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Austin Hsin
Ms. Dormer
AP Chem
23 May 2014
Erythropoietin
In 1905, Paul Carnot and his assistant, Clotilde Deflandre, made a connection between
hormones and the regulation of red blood cell production. After conducting experiments on
rabbits and bloodletting, Carnot and Deflandre connected an increase in red blood cells in rabbits
to a hemotropic factor called hemopoietin. Eva Bonsdorff and Eeva Jalavisto further studied the
idea and decided to call the hemopoietic substance 'erythropoietin'. Investigations by K.R.
Reissman and Allan J. Erslev, separately, demonstrated that a certain substance, circulated in the
blood, is able to stimulate red blood cell production and increase hematocrit, the percentage of
blood in the blood stream. (Jelkmann 78)
While studying sheep and other animals in the 1970s, Haematologist John Adamson and
nephrologist Joseph W. Eschbach looked at the role of the natural hormone EPO in the formation
of red blood cells. Both scientists helped establish that EPO stimulates the production of red cells
in bone marrow and could lead to treatment for kidney diseases such as anemia. (Cera)
In 1968, Goldwasser and Kung began work to purify human EPO. They were able to
purify milligram quantities of over 95% pure material by 1977. The breakthrough in the
purification allowed the amino acid sequence to be partially identified and the gene to be
isolated. Later, an NIH-funded researcher at Columbia University discovered a way to
synthesize EPO. Columbia University patented the technique, and licensed it to Amgen. Since
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then, controversy has ensued over the fairness of the rewards that Amgen reaped from NIH-
funded work, while Goldwasser was never financially rewarded for his work. (Cera)
In the 1980s, Adamson, Joseph W. Eschbach, Joan C. Egrie, Michael R. Downing and
Jeffrey K. Browne conducted successful clinical trials at the Northwest Kidney Centers for a
synthetic form produced by Amgen. In 1985, Lin isolated the human erythropoietin gene from a
genomic phage library and were able to characterize it for research and production. The
industrial production of recombinant human erythropoietin (RhEpo) for treating anemia patients
would begin soon after. (Cera)
In 1989, the US Food and Drug Administration approved the hormone Epogen, which
remains in use today. Since then, more synthetic forms have been synthesized and produced for
various medicinal purposes. (Cera)
General Info
Erythropoietin is a cytokine hormone, a type of hormone that are important in cell
signaling. Erythropoietin and other cytokines affect other cells through receptors, erythropoietin
specifically regulates the production and growth of red blood cells. Erythropoietin is produced
primarily in the kidney, about 90%. The rest is produced in the liver. In the kidney,
erythropoietin is produced in the renal cortex by peritubular interstitial cells. Once erythropoietin
is produced, the production of red blood cells can begin through a process called erythropoiesis.
(Nabili)
Erythropoiesis
Erythropoietin promotes the formation of red blood cells through a process called
erythropoiesis.
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Erythropoietin stimulates bone marrow through its receptors on the surface that basically send a
signal to begin the development of erythrocytes into red blood cells. EPO binds to EPO
receptors, erythroid precursors, on the surface of bone marrow which results in replication of red
blood cells. (Fibrogen)
Receptors
The predominant pathway activated by the EPO receptors, and other cytokine receptors,
is the Jak/STAT signaling cascade. Jak tyrosine kinases (JAKs) are generally associated with the
membrane of cytokine receptor intracellular domains and are activated upon ligand binding and
receptor reorientation. The EpoR associates specifically with the Jak2 kinase.

After EpoR
activation, Jak2 phosphorylates tyrosine residues in the intracellular region of the EpoR,
providing a placeholder for signaling molecules with phosphotyrosine binding motifs, including
the signal transducer and activator of transcription protein STAT5, which mediates the principal
intracellular signaling pathway elicited by the EpoR. (Lappin) Specifically in erythropoiesis, the
phosphorylated tyrosine residues help the development of proerythrocytes that will develop in to
red blood cells.
Neuro-Protection
However, mRNA and protein of EPO and EPOR have been detected in brain
(hippocampus, internal capsule, cortex, midbrain), as well as in neurons and other kinds of cells.
Expression of EPO and its receptors have shown to increase during brain development. EPO is
also vital in cell replication and neruonal survival in embryonic brain. Expression of EPO and
EPOR in the adult brain is stress-responsive and is regulated by oxygen supply: Both receptor
and ligand expression decreases after hypoxia or ischemia. Other stimuli such as hypoglycemia,
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insulin release, reactive oxygen species and insulin-like growth factor activate hypoxia-inducible
factor and lead to increased expression of EPO. (Bytes: Sirn)
EPO and its receptor are abundantly expressed in the developing human brain and in
response to negative changes. Hypoxia rapidly induces expression of brain EPO as evidenced
by the increased expression of EPO in cerebrospinal fluid (CSF) or postmortem brain tissue in
humans with traumatic brain injury, SAH, stroke and hypoxia. Expression of EPOR has also
been detected in the human peripheral nervous system. (Bytes: Sirn)
EPO holds a host of protective mechanisms including protection against oxidative
damage, inflammation. EPO attenuates inflammation by reducing reactive astrocytosis and
microglia activation and by inhibiting immune cells recruitment into the injured area (Bytes:
Sirn) EPO also protects vascular integrity and preserves blood-brain barrier integrity during
injury by restoring expression of tight junction proteins, by reducing vascular inflammation and
reactive free radical expression.(Bytes: Sirn)
Traumatic brain and spinal cord injury
EPO also helps certain areas of injury in the brain and spinal cord, the neural system in
general, through active transport across the blood-brain barrier. Administration of EPO and
EPO-analogs in experimental models of traumatic brain and spinal cord injury leads to recovery
that can be attributed to some of EPOs cytoprotective mechanisms including inhibition of
apoptosis, anti-inflammatory and anti-oxidant actions, restoration of blood-brain barrier integrity,
stimulation of neurogenesis and angiogenesis. (Bytes: Sirn)
Interestingly enough, it is unknown whether EPO is responsible for the long-term
prevention of trauma-induced brain atrophy, cognitive and neurobehavioral, Chronic peripheral
administration of EPO has been reported to improve spatial memory function and cognitive
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functioning in the context of an aversion task also in healthy mice Improved hippocampal
functioning after a single intravenous bolus of EPO was recently shown in a study using
functional magnetic resonance imaging in healthy human volunteers. (Bytes: Sirn)
Degeneration & Neuro-inflammation
EPO also offers protection against neurodegenerative and neuro-inflammatory disease.
In experimental autoimmunencephalitis (EAE), an animal model for multiple sclerosis (MS),
treatment with EPO and EPO analogs can improve functional recovery, reduce tissue damage,
inflammatory responses and blood-brain barrier leakage. Beneficial effects of EPO have also
been reported in models of peripheral axonal nerve injury, injury-induced Wallerian
degeneration and HIV-associated sensory neuropathy. (Bytes: Sirn) Here, EPO seems to play
an important role in reducing inflammation and preserving neuronal function. Chronic
neurodegeneration might also be a target for EPO therapy as EPO and its analogs can counteract
degenerative processes in experimental models of Parkinson disease and amyotrophic lateral
sclerosis (ALS) (Bytes: Sirn)
Medicinal Uses
Normal Erythropoietin levels range from 4 to 24 mU/mL in the human body and anything
higher or lower than such a range can indicate certain conditions or diseases. Low levels of
erythropoietin can indicate kidney diseases such as anemia, can be treated using synthetic EPO.
Hypoxia, a lack of oxygen, can also be treated. High levels of erythropoietin can indicate a
kidney tumor, EPO abuse, or polycythemia. Recombinant forms, Alpha, Beta, Omega, Delta, and
darbepoetin-alpha are the current forms of synthetic erythropoietin available. (Ritter) Using
recombinant DNA technology, the synthetic forms of EPO are nearly identical to the natural
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form which has caused huge problems in the athletic world as has not been a fool-proof test to
distinguish synthetic EPO from natural EPO.
