Abstract
Erythropoietin was first
hypothesized in the early 20th century and within the same century
its composition, function, mechanism, and synthesis were all discovered. This rapid rise in scientific knowledge parallels the rise in technological development.
In 1906 at the turn of the century, Professor Carnot hypothesized a compound
named “hemopoietine” to be responsible for red blood cell proliferation in
animals. Less than 100 years later, Amgen, a rising California biotech company,
produces a DNA recombinant version of erythropoietin available on the market as
Epogen®. The tale behind the discovery of erythropoietin and its production
into one of the most successful biotech ventures in the world is further
complicated by later abuse of erythropoietin recombinants by professional
athletes. In the world of professional sports, with millions on the line for
contracts, some athletes would do anything to win. They turned to tampering
with red blood cell production in their own bodies. However, as with many
biological functions there exists a thin line between legitimate training and detrimental
side effects.
Introduction
A glycoprotein of 165 amino acids
and 4 sugar side chains in the blood at less than 10 picomolar concentration,
erythropoietin appears, at first, quite insignificant in the body. Yet this one
hormone plays a vital role in red blood cell formation as well as tells a
fascinating story of both scientific accomplishment and drug abuse. On one hand, you have the scientific miracle of one of the world’s most successful biotech
companies. On the other hand, you have a drug being used illegally by athletes for
doping. It is a hormone found naturally in your body that keeps the red blood
cells going. However, as is the case for most healthy body products, too much
of a good thing can turn out to be detrimental to your health. This story of
fame, fortune, and notoriety starts out at the turn of the 20th
century.
Discovery of Erythropoietin
The story starts in the early 20th
century when Paul Carnot, Professor of Medicine at the University of Paris, and
his colleague Madame Cl DeFlandre published their paper on increased red blood
cell production in rabbits. They noticed that injecting serum collected from
bleeding rabbits, increased red blood cell production in normal rabbits. They
suspected a hormone that they dubbed “hemopoietine” increased bone marrow
activity and was responsible for increasing red blood cell proliferation.
However, there were issues with their paper. The experimental results reported
were difficult to reproduce. While Carnot had to inject less than 10mL of serum
into normal rabbits to experience red blood cell proliferation, other
researchers had to inject substantially large quantities of serum (Fisher,
2010). This discrepancy and other possible issues, lead to the paper being
ignored for the following decades. Then in 1948, following a series of
reticulocyte discoveries, Bonsdorff and Salavisto renamed the suspected
“hemopoietine” into the modern name, “erythropoietin.”
Mechanism of Action
What Paul Carnot originally
hypothesized as “hemopoietine” is now known to be a vital hormone in red blood
cell formation. The development of different cells found in the blood falls under
the process named hematopoiesis (also known as hemopoiesis). All cells in the
blood original arise from a common ancestor in a manner similar to human
beings. In the case of blood, a hematopoietic stem cell gives rise to myeloid
or lymphoid. These two classes are further broken down into red blood cells,
white blood cells, and platelets. For red blood cells, hematopoietic stem cells
undergo development to become common myeloid progenitor cells. These common
myeloid progenitor cells later develop into proerythroblasts. Further
maturation and chemical reaction transform proerythroblasts into mature
erythrocytes, also known as red blood cells.
Figure 1: Hematopoiesis – Common stem cell can
differentiate into different types of cells in the body. Image courtesy of
Wikipedia commons: https://en.wikipedia.org/wiki/Haematopoiesis#/media/File:Hematopoiesis_simple.svg
The process of red blood cell
maturation and formation does not occur if erythropoietin is absent. Why does
the process red blood cell production halt when erythropoietin is not
available? Erythropoietin is needed to bind to the aptly named erythropoietin
receptor in order to set off a series of kinases and other secondary messengers.
These secondary messengers and associated transcription factors are needed to
activate red blood marrow and start the erythrocyte stem cell differentiation
process. In addition to stem cell differentiation, erythropoietin also promotes
the survival of existing red blood cells by protecting these cells from
apoptosis. Thus, impeding or eliminating the production of erythropoietin not
only prevents the production of new red blood cells, but also rapidly increases
the breakdown of existing red blood cells.
Further Development of Erythropoietin
In the present day, we have the
benefit of hindsight to be able to see the function of erythropoietin as well
as its chemical composition. However, researchers of the past had to gather the
pieces of information through various experiments. In 1950 Reissman, K.
reported the link between oxygen consumption, erythropoietin production, and
erythropoiesis. Interest in the compound increased as scientist collaborated to
figure out the details of erythropoietin (Ribatti, 2008). In 1953 Erslev, A
performed a similar rabbit experiment to Professor Carnot, only this time
taking plasma and adjusting for more possible error. The plasma from the
bleeding rabbits, when injected into the normal rabbits resulted in increased
hematocrit and reticulocyte counts. This was followed in 1955 by the
development of the first quantitative and specific assay for erythropoietin.
Now scientists could test to determine the hormone’s concentration in various
samples. The specific production site of erythropoietin was later discovered in
1961 when Kuratowska et al. isolated erythropoietin in isolated dog
kidneys. Enough information had been compiled for erythropoietin by that point
for an entry in the 1966 international reference standard. Within a span of
about six decades, erythropoietin has gone from a purely speculated hormone to
one with a specific index and reference.
Science continued to race ever
forward as the 1970s brought another round of vital discoveries. In 1973
erythropoietin stimulation was linked to prostaglandins a diverse family of
cell signaling molecules. Just four years later erythropoietin was extracted
from over 1,000L of human urine through painstaking work (Fisher, 2010). That
same year scientists reported that the liver was the primary erythropoietin
production site for fetuses opening a new door in the research of human
development. With knowledge of where the hormones were produced as well as the
purified compound itself; work could be started towards applying erythropoietin
towards medical treatments.
Erythropoietin Involvement in Medical Treatments
Erythropoietin plays a vital factor in red blood
cell formation. Therefore, losing erythropoietin causes a loss in the ability
to regenerate red blood cells, eventually leading to anemia. This is especially serious in patients
suffering from severe chronic kidney disease (CKD), as the primary producer of
erythropoietin is the renal cortex.
Table 1: Stages of Chronic Kidney
Disease (1-5)
|
Stages of Chronic Kidney Disease (CKD)
|
|
|
Stage 1
|
Kidney damage with normal
kidney function (estimated GFR ≥90 mL/min per 1.73 m2) and
persistent (≥3 months) proteinuria.
|
|
Stage 2
|
Kidney damage with mild loss of
kidney function (estimated GFR 60-89 mL/min per 1.73 m2) and
persistent (≥3 months) proteinuria.
|
|
Stage 3
|
Mild-to-severe loss of kidney
function (estimated GFR 30-59 mL/min per 1.73 m2).
|
|
Stage 4
|
Severe loss of kidney function
(estimated GFR 15-29 mL/min per 1.73 m2).
|
|
Stage 5
|
Kidney failure requiring dialysis
or transplant for survival. Also known as ESRD (estimated GFR <15 mL/min
per 1.73 m2).
|
Directly taken from
government website: https://www.niddk.nih.gov/health-information/health-statistics/kidney-disease
If kidney function is limited or
nonexistent, as is the case for patients undergoing dialysis[*]
there will be limited to no erythropoietin being produced. The lack of
erythropoietin, in turn, causes issues during hematopoiesis resulting in halted
production of red blood cell production from red bone marrow. With no new red
blood cells to replace damaged and old red blood cells, anemia kicks in. The
body starts to suffer from reduced oxygen capacity as the number of mature red
blood cells present to capture oxygen via hemoglobin is significantly reduced.
The decrease in oxygen induces hypoxemia, a state of low arterial oxygen supply
(Saladin, 2012). Activities linked to oxygen such as the electron transport
chain (ETC) during adenosine triphosphate (ATP) production are significantly hindered,
as oxygen is not available to be reduced. Expected symptoms are weakness,
fatigue, headaches, paleness, dizziness, shortness of breath, and chest pain. If
severe enough, the body tissues of certain vital organs will suffer from
weakness that could impede function (especially the heart).
With the rise in medical
technology, patients with chronic conditions such as CKD continue to increase. Considering
the fact that more patients continue to live with chronic kidney disease as well
as kidney transplants, erythropoietin was needed more than ever. Unfortunately,
only minute quantities of the hormone could be purified from human urine of
patients suffering from aplastic anemia. This process was too inefficient for
mass medical consumption. The answer to the erythropoietin shortage was
recombinant DNA production.
Biotech Success Amgen and Epogen®
In 1980 a new biotech company AMGen
(Applied Molecular Genetics Inc.) was founded with an ambitious CEO George B.
Rathmann. The company had one specialized focus, the application of recombinant
DNA technology (Medicosis Perfectionalis, 2019). Recombinant DNA technology
takes human genes needed for the production of certain proteins and incorporates
them into bacterial genes. The goal is to have the bacteria produce human
hormones as their growth is exponential and more efficient than humans.
Using recombinant DNA technology as
their base model, the company applied its specialty production towards many
different possible business ventures ranging from oil extraction to the
production of indigo dye. Eventually, they focused on medical treatments,
specifically the production of necessary human hormones. They set their sights
on erythropoietin and its responsible genes. In 1985 after two years of tedious
effort, an AMGen team lead by Taiwanese researcher Fu-Kuen Lin finally succeeded
in isolating the human gene responsible for erythropoietin. By 1989, they
produced their first FDA-approved medication Epogen® (AMGen, 2015). Later other
biotech companies would produce alternatives such as Procrit® and Aranesp®.
Epogen® and Erythropoietin Recombinant Side
Effects
As with all medications, Epogen®
and other erythropoietin recombinants have unintended side effects that may
prove to be more detrimental than beneficial. The common side effects are flu-like symptoms, cough, rash, nausea, soreness of the mouth, and redness and pain in the skin where Epogen® injected. The more severe possible adverse effects are
anaphylactic shock, hyperviscosity, thrombosis, hypertension, and pure red cell
aplasia (John, Jaison, Jain, Kakkar & Jacob, 2012). The more severe issues
are caused by the rapid proliferation of red blood cells in the blood. Increase
in the red blood cells changes the viscosity of blood, making the already viscous
blood even thicker. Increased viscosity of blood has additional rippling
effects of increased clotting owing to the slower blood flow. This, in turn, has
the devastating effect of possibly causing myocardial infarctions,
cerebrovascular accidents, and pulmonary embolisms. With all these frightening possible
adverse effects, why would the doctors prescribe erythropoietin recombinants? Based
on the physician judgment that the benefits of increased red blood cells to
oxygenate the body outweigh the possible detriments of blood clots. Also, the
blood clot risk is minimized through careful measured levels of red blood cell
counts, ensuring that the hematocrit[†] is
just slightly below the average.
Medical Drug Turned into Illegal Doping
Although originally intended to
treat patients suffering from anemia due to CKD, Epogen® and other red blood
cell proliferation-inducing medications became a source of illegal sports
doping. Increasing the amount of red blood cells in the body increases the
amount of oxygen your body tissues can possibly receive, thereby increasing an
athlete’s maximum oxygen capacity. Sports athletes in endurance exercises would
benefit from boosts of extra red blood cells. Yet the adverse side effects of
erythropoietin recombinant abuse, especially for normal people without severe
anemia, are fatal. In 1996, an article in Nature, titled “Erythropoietin abuse
in athletes” reported that German weekly magazine Der Spiegel recorded 18
cyclist deaths caused by erythropoietin abuse (Gareau et al., 1996). The
article went on to lament that despite being on the International Olympic
Committee’s list of banned substances, erythropoietin continued to have no
reliable tests. Without the tests, the ban was unenforceable.
Several factors made testing for
erythropoietin difficult. Artificial or recombinant erythropoietin closely
resembles the body’s natural erythropoietin. The drug also has a relatively
short half-life in blood serum and urine, about 8.5 hours and 2 days
respectively (John et al., 2012). Despite these difficulties in detecting
erythropoietin in blood, scientists managed to develop techniques both direct
and indirect for determining erythropoietin abuse. The direct method involves using
electrophoresis tests to detect recombinant erythropoietin proteins. However,
to actually run these types of tests extensively on site at different sporting
locations is challenge owing to the different infrastructure and varying level
of staff training of various nations. Indirect methods revolved around testing
specific cell counts and comparing the previous cell count numbers to the
current. This method is dubbed the “hematological passport” as the athletes must
carry their blood count reports to various events. Indirect methods are cheaper
and easier to implement, but are more open to possible fraud or
misinterpretation.
Away From Blood Transfusions and Medication and
Towards Altitude Training
With most forms of blood doping
through the use of medications and transfusions now banned by international
sports communities, attention was a shift to a more “natural” way to induce
higher levels of red blood cells in the body. As discovered earlier during the
search for erythropoietin, induced hypoxia in animals raises the erythropoietin
levels through negative feedback. The same was found in humans that lived in
high altitude environments and the 1968 Mexico City Olympics gave a perfect
testing ground to some scientific theories regarding erythropoietin (Eicher,
2007). During that Olympics, athletes performed better in endurance sports
hinting at the presence of erythropoietin due to the low oxygen environment. This
opened up the idea of inducing increased erythropoietin through carefully
planned oxygen deprivation at high elevation locations or artificially
generated atmospheres. Since there is no injection of foreign recombinant
erythropoietin, the elevated red blood cells are more difficult to directly
detect. Indirect methods will successfully catch the difference in red blood
cell concentration though.
Erythropoietin Legacy
Despite the negative reception of
erythropoietin abuse, erythropoietin remains a vital tool in the doctor’s
arsenal. The primary clinical use of erythropoietin is to treat anemia in
chronic kidney disease patients. However, it has an additional range of uses
for other types of secondary anemia. It also has been used as a preoperative
medication to decrease or avoid the application of blood transfusions (John et al.,
2012). These successful medical treatments outweigh the possible negative
effects of blood doping in sports as the elite athlete population is but a
small fraction of the human population compared to those with chronic diseases.
