BLOOD DOPING (REPORT)

NOVEMBER 12, 2015

GROUP 7

Outline
I.
II.

III.

IV.
V.

Introduction
Body
A. Hemopoiesis
B. Blood
C. Increasing RBC Amount
Blood Doping
A. Blood Doping Scandals
B. Substances and Methods Used in Blood
Doping
1. Erythropoietin
2. Synthetic Oxygen Carriers
3. Blood Transfusions
Conclusion
Sources

I.

Introduction

Increasing the amount of red blood cells in the

body can be a powerful ergogenic aid acting in
multiple physiological mechanisms.
o Ergogenic enhancing physical
performance
o Can be natural or artificial
Natural means:
Training in areas of high altitude hypoxic
condition induce production of RBC
secures high hematocrit
o haematocrit ratio of the volume of
RBC to total volume of blood
Artificial means:
recombinant human EPO (rHuEPO)
Artificial Oxygen Carriers (AOC)
Blood Transfusion
- deemed unethical by World Anti-Doping Agency
- banned due to serious health risks to users

II. Body
A. Hemopoeisis
Generative process by which mature blood cells
develop from precursor cells.
Production per day:

o 1010 erythrocytes
o 4x108 leukocytes
Main sites of hemopoeisis (adult): bone marrow
(in medullary cavities of long bones and in the
vertebrae, ribs and sternum)

Blood cell formation:

o Hematopoietic stem cells may undergo:
self-renewing divisions
differentiating divisions
o hematopoietic stem cells
differentiation production of cells that
proliferate into progenitor cells (CFUs)
4 kinds of colony-forming unit:
o colony-forming unit granulocytemonocyte (CFU-GM)
o colony-forming unit erythrocyte (CFU-E)
o colony-forming unit lymphocyte (CFU-L)
o colony-forming unit megakaryocyte
(CFU-Me)
B. Blood
Tissue composed of 45% formed elements and
55% blood plasma
Functions:
o distribution of oxygen and nutrients
o logistical support
o communication between the tissues
o Waste disposal
3 Major Functional Classes:
o Erythrocytes
o Leukocytes
o Platelets
Erythrocytes - have iron-containing respiratory
pigment haemoglobin for the transport of
oxygen and carbon dioxide in the body
Leukocytes
o Granulocytes

neutrophils, eosinophils and

basophils

contains specific granules

important for immune responses
o Agranulocytes (AKA: Mononuclear
Leukocytes)
Lymphocytes (B or T)
Monocytes Macrophages
(peripheral tissues and organs)
Platelets from megakaryocytes
o For blood clotting (hemostasis)

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C. Increasing RBC amount
To gain more O2 capacity, athletes train under
hypoxic conditions (e.g. high altitude areas) to
induce RBC production
O2 capacity = time takes to fatigue =
advantage over competitors
Some athletes resort to BLOOD DOPING
(illegal) to increase O2 capacity since legally
increasing O2 capacity is more difficult.

III. Blood Doping

A. Blood Doping Scandals
Lance Armstrong
o 7-time Tour de France winner
o Stripped of his 7 titles after the scandal
o Confessed in 2013
80% of the Tour de France medalists between
1996 and 2010 have been "similarly tainted by
doping.
B. Substances or Methods Used for Blood Doping
Erythropoietin (EPO)
SYNTHESIS
o Glycoprotein hormone
o Naturally produced by the kidneys: renal
peritubular interstitial cells (90%), liver and
brain (~10%)
o Stimulates erythropoiesis in response to
hypoxia
o Kidney failure loss of EPO loss of
RBCs anemia
ISOLATION, EXPRESSION & PRODUCTION
o EPO: first isolated and purified from the
urine of anemic patients lead to
production of rHuEPO for treatment of
anemia
o Recombinant Human EPO (rHuEPO)
Exogenous
Cloned form EPO and mass produced
through recombinant human technology
Commercially produced from Chinese
Hamster Ovary (CHO) cells
Functionally equivalent to EPO
Differ from the endogenous EPO (native
human EPO) in that:
Molecular weight is higher
Neutral glycan content is lower

BLOOD DOPING

METHODS OF ADMINISTRATION
o Injections
Intravenous
Subcutaneous
Intramuscular
Intradermal
o Subcutaneous Injection:
Advantages: longer half-life more
sustained plasma erythropoietin
concentrationlower dose lower risk
of erythropoietin-induced hypertension
Can be self-administerd without the
need for a clinic visit and intravenous
access
! Low dosages work better than intermittent
peaks from high dosages
! Selection of the route depends on
pharmacokinetic considerations and
practicability.
DETECTION
o rHuEPO can be differentiated from
endogenous EPO by the ff:
urine tests: detects traces of longer
lasting versions of EPO
counting the number of immature
RBCs (reticulocytes) in the blood:
detectable for several weeks
CLEARANCE
o Half-life: 6 hours
o Cleared from the body in 1-3 days (effects
remain for a longer time)
o Hemoglobin values are back to normal after
approx. 4 weeks
MECHANISM
1. Kidney cells detect tissue hypoxia.
2. Kidney cells produce and secrete EPO into
plasma.
3. EPO is carried to the bone marrow.
4. In the bone marrow, EPO binds EPO receptors
on its target cells: CFU-E, proerythroblasts, and
basophilic erythroblasts.
5. Binding of EPO to these cells increases their
ability to survive and reach the reticulocyte stage
(by preventing apoptosis)
6. Once reticulocyte stage is reached, amount of
circulating RBCs is increased.

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7. With more RBCs in the circulation, more oxygen
is delivered to tissues.
8. Kidney cells detect increased oxygen delivery,
inhibits production of EPO.
EFFECTS
o Beneficial Effects/Advantages
! Epoietin first commercially available rHuEPO

Epoietin can correct low hemoglobin

levels in anemia.
O2 capacity = maximal uptake for
oxygen = time takes to fatigue
O2 in the body and muscles yields =
ATP = muscle contractions =
athletic performance
rHuEPO may be obtained and
administered without medical
supervision
o Harmful Effects
EPO = RBCs = high hematocrit =
cardiac stress and elevated arterial
blood pressure
May lead to heart disease, stroke,
cerebral or pulmonary embolism
With EPO use, dehydration from
exercise, enhanced platelet activation,
endothelin production and augmented
vascular smooth muscle in response to
norepinephrine and angiotensin II will
increase the risk for thrombosis
Misuse of rHuEPO may also cause
autoimmune diseases (WADA, 2013),
hyperkalemia and mild flu-like
symptoms (WebMD, 2014).
Synthetic Oxygen Carriers
AKA Artificial Oxygen Carriers (AOCs)
synthetic solutions capable of binding,
transporting, and unloading oxygen into
the body
NOT substitutes for blood
function as bridge oxygenators, where
they serve as alternatives to blood
transfusion (to delay transfusion of
allogeneic blood)
improve tissue oxygenation of organs
with poor blood supply improve
sports performance

BLOOD DOPING

2 Types of AOCs:
1. hemoglobin-based
2. perfluorocarbon-based

Hemoglobin-based AOCs

SYNTHESIS
Hemoglobin: may be artificially synthesized
via genetic engineering
o Purified human, bovine or recombinant
hemoglobin - used as raw materials for
making Hb-based AOCs
either then altered chemically or
microencapsulