker of liver status than albumin because it
has a shorter half-life (2 days compared 2 weeks)
c) Drug dosage has to be decreased because there is less of an ability of the liver to
metabolize the drug and thus the active drug has a longer circulation lifetime
d) Coagulation time is increased (Need to know the two reasons for this – see
chapter notes)
5
B. Bilirubin Formation and Metabolism
1. Know the degradation pathway of heme:
a) Reactions that occur in reticuloendothelial (RE) cells of spleen: (also can occur in RE cells
of liver:
globin
dissociation
+
Hemoglobin
→
heme
(CO + Fe3+)
heme oxygenase
+
biliverdin reductase
→
biliverdin
→
bilirubin
3O2 + 5NADPH
b) Reaction that occurs in hepatocyte
glucuronyl transferase
glucuronyl transferase
Bilirubin
→
bilirubin monoglucuronide
→
bilirubin diglucuronide
UDP glucurononic acid
UDP glucuronic acid
c) Reaction in ileum (last part of small intestine) and colon
Bilirubin diglucuronide
2 glucuronic acids
Β-glucuronidase
+
→
bilirubin
→
urobilinogens
d) Reaction in colon
urobilinogens →
urobilins
2. Know the following general features about the structure of porphyrin: 4 pyroles, 4 methylene
bridges (know these structures)
3. Know that protoporphyrin has the general porphyrin structure + the following attached to 8
available positions on the 4 pyroles: 4 methyl groups, 2 vinyl groups, 2 propionic acid groups
(know these structures)
4. Know that the only natural occurring porphyrin is protoporphyrin IX, which is one of the 15
ways to arrange the side chains given in point 3
5. Know that protoheme consists protoporphyrin IX and Fe2+
6. Note: Do not need to know point 6 for this test Given the structure of protoporphyrin IX
and glucoronic acid (do not need to memorize it, will be given to you) be able to identify:
a) Identify the methylene bridge that is broken by the oxidation reaction to convert
heme to biliverdin (i.e. be able to identify the α bridge)
b) Identify the chemical change on the pyrole and the opening up of the cyclic
structure in going from heme to biliverdin
c) Identify the exact chemical change (the one main reduction reaction) in going
from biliverdin to bilirubin
d) Identify the place and the chemical structure of the attached glucuronic acids
e) Identify the various places of reduction (in which 2H are added) in going from
bilirubin to the various urobilinogens (Note: you do not have to know what
changes correspond to any one urobilinogen)
6
f) Identify the place of oxidation (the place where two H are taken away) in going
from urobilinogen to the corresponding urobilin
g) Identify the double bonds that change from Z to E confirmation with light, freeing
the propionic acid groups from the intramolecular H bonds making bilirubin
soluble after light treatment (Z confirmation around the double bond is the
following: the groups of highest molecular weight attached to the double bond are
on the same side of the double bond. E confirmation around the double bond is
the following: the groups of highest molecular weight attached to the double bond
are on opposite side of the double bond).
7. Identify the type of chemical reaction in going from:
a) Heme → biliverdin (oxidation)
b) Biliverdin → bilirubin (reduction)
c) Bilirubin → bilirubin glucuronide (conjuagation)
d) Bilirubin glucuronide→bilirubin (deconjugation)
e) Bilirubin →urobilinogen (reduction)
f) Urobilinogen →urobilin (oxidation)
8. Know that heme is not stable unless attached to a protein. It is most often attached through a
coordinate bond (not as strong as a covalent bond) by an amino acid of the protein to the iron
There are 6 coordination positions of the Fe2+. Four are planar consisting of the four
nitrogens of heme, with the fifth and sixth being taken up by amino acids in the proteins (for
cytochromes) or by a histidine of the protein and carrier species, such as O2 and CO2, for
hemoglobin and myoglobin. Besides these proteins, another heme protein is catalase, which
is involved in H2O2 utilization.
9. Know that functional hemoglobin requires a reduced Fe (Fe2+) to be functional. Hemoglobin
with Fe3+ is not functional and is called methemoglobin and is normally present < 1% of the
hemoglobin in RBCs.
10. In bilirubin formation 80% is from breakdown of senescent (old) RBCs. RBCs have an
average lifetime of 120 days. The remaining 20% is called early labeled faraction is from the
breakdown of other heme proteins or defective red blood cells It is named early labeled
fraction because in experiments in which radioactive labeled reactants to heme synthesis (∆aminolevulinic acid and glycine) radioactivity was excreted early (3-5 days) compared to the
expected 120 days.
11. Know the five functions of the spleen:
a) Defense – major location of macrophages which ingest bacteria, micro-organisms,
parasites, antibody- bound antigens.
b) Blood cell production – produces non-granular cell leukocyte (white blood cells)
which are monocytes and lymphocytes
c) Blood reservoir – can either hold (upto 350 mL) or release blood (200 mL)
d) Immunologic – one of the places in the body in which antibodies are produced)
e) Destruction of senescent and iperfect blood cell components
12. Know that the spleen has tortuous network which contain a high concentration of
macrophages for blood to naviagate through, which presents very harsh conditions to the
blood and its cellular components as it navigates through it. The channel ways are 3 µm in
diameter (RBCS are 7 µm in diameter, which means RBCs must squeeze through passage
ways half their size). Old RBCs are less flexible and take minutes to hours (as opposed to
7
viable RBCs which take 30 sec) to get through this meshwork of narrow channels, meaning
they get significant exposure to the macrophages for eventual destruction
13. Know the following steps in bilirubin processing. Know which steps, when defective, lead to
unconjugated hyperbilirubinemia (steps a-e) and which, when defective, lead to conjugated
hyperbilirubinemia (step f).
a) Formation of bilirubin – occurs in spleen (to a smaller extent liver and bone
marrow) in the reticulendothelial cells principally (80%) from the breakdown of
RBCs
b) Delivery of bilirubin in blood- bound to albumin since unconjugated bilirubin is
not water soluble
c) Uptake of bilirubin by hepatocyte- through a 54K dalton carrier protein (place of
competition with particular organic anions, however, not bile acids). Although
this binding displays Michaelis Menton kinetics, the point of saturation is not
close to conditions that are present in health or hyperbilirubinemic conditions.