Doping
Since the production of synthetic EPO, medically it has been very beneficial. However,
the known increase in oxygen has involved EPO in a long history of doping in athletes. Before,
officials were unable to detect the differences between synthetic and natural EPO due to the
similarities of their structures. Officials tried to counter the surge of doping by limiting athletes
hematocrit, percentage of red blood cells in blood, to around 50% in order to contain the doping
however it does not stop the practice. (Smith) Subsequently an EPO test was developed that
takes advantage of slight structural differences between the natural form and the synthetic form,
both of which are excreted in the urine. The structural differences lead to different electrical
charges on the two types of EPO, so that in an electrical field they can be separated and
analyzed. However, the test is not always accurate since the two consist of a group of similar but
slightly different molecules. Each type of molecule behaves slightly different in an electrical
field, so distinguishing natural from synthetic has some difficulties. Not only that, but the test has
a very large number of false negatives in the patterns. (Swiss Lab for Doping)
Properties and Structure
The chemical formula of Erythropoietin is C815H1317N233O241S5 with a molecular
weight 18396 g/mol. It has a melting point of 53 C and a boiling point of 100 C with an
isoelectric pH of about 4.4 and is water soluble. (Amgen) Chemically, erythropoietin is a
glycoprotein, a sugar attached to a protein. EPO consists of 165 amino acids, which are anime
and carboxylic acids put together. The amino acids form peptide bonds with each other through
combination of carboxyl and amine groups. EPO consists of 4 carbohydrate chains, 3 N-linked, 1
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O-linked and 2 disulfide bonds and a hydrophobic protein core keeps EPO stable. (Marcey) The
hybridization of the important cysteines in erythropoietin would be sp3 at the core due to its
carboxyl group, amine group, and the rest of the chain.
An important structural feature of erythropoietin is that it has two di-sulphide bonds, one
links the cysteine at amino acid 6 with the cysteine at amino acid 161, and the other links
cysteines 29 and 33. The link between 29 and 33 is more important because it acts as a tether,
ensuring that the whole molecule is held in the correct shape for binding to the erythropoietin
receptor. If this bond breaks, the molecule loses its biological function. From the structure of
the erythropoietin molecule, and from experimental evidence, one can infer that molecules of
erythropoietin aggregate together through a process known as hydrophobic interaction. When
this happens to any degree, perhaps as a result of improper storage, erythropoietin becomes
significantly less potent. (Lappin)
There is also great importance in EPOs hydrophobic core inside the folding of its
helixes. Without the interaction of the hydrophobic core and the residues nearby the structure of
EPO would not fold the same way. Similarly an orientation of helix D of about 18 degrees is
caused by a discontinuity of the chemical shift index along the helix. The chemical shift index is
used to determine the helices, sheets, and random coil that make up the secondary structure.
(Rutgers, MAP) This kink is of great structural importance. It allows certain side chains to come
within hydrophobic contact with the center of the protein thereby enhancing the conformation of
the EPO structure.
Approximately 60% of the erythropoietin molecule is protein and 40% is carbohydrate.
Each of the four carbohydrate groups consists of a small, branched chain of sugars, some of
which terminate in sialic acid. The presence of sialic acid on the carbohydrate slows the rate of
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clearance of erythropoietin by the liver. (Lappin) Clearly, the longer erythropoietin remains in
the circulation, the greater the opportunity for it to stimulate erythroid precursors located in the
bone marrow.
Why I chose this molecule
As a member of the Mock Trial team, I had to read a case this year on a case involving a
high school track runner who died from EPO intake. I had to do some research since the case
went into drug testing and how because of the similarities between synthetic and naturally
produced EPO structure. So it piqued my interest and after dealing with it for the rest of the
Mock Trial season, I decided that it would be fun to really research it.
How it illustrates chemical principles, theories, or concepts learned this year
The structure of EPO best illustrates the complexity of intermolecular forces and its
relationship to its function. The di-sulphide bonds and the N and O linked carbohydrate chains
along with the hydrophobic protein core all attribute to its function when leaving the kidney and
binding to its receptors. The same goes with the small, but nice amount of organic chemistry
with the addition of all the amino acids and classification of carbohydrate chains and functional
groups.
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