Cheating athletes, regardless of how clever they believe themselves to be,
cannot cheat natural body reactions. Thus, our erythropoietin tale comes to an
end with more than a century of development. Yet the biochemical properties of
the hormone remain vital to life and will continue to remain an important
hormone to study for human advancement.
References
Amgen. (2015). The amgen story. Retrieved
from https://www.amgenhistory.com/
Eichner, E. (2007). Blood doping. Sports
Medicine, 37(4), 389-391. doi:10.2165/00007256-200737040-00030
Ekblom, B. (1996). Blood doping and
erythropoietin. the effects of variation in hemoglobin concentration and other
related factors on physical performance. The American Journal of Sports
Medicine, 24(6), S40.
Elliott, S. G. (., Foote, M., &
Molineux, G. (. (2009). Erythropoietins, erythropoietic factors, and
erythropoiesis molecular, cellular, preclinical, and clinical biology (2nd
rev. and ext. ed.. ed.). Basel; Boston: Basel; Boston : Birkhäuser.
Fisher, J. W. (2010). Landmark advances
in the development of erythropoietin. Experimental Biology and
Medicine, 235(12), 1398-1411. doi:10.1258/ebm.2010.010137
Gareau, R., Audran, M., Baynes, R. D.,
Flowers, C. H., Duvallet, A., Louis Senécal, & Brisson, G. R. (1996).
Erythropoietin abuse in athletes. Nature, 380(6570), 113.
doi:10.1038/380113a0
John, M. J., Jaison, V., Jain, K.,
Kakkar, N., & Jacob, J. J. (2012). Erythropoietin use and abuse. Indian
Journal of Endocrinology and Metabolism, 16(2), 220-227.
doi:10.4103/2230-8210.93739
Lamberti, N., Finotti, A., Gasparello,
J., Lampronti, I., Zambon, C., Cosenza, L., . . . Manfredini, F. (2018).
Changes in hemoglobin profile reflect autologous blood transfusion misuse in
sports. Internal and Emergency Medicine; Official Journal of the
Italian Society of Internal Medicine, 13(4), 517-526.
doi:10.1007/s11739-018-1837-7
Lasne, F., Crepin, N., Ashenden, M.,
Audran, M., & de Ceaurriz, J. (2004). Detection of hemoglobin-based oxygen
carriers in human serum for doping analysis: Screening by electrophoresis. Clinical
Chemistry, 50(2), 410-5. doi:10.1373/clinchem.2003.026583
Medicosis Perfectionalis (Producer),
& Medicosis Perfectionalis (Director). (2019, Jan 10,). Erythropoietin
(EPO). [Video/DVD] Youtube.
National Institute of Diabetes and
Digestive and Kidney Diseases. (2014). Anemia in chronic kidney
disease Retrieved from https://www.niddk.nih.gov/health-information/kidney-disease/anemia
National Institute of Diabetes and
Digestive and Kidney Diseases. (2016). Kidney disease statistics for the united
states. Retrieved from https://www.niddk.nih.gov/health-information/health-statistics/kidney-disease
Revers, L., & Furczon, E. (2010). An
introduction to biologics and biosimilars. part I: Biologics: What are they and
where do they come from? Canadian Pharmacists Journal, 143(3),
134-139. doi:10.3821/1913-701X-143.3.134
Ribatti, D. (2008). Erythropoietin, the
first century. Leukemia Research; Leukemia Research, 32(8),
1169-1172. doi:10.1016/j.leukres.2008.01.018
Saladin, K. S. (2012). Anatomy
& physiology (6. ed., internat. ed. ed.). New York, NY:
McGraw-Hill.
Sanchis-Gomar, F., Martinez-Bello, V.,
Domenech, E., Nascimento, A., Pallardo, F., Gomez-Cabrera, M., & Vina, J.
(2009). Effect of intermittent hypoxia on hematological parameters after
recombinant human erythropoietin administration. European Journal of
Applied Physiology, 107(4), 429-436. doi:10.1007/s00421-009-1141-3
Spivak, J. L. (1986). The mechanism of
action of erythropoietin. The International Journal of Cell
Cloning, 4(3), 139-166. doi:10.1002/stem.5530040302
Varlet-Marie, E., Ashenden, M., &
Lasne, F. (2004). Detection of hemoglobin-based oxygen carriers in human serum
for doping analysis: Confirmation by size-exclusion HPLC. Clinical
Chemistry, 50(4), 723-31. doi:10.1373/clinchem.2003.026591
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