This is NOT a rate limiting step in bilirubin processing.
d) Storage of bilirubin by hepatocyte- bound to ligandin
e) Conjugation of bilirubin by hepatocyte – conjugation of bilirubin in the
endoplasmic reticulum of the hepatocyte (microsomes), with two glucuronic acid
groups via the enzyme glucuronic acid transferase. There is a remarkably high
reserve of this enzyme in the hepatocyte, meaning that even in high bilirubin
conditions presented by pathologic conditions, the bilirubin amount does not
exceed the hepatocyte’s capability to conjugate this bilirubin. The exception to
this is the neonate and congenital deficieciecies of the enzyme.
f) Excretion of conjugated bilirubin into bilecanaliculi – this is mediated by 100K
Dalton protein in the canalicular membrane, it is an active process (requires
energy) and is the rate limiting step in bilirubin processing after its formation
(after step a) Since it requires energy it is a place of susceptibility in conditions of
hypoxia, shock, circulatory insufficiency). Other organic anions use this 100K
transport protein for excretion in the bile (But not bile acids which has a different
system)
g) Catabolism of bilirubin in GI tract – in terminal ileum and colon, there is
deconjuagation of bilirubin by bacterial β-glucuronidase and reduction of
bilirubin to urobilinogens. Subsequently in the colon urobilinogens are oxidized
to their corresponding urobilins
h) Enterohepatic circulation of urobilinogens - 20% of formed urobilinogens are
resabsorbed and 90% is re-excreted in the bile. The remaining 10% enters the
general circulation which is how urobilinogen makes its way into the urine.
i) Elimination of urobilins through the feces
14. Know that unconjugated bilirubin is not soluble in aqueous solution because the propionic
acid groups that what normally make it soluble are tied up by 6 intramolecular H bonds.
(You DO NOT need to write this structure).
15. Know the various means by which bilirubin is made soluble in the body:
a) Unconjugated bilirubin requires binding proteins (albumin in blood, ligandin in
the hepatocyte)
b) Conjugation with glucuronic acid
8
c) Light treatment cause a change of the Z to the E confirmation of one or both of
the double bonds in bilirubin
16. Know the principle of distribution of bilirubin between the plasma and tissue components
depends on the concentration of binding proteins in the particular components (an increase in
binding protein concentration in a particular compartment will lead to a shift of bilirubin into
that compartment, conversely a decrease in binding protein concentration in a particular
compartment will lead to a shift of bilirubin out of that compartment into the other
compartment). Thus an increase in [albumin] in the blood will lead to an increase in
[bilirubin] in the blood. Remember that practically all bilirubin in the blood is bound to
albumin. A lab test of total bilirubin includes what is bound to albumin.
17. Also an increase in substances that displace bilirubin from the binding protein in a particular
compartment will lead to a shift of bilirubin out of that compartment into the other
compartment. As an example increase in fatty acids or other organic anions will displace
bilirubin from the albumin of blood and decrease the [bilirubin] in blood, even though it is
increasing the amount of free bilirubin (not bound to albumin).
18. Know that conjugated bilirubin also binds to albumin, however with a much weaker affinity
constant. This means that there is a higher amount of conjuagated bilirubin that is unbound
(0.6%) which can be filtered by the kidneys.
19. Know the two important aspects of the conjugation reaction of bilirubin:
a) It solubilizes bilirubin in aqueous solutions so it can be excreted into the bile
b) It impairs reabsorption of bilirubin from the intestinal tract and gallbladder
(because it makes bilirubin hydrophilic)
20. Know the effect of the following types of compounds on the activity of glucuronyl
transferases:
a) Microsomal enzyme inhibitors (best example, phenobarbital) causes an increase
in enzyme activity
b) Glucuronide forming compounds that use the same subset of glucuronyl
transferase enzymes (note: different compounds will use different subsets of the
enzyme) will compete with bilirubin for the enzyme and thus decrease its
capability of conjugating bilirubin
21. Know the three differences in bilirubin metabolism between neonates (newborns) and nonneonates:
a) Neonates have decreased levels of glucuronyl transferse, which is the main cause
of physiologic jaundice in the newborn
b) Neonates have decreased binding of unconjugated bilirubin to albumin due to an
unidentified chemical agent present in the blood which competes with bilirubin
for binding to albumin
c) Neonates do not have bacteria in GI tract and thus bilirubin is directly excreted
because of the absence of bacterial glucoronidase does not remove glucuronic
acid from conjugated bilirubin (This happens also with the administration of
broad spectrum of antibiotics)
22. Know that not all of the conjugated bilirubin entering the GI tract from bile is processed to
urobilinogen/urobilin. 50% of the conjugated bilirubin is converted to other unknown
metabolites.
9
23. Elevated urobilinogens/urobilin in feces indicates an overproduction of bilirubin (hemolytic
anemia). Note that it is not always elevated in these conditions because of the presence of the
alternate pathway given in point 22 above (also difficulty in its measurement).
24. Know that the concentration of urobilinogen/urobilin in serum is elevated in both liver
disease and hemolytic anemias.
25. Know the following:
a) Serum

Unconjugated – 96%

Conjugated – 4%
b) Bile
 Unconjugated – trace
 Conjugated – 100%
 Glucuronides – 95%
 Glucosides, Xylosides – 5%
C. Laboratory Measurement of Bilirubin
1. Know the following about the diazotized sulfanilic acid testing for bilirubin:
a)
Memorize the name and know that it is a colorimetric method in which a
diazonium group covaently attaches to the pyrole group on either side of the
middle methylene bridge of bilirubin, releasing the bridge carbon as a CO2
group . (This reagent reacts with both conjugated and unconjugated bilirubin,
however for the unconjugated bilirubin an accelerator has to be added to
solubilize the bilirubin, because the reagent will not react with the
unconjugated bilirubin bound to albumin)
b)
Know that the Jendrassik-Grof is the most utilized test diazotized sulfanilic
acid, that the reaction is done at pH 6.5, and the product is measured at pH 13
at 600 nm
c)
Total bilirubin (direct and indirect) is measured by adding an accelerator
(alcohol or benzoate/caffeine) to the assay mixture, which solubilizes the
unconjugated bilirubin (the conjugated fraction is already soluble in the assay
mixture)
d)
Direct bilirubin is determined by NOT adding accelerator to the assay mixture
e)
The indirect is determined by calculation, obtained by subtracting the direct
from the total
f)
Know that the designations of direct and indirect are terms that apply to what
is determined by the diazotized sulfanilic acid testing method:
 Direct – conjugated bilirubin and δ-bilirubin
 Indirect – unconjugated bilirubin
2. Know that the normal values for the direct bilirubin determined by the diazotized sulfanilic
acid method is ≤ 15% direct. This is higher than the 4% that is determined by more accurate
methods. The reason for this overestimate of the conjugated fraction by this colorimetric
method are:
 There are diazo-reacting substances in serum
 Some unconjugated bilirubin reacts, and the amount that reacts increases with
increased time and temperature of the assay
10
3. Know that a direct spectrophotometric technique is done for neonatal and amniotic samples
because these samples do not have caretenoids (which would absorb 454 nm light) or lipemia
(which scatters incident light which will register as an absorption) because the neonates have
not ingested food and these substances are not in amniotic fluid. The technique is a dual
wavelength technique making measurement at 454 nm (in which both bilirubin and
hemoglobin absorb) and 540 nm (in which only hemoglobin absorbs, having the same
absorptivity coefficient as hemoglobin at 454 nm). Thus the absorbance value can be
corrected for hemoglobin interference as given below:
Abilirubin = A454 nm – A540 nm
D. Hyperbilirubinemia – General Information
1.
2.
3.
4.
Know the definition of :
Hyperbirubinemia - bilirubin in serum exceeds 1 mg/dL
Jaundice – yellow pigmentation of skin and sclera (occurs when serum bilirubin > 2
mg/mL)
Kernicterus – condition of severe neural symptoms due to binding of bilirubin to
lipophilic components of the brain, leading to cell destruction and encephalopathy
(degenerative condition of the brain)
Know that hyperbilirubinemia is only a problem in the neonate in which there is
underdeveloped blood-brain barrier where unconjuagate bilirubin has access to neural
cells in the brain leading to their cell destruction and development of kernicterus.
Know what the laboratory results are for:
a) Unconjugated hyperbilirubinemia – elevated unconjugated bilirubin in serum (but
no more than 15 % direct)
b) Conjugated hyperbilirubinemia – elevated bilirubin in serum, both conjugated and
unconjugated , > 30% (usually > 50%) direct [reason for elevation of
unconjugated levels is that there is hydrolysis of the glucuronic acids by tissue
glucuronidases of a significant portion of the conjugated bilirubin that would be in
the blood, or for liver disease, there is a compromise of the hepatocyte in liver
disease which not only affects the excretion of conjugated bilirubin by the
hepatocyte into the bile (which increases the conjugated bilirubin backing up into
the blood) but also compromises the ability of the liver cell to uptake and storage
unconjugated bilirubin from the blood].
Know that for unconjugated hyperbilirubinemia there is NO bilirubin in the urine and for
conjugated bilirubin there is bilirubin present in the urine. No why this is the case [in
unconjugated hyperbilirubinemia most all of the bilirubin in the blood is unconjugated
bilirubin, which is tightly bound to albumin and thus NOT filtered in the glomerulus,
while in conjugated hyperbilirubinemia the conjugated bilirubin is weakly bound to
albumin, which means there is free conjugated bilirubin (not bound to albumin) present in
the blood that can be filtered, and once this is removed into the tubular system, more free
conjugated bilirubin dissociates from the albumin, which can then be filtered at the
glomerulus, and so forth].
11
E. Hemolytic Anemias – General Aspects
1. Define:
Hemolytic anemia – disorder that leads to premature breaking up of red blood cell
Anemia – reduction in the number of red blood cells in the blood that can occur by either one of
three causes: blood loss, hemolysis, diminished erythropoiesis. This is diagnosed either by
decreased in hematocrit or by decrease in hemoglobin content in the blood
Hematocrit – percentage of blood volume that is due to cells (normally 40-50%)
Erythropoiesis – production of new red blood cells by the action of the hormone erythropoetin on
stem cells
Reticulocyte - the immediate precursor to the mature erythrocyte (red blood cell)
Reticulocytosis – increased number of reticulocytes in blood (normally 0.5 – 1.5 % of the RBC)
Hemosiderosis – increased storage of iron in tissues. The storage occurs in the reticuloendothelial cells (macrophages) principally in the liver, spleen, lymph nodes, and bone marrow.
Iron is stored associated with the protein hemosiderin, which is an aggregate, precipitated form
of the protein ferritin. Hemosiderosis is not a pathologic condition.
Hyperplasia – increased number of cells in tissue arranged normally (non-cancerous)
Neoosteogenesis – expansion of bone marrow volume
2. Know that there are two classification systems of hemolytic anemias:
a) Classified by where hemolysis takes place:
 Intravascular – takes place in the vasculature without needing the blood to
go through the spleen for hemolysis to occur. This is the most severe
hemolysis. One example of this type is glucose-6-phophate dehydrogenase
deficiency
 Extravascular – hemolysis takes place in spleen. Most hemolytic anemias
are of this category
b) Classified whether it is congenital (birth defect) or acquired (outside agent causes
hemolysis)
3. Name the blood cells that arise from stem cells (a stem cell can differentiate into any one of
the following):
a) Erythrocytes (red blood cells)
b) White blood cells
 Monocytes (which mature into macrophages)
 Granulocytes
 Lymphocytes
c) Thrombocytes (platelets) (serves as plugging material for cuts in the vasculature)
4. Know the following facts about the development of blood cells in the fetus:
a) Stem cells form in the third week
b) Stem cells migrate to the liver. By the third month the liver is the principal place
of production of blood cells
c) By the fourth month, some production of blood cells is seen in the bone, although
the principal location for blood cell continues to be in the liver
d) From the fourth month to the ninth month the production of blood cells
progressively increases for bone marrow and progressively decreases for the liver
e) From approximately the third to the 9th month minor sites of blood cell production
include the spleen, lymph nodes, and thymus.
12
It is important to know these sites of development in the fetus because these sites can be
“turned on” (a process called extramedullary hematopoesis) in hemolytic anemia
conditions, in which the body needs to rapidly produce red blood cells.
5. Know that from birth to puberty that all the blood cells are produced by the bone marrow in
the bone. At puberty, the process begins in which a large portion of the bone becomes
inactive (yellow and fatty) while certain bones have red marrow and are active (see slide for
which bones, for the test you DO NOT need to know which bones are active). This
transformation takes place until the age of 18 when the bones are set as to whether they have
active red marrow or are inactive.
6. Know the general manifestations of hemolytic anemias:
a) pallor skin
b) hemosiderosis
c) fatty increases in heart, liver, proximal tubule cells, and ganglion cells
d) hyperactive bone marrow (hyperplasia, neoosteogenesis, activation of fatty
marrow; bone marrow can increase its production of RBCs by a factor of 7 to 8)
e) extramedullary hematopoesis
f) decreased hematocrit
g) reticulocytosis
h) jaundice, unconjugated hyperbilirubinemia
7. Anemia only happens if the destruction of red blood cells outstrips the compensatory
mechanisms increasing red blood cell production
8. Know the distinquishing features in the laboratory between intravascular and extravascular
hemolysis Intravascular hemolysis has a greater degree of breakdown of RBCs leading to
greater release of hemoglobin in the blood, leading to the following:
a) Conditions that are present in intravascular but not extravascular hemolysis
 Hemosiderinuria (hemosiderin in sediment of urine)
 Hemoglobinuria (hemoglobin in the urine)
 Hemoglobinemia (↑ hemoglobin in the plasma)
 Methemalbuminemia (oxidized heme bound to albumin)
b) Conditions in which there is a greater change for intravascular condition than the
extravascular condition
 Greater decrease in haptoglobin for intravascular hemolysis
c) Condition in extravascular but not intravascular hemolysis
 Enlargement of spleen (splenomegaly)
9. Know why haptoglobin decreases in hemolytic anemias
Haptoglobin is a 100K Dalton binding protein in blood that binds hemoglobin. When
hemoglobin binds to hatoglobin the complex is rapidly removed from circulation
leading to a decrease in haptoglobin.
10. Know the process by which hemosiderin appear in the urine
 Hemoglobin (Hb) exceeds the stoichiometric amount of haptoglobin present in
the blood
 Dimer Hb (normally a tetramer) is filtered in the glomerulus into the proximal
tubule
 Hb is reabsorbed by the proximal tubule
 Iron from Hb is stored as associated with the proteins ferritin and hemosiderin
13

Proximal tubule cells periodically are sloughed off (natural process), which
appears in the sediment. Iron in the sediment is detected with Prussian blue dye
11. Know the process for hemoglobinuria – same as above (point 9) upto and including
absorption of Hb by the proximal tubule. For intravascular hemolysis the Hb concentration in
the tubular fluid is so high that it exceeds the capacity of the proximal tubule to reabsorb it
12. Know the mechanism of methemalbuminemia in intravascular hemolysis
Free hemoglobin in the blood (appears in blood when the haptoglobin binding capacity is
exceeded) is very unstable. The heme dissociates from the Hb is oxidized and then binds
to albumin
F. Hemolytic Anemias – General Points about Congenital Disorders
1. Congenital (inherited defects) hemolytic disorders affect the red blood cell causing premature
lysis (breaking up) of the red blood cell. There are three categories of these disorders each
affecting a different aspect of the red blood cell (given below):
a) Defects in the RBC membrane
 Hereditary Spherocytosis
 Hereditary Eliptocytosis
b) Defects in enzymes in RBCs
 Glucose-6-Phosphate-Dehydrogenase Deficiency
 Pyruvate Dehydrogenase Deficiency
c) Defects in hemoglobin structure or synthesis in the RBC
 Sickle Cell Disease
 Thalassemia (α and β)
2. The normal shape of a RBC is a donut with the middle being indented (instead of being a full
hole)
G. Hemolytic Anemias – Membrane Disorders
1. Know that spectrin is a protein in the RBC membrane, which forms a network along the entire
membrane through self-association, giving the RBC structural support.
2. Facts to know about Hereditary Spherocytosis
 Involves a defect in spectrin (in either structure or amount) or another membrane
protein
 Causes the RBC to have a spherical shape and have a fragile membrane leading to
their being lysed by the spleen (extravascular hemolysis)
 Clinical course – excessive jaundice in neonates sometimes requiring transfusion.
Most adults have a partially compensated anemia with intermittent jaundice and
an enlarged spleen. The course can involve episodes of three crises:
a) Hemolytic crisis – acute hemolysis brought about by activation of
reticuloendothelial cells by an infection (most common crisis)
b) Aplastic crisis - caused by parvovirus which affect stem cells leading to ↓
in RBC synthesis
c) Megaloblastic crisis – inadequate folic acid intake
 Distinguishing lab tests
a) Presence of spherocytes
14
b) Osmotic fragility demonstrated (cells cannot tolerate swelling in hypotonic
solutions)
c) Increase Hb concentration in RBC
 Treatment
a) Spenectomy in patients with significant hemolysis
b) Folic acid supplementation
3. Facts about Hereditary Elliptocytosis (HE)
 Caused by defect in spectrin or other RBC membrane protein causing elliptical
shape in the RBC
 Three types:
a) Common HE – no symptoms, tests show elliptocytes, normal osmotic
fragility, absence of hemolysis test indicators (except slight rise in
reticulocytes)
b) Hereditary Pyropokilocytosis – hemolytic anemia, shows usual test results
of hemolysis, with added indicators of increased osmotic fragility,
poikilocytes, decreased RBC thermal stability
c) Spherocytic HE - hemolytic anemia, usual test results of hemolysis, with
added indicators of increased osmotic fragility, elliptocytes
 Extravascular hemolysis
H. Hemolytic Anemias – Enzyme Defects
1. Know the following for glucose-6-phosphate-dehydrogenase (G-6PD) deficiency:
a) The steps of the hexose monophosphate shunt pathway [chemical species name or
abbreviations (not formulas) and the enzymes involved]
b) Know the importance of hexose monophosphate shunt pathway:
 Generates NADPH which is used in biosynthesis of cholesterol and fatty
acids (reductive biosynthesis)
 Generates glutathione in the reduced form (GSH) which is the redox
buffer of the red blood cell, which acts as an antioxidant protecting the
RBC components from free radical oxidation and thus protecting cells
viability
c) In G-6PD deficiency hemolysis only happens when there is a high generation of
free radicals brought on in response to infection or administration of oxidant
drugs.
d) There is a difference for males and females in the presentation of the disease
because the gene for G-6PD is on the X chromosome
 Females (XX*, where X* indicates the chromosome that has the
defective G-6PD gene). According to Lyon’s Principle only one X
chromosome is active in a particular cell. Thus the G-6PD activity for a
particular red blood cell could be normal (if X is active) or deficient (if
X* is active), and thus there is a mosaic of the RBC population with
respect to activity of G-6PD in the RBC. The proportion of RBC in
which the X* G-6PD gene is activated varies for different female
individuals so the enzyme maybe normal, moderately reduced (normal
15
case), or significantly reduced. In most cases the severity of the
hemolysis is less than that for males.
 Males have X*Y genotype. So all the RBCs have the affected X
chromosome and thus the defective G-6PD gene. Thus all the cells have
a deficiency of G-6PD activity.
e) Know the characteristics of the two disease variant of G-6PD deficiency
1’) GdA Labile enzyme with half-life of G-6PD of 13 days (normally 60
days), thus young RBC have normal G-6PD activity but old RBCs have
greatly diminished activity
 Thus hemolysis occurs with old RBCs only, meaning a less severe
hemolysis that the GdMed
 Prevalent in black populations
2’) GdMed
 Defective enzyme, all RBCs regardless of age are affected
 Severe hemolysis, sometimes requires transfusion
 Prevalent in Mediterranean, India, and SE Asian populations
f) Know the mechanism of hemolysis
Free radicals present in the RBC because of the lack of G-6PD antioxidant
activity causes:
 Oxidation of hemoglobin which leads to the precipitation of the oxidized
hemoglobins as Heinz bodies, which adhere to membranes causing
decreased RBC deformability and leaky membranes; the cells are lysed
 Oxidative cross-linking of spectrin causing decreased flexibility and
lysing
 Peroxidation of membrane lipids compromising membrane structure
g) It is an intravascular hemolytic condition
2. Pyruvate Kinase Deficiency – Deficiency in pyruvate kinase leading to chronic extravascular
hemolytic anemia. This enzyme is in the glycolytic pathway. The mechanism of the hemolysis
is that ATP production is decreased leading to less viability of the RBC and thus premature
lysis.
I. Hemoglobin Structure and Forms; General Comments about Hemoglobinopathies
1. Hemoglobin consists of a tetramer consisting of four globin chains, comprising two pairs of
similar chains. The normal forms of hemoglobin are the following, with the normal
percentage in red blood cells given:
 HbA – α2β2 (96%)
 HbA2 – α2δ2 (3%)
 HbF - α2γ2 (1%)
2. Know the timing of the appearance of the various hemoglobin chains:
a) α chain synthesis begins early in the fetal life and continues throughout the life
both before and after birth
b) γ chain synthesis begins early in the fetal life (replacing the embryonic synthesis)
and is maintained at a high level throughout the fetal life. At birth the γ chain
16
synthesis steadily falls such that the percentage of HbF goes from 70-90% at
birth, to 2% after one year after birth, and below 1% after the third year after birth
c) β chain synthesis is at a insignificantly low level from the third month of the fetal
life and starts to increase in the last 6 weeks of fetal life such that HbA begins to
appear. HbA continues to increase after birth, replacing HbF until it reaches adult
levels of 95-98% at 6-12 months after birth.
d) HbA2 first appears at birth and is at a low level (2-3%) throughout life
3. Know the embryonic forms of hemoglobin. In the first months of fetal life embryonic forms
(Gower I and II) of fetal hemoglobin predominate (ζ2ε2 and α2ε2). These forms decrease in
synthesis after this to undetectable levels. These forms of hemoglobin have a higher affinity
constant and can bind O2 at lower pH. Portland hemoglobin (ζ2γ2) is an embryonic form that
persists throughout the fetal life.
4. Know that a hemoglobinopathy is a genetic disorder leading to hematologic disease from:
 a structural defect (i.e., addition, deletion, substitution of at least one amino acid
in at least one of the globin chains)
 a quantitative effect in which there is a decrease in synthesis of either an α or β
chain
5. Know that a mutation in the chain does not necessarily result in disease. Mutations that do
cause disease (most occur in the β chain) manifest in one of four ways:
a) Hemolytic anemia
b) Cyanosis – which is lack of oxygen delivery to tissues. This can result from either
the mutant causing methemoglobinemia (oxidized iron in the heme) or other
causes leading to decreased oxygen affinity
c) Polycythemia – increase in the number of RBCs
d) Hypochromic anemia – reduced output in hemoglobin
J. Sickle Cell Disease
1. Know that HbS, the hemoglobin in Sickle Cell disease, is the same as HbA EXCEPT that
there is substitution of glutamic acid with a valine in the 6th position in the β chain.
2. Know that the mutant β chain gene is recessive and that disease needs a homozygous
geneotype. Know that sickle cell trait (heterozygotes) has a high prevalence in AfricanAmericans (10%) and that 1 in 400 African-Americans have the disease.
3. Know that the disease is a hemolytic anemia and that the mechanism of hemolysis and
disease is as follows:
a)
HbS in the deoxygenated form aggregates to from a viscous gel causing the
RBC to sickle
b)
When HbS binds oxygen then the HbS aggregate dissociate from one another
and the RBC resumes its normal shape
c)
This cycling between the sickle and the normal shape occurs many time
damaging the membrane such that the cells lose water.
d)
Eventually the cell is irreversibly maintains its sickle shape
e)
The sickle cells get clogged in capillaries, which leads to cutting off of blood
flow in the tissues and subsequent tissue damage
f)
Irreversibly sickled cells are broken down by the spleen – avg lifetime of RBC
is 20 days
17
4. Know that Sickle Cell Trait (heterozygote) is basically asymptomatic (no symptoms) except
that not all patients are able to concentrate urine because there is micro-infarction of the renal
medulla (more cells sickle in the renal medulla because the hyperosmolarity draws water out
of the RBCs causing a ↑ [HbS]) Also hemolysis will result in cases of extreme oxygen
deficiency (depressurized aircraft).
5. For Sickle Cell Disease, hemolysis begins 3-6 months after birth (why?) and many die within
2-3 years. For those who survive, there are many tissues that can be affected by vasoocclusion (do NOT need to memorize these). Need to know that the pain of Sickle Cell
Disease results from vasoinclusion occurring in bones, with the pain lasting for hours to days.
Also know that polyuria and hematuria (RBCs in urine) can result because of renal medulla
infarction and that there is delayed growth and development. The mean survival is to the age
of 40.
6. Know that lack of oxygen and an increase in HbS concentration leads to sickling.
7. Know testing aspects:
a)
Sodium metabisulfate test – sodium metabisulfate deoxygenates blood which
cause cells to sickle, which can be observed under the microscope
b)
Solubility test – blood sample is placed in solution of high ionic strength,
which causes sample with sickle cells to become turbid (normal cells are
clear)
c)
Electrophoresis – HbS with the substitution of a glutamic acid (which has a
negative charge) with a valine (neutral charge)migrates slower in cellulose
acetate (pH 8.4) electrophoresis than HbA. Electrophoresis separates many
variants of hemoglobin and an estimate of percentages can be made. Given
below are the Hb values for Sickle Cell Trait and Disease:
 Sickle Cell Trait (Hetreozygous)
 HbS – 30-40%
 HbA - 60-70%
 Sickle Cell Disease (Homozygous)
 HbS – 80-90%
 HbF - 5-15%
K. Thalassemia
1. Know that thalassemia is a hemoglobinopathy which is characterized by decreased synthesis
in either the α chain (α-thalassemia) or the β chain (β-thalassemia). α-thalassemia affects
predominantly African-Americans (30% are carriers) and β-thalassemia affects
Mediterranean and Asian populations.
2. For β-thalassemia there are many specific mutations (approximately 25) of the β-chain gene
that lead to decrease synthesis of the β chain. These mutations are classified into three
categories:
 Complete absence of β-chain synthesis (β0)
 Half-normal amount of β-chain synthesis (β+)
 2/3 normal amount of β-chain synthesis (β++)
3. There are three clinical classifications of β thalassemia depending on the pairing of the genes
inherited from the parents:
 β-Thalassemia Trait – is for the most part asymptomatic, results from the
genotype of any of the defective genes (β0, β+, β++) paired with a normal gene (β)
18

4.
5.
6.
7.
β-Thalassemia Major – full blown disease hemolytic anemia disease, involving
accumulation of iron in liver, endocrine glands, and heart leading to death by age
of 17 due to cardiac dysfunction (congestive heart failure and arrhythmias) if
transfusions are given, or by age 2-3 if no transfusions given. This results from a
genotype of β0 paired with either β0 or β+ or in a small percentage (10%) of
homozygous in β+ (β+ β+)
 β-Thalassemia Intermedia - is an intermediate disease in which there is
hemolytic anemia leading to an accumulation of iron causing cardiac and
endocrine dysfunction but longer life and is less severe in its presentation. The
genotype is β++ paired with any defective gene (β0,β+, β++) and for most of the
homozygous β+ (β+ β+)
Know the mechanism of hemolysis in thalassemias. Decreased synthesis of one of the chains
leads to a relative excess of the counterpart chain that is synthesized in normal amounts. This
leads to the formation of a homotetramer (β4 for α-thalassemia and α4 for β-thalassemia)
which eventually precipitates out causing damage to the RBC and its ultimate sequestration
(taking up) by the spleen and hemolysis. β-thalassemias are much worse in their presentation
than the corresponding α-thalassemia because α4 present in the β-thalassemia is less soluble
than β4 in α-thalassemia.
The mechanism iron overload cause and effects in β-thalassemia is that the body does not
excrete iron efficiently. The multiple transfusions and the increased iron absorption in the GI
tract (due to the hemolytic condition triggering erythropoesis) leads to iron buildup in the
body which initially present as hemosiderosis but which turns to hemochromatosis (exceess
iron leads to tissue damage) in endocrine, heart, and liver tissues, which are particularly
susceptible to iron.
Know the designations and clinical features of α-thalassemias:
a) α-thalassemia syndrome – one defective gene – asymptomatic
b) α-thalassemia trait – two defective genes –mild microcytoisis (small
RBCs) no other manifestions
c) Hemoglobin H Disease – three defective genes- mild hemolysis
d) Hemoglobin Bart’s Disease – four defective genes –fetus does not
survive or dies immediately after birth, severe anemia with congestive
heart failure [no α synthesis, thus no HbA, HbF, HbA2 ; only γ4 present
(although β4 is produced at end of gestation), which has such a high
affinity for O2 it is functionally useless]
Know the distinguishing laboratory hemoglobin profiles of thalassemias (know why –
explained by knowing the chain composition of the hemoglobin, knowing that those
hemoglobins having the deficient chain will be decreased and other Hbs not consisting of a
deficient chain will be increased). In parentheses is how it compares with normal
a) β-thalassemia major
 HbF (α2γ2) – 70-90% (increased)
 HbA2 (α2δ2) - upto 20% (increased)
b) β-thalassemia intermedia
 HbF (α2γ2) – large amounts (increased)
 HbA2 (α2δ2) - significant amounts (increased)
 HbA (α2β2) – variable reduced amounts (decreased)
c) β-thalassemia trait
19
 HbA2 (α2δ2) - 4-8% (increased)
 HbF (α2γ2) – 1.5 -2.5 % (increased)
 HbA (α2β2) – remainder (decreased)
d) α-thalassemia trait
 HbA2 (α2δ2) and HbF (α2γ2) are normal or slightly decreased
e) Hemoglobin H disease
 HbA (α2β2) – 70-90% (decreased)
 HbH (β4) – 10-30% (not normally present)
 HbA2 (α2δ2) - decreased
L. Electrophoresis and Hemoglobin Electrophoresis
1. Know the general mechanism of electrophoresis. Sample is put on a gel that is saturated with
a buffer that has an electric field imposed upon it. The buffer serves to establish a charge on
the protein and also establish electrical contact between the electrodes (anode is + electrode,
cathode is – electrode). In most cases a pH is chosen such that most proteins are negatively
charged (for example pH 8.4) moving toward the anode. Proteins move at different velocities
depending on their charge/size ratio thus causing separation.
2. Know the general equation describing the rate of migration in electrophoresis:
Q/(6πrη) = υ/E = μ
Where Q is the charge of the protein, r is the ionic radius of the protein, η is the viscosity of
the liquid medium, velocity of protein, E is the electric field strength, µ is the rate of
migration per unit electric field strength.
3. In the above equation note that:
 The velocity is dependent on both the charge/size ratio and the magnitude of the field.
This latter factor is significant in that separationns can occur faster at higher electric
fields
4. Know what electo-osmosis is and the mechanism of it.
a) Electro-osmosis is a process by which there is a slow movement of fluid in a certain
direction causing neutral proteins to move away from the origin, and proteins with
charges to either increase in velocity (if the electro-osmosis force is in the same
direction of the protein movement in the electric field) or decrease in velocity (if the
electro-osmosis is in the opposite direction of the protein movement).
b)
Mechanism
The movement of fluid arises because the gel has charges on it. In the usual case the
gel has a residual negative charge (these are attached to the gel and ARE THUS
FIXED and cannot move toward its oppositely charged electrode). Now the liquid
phase also has negative charges of the buffer which are counterbalanced by an equal
number of positive charge. However there are excess positive charges in the liquid
phase balancing the fixed negative charges on the gel . In an electric field all ions in
the liquid phase move to its oppositely charged electrode. As noted, there are more
positive charges in the mobile phase than negative charge. Thus there are more ions
moving toward the cathode than ions moving toward the anode. Water follows the
movement of solute (osmosis). Since there are more ions (solute) moving toward the
20
cathode, there is thus more water moving toward the cathode. Thus the flow of the
water is towards the cathode.
5. Know the gel types used in electrophoresis and their attributes, advantages and disadvantages
as given in the table below:
Gel type
Cellulose Acetate
Attributes
Used in clinical lab
Advantages
Shorter analysis
time, reproducible,
Agarose
Large pore size
Low electro-osmotic effect
Used in clinical lab
Pores give an additional size
separation effect (smaller
molecules are less restricted in
their movement and thus move
at faster velocity than bigger
molecules)
Used in research tool
Higher resolution
than cellulose
acetate
High resolution
(serum proteins
separated into 20
bands)
Polyacrylamide
Disadvantages
Lower resolution (serum
proteins separated into 89 bands)
Reproducibility
Labor intensive gel
preparation
6. For polyacrylamide gel electrophoresis (PAGE) with addition of the detergent sodium
dodecylsulfate SDS (termed SDS-PAGE). In the normal procedure the disulfide bonds are
cleaved. Significant amounts of SDS adsorbs to the protein causing the protein to assume a
rod shape (so many negative charges on the protein that this is the most favorable shape to
separate the charges). Since all proteins have the same charge to size ratio (since the
negatively-charged SDS groups overwhelm any “natural” charges of the protein and there is a
constant amount of SDS absorbed per gram of protein) the separation is based solely on size
of the denatured protein. The proteins migrate at a distance proportional to the logarithm of
their molecular weight.
7. Facts to know:
 Highest resolution is obtained at the highest voltage since:
 The higher voltages leads to proportionally higher velocities and thus greater
separation of peaks for a given time
 Lower analysis time and thus less diffusion of the bands (which increases with
the square root of time).
However there is a practical limitation in that with increased voltage there are high
currents which causes heating which severely distorts the bands. Thus cooling is a
must. This is the advantage of capillary electrophoresis because the capillary can be
efficiently cooled, allowing extremely high voltages and thus very short analysis
times.
 The two most widely-used routine modes of electrophoresis operation are constant
current (which gives the fastest separation – but the poorest resolution because of the
heat generated) and constant voltage (which gives the best separation but takes a
longer time).
8. For hemoglobin electrophoresis red blood cells are lysed and the sample is applied to
electrophoresis gel. Staining is required to identify and quatitate the hemoglobins. Cellulose
21
acetate electrophoresis at pH 8.4 is done. Citrate agar electrophoresis (pH 6) is done to
separate co-eluting variants.
9. Know the order as given below for the following hemoglobins in cellulose acetate
electrophoresis (pH 8.4) [as proceed to the right closest to the anode (fastest migration rate in
the gel)]:
HbA2 → HbS → HbF → HbA → HbH (this is the fastest of all Hbs)
10. Know why HbS migrates slower than HbA
11. Know the mechanism of the citrate agar electrophoresis.
 The gel consists of agraose (which is the support of the gel) and agaropectin (which is
a negatively-charged sulfated polysaccharide)
 The agaropectin bind to some hemoglobins selectively imparting a negative charge on
them so that they migrate toward the anode. Most others hemoglobins that don’t bind
agaropectin are neutrally charged and migrate slightly to the cathode.
 This is used to separate S from D and G (which all co-migrate in cellulose acetate
electrophoresis) and C from E and O (which all co-migrate in cellulose acetate
electrophoresis)
12. Know what amino acids are potential charge sites of the protein (just need to know the names
and whether it is acidic (potential negative charge) or basic (potential positive charge).
 Basic amino acids: arginine, lysine, N terminus amino acid
 Acidic amino acids: aspartic acid, glutamic acid, C terminus amino acid
13. Know how the protein charge generally changes with change in pH:
 As pH decreases (starting from high pH) the protein becomes less negatively charged,
becomes neutral with further decrease in pH, and then becomes positively charged
with further decrease in pH
 As pH increases (starting from low pH) the protein becomes less positively charged,
becomes neutral with further increase in pH, and then becomes negatively charged
with further increase in pH
Other (Miscelleneous)
1. δ-bilirubin is a form where conjugated bilirubin is covalently attached to albumin
2. The storage forms of iron in the body is hemosiderin and ferritin (which are proteins that iron
binds to.
3. Know the following: In the enterohepatic circulation of urobilinogen, 20% of urobilinogen is
absorbed by the intestines entering the blood system. 90% of this absorbed urobilinogen
reneters the GI tract through the hepatocyte uptake and its excretion in the bile, while the
remaining 10% of the absorbed urobilinogen is filtered in the kidney, where it is excreted in
the urine. Increased urobilinogen in the urine or blood can reflect an overproduction of
bilirubin (hemolytic anemias) or liver disease (since it does not recycle urobilinogen into the
bile as well as a healthy liver).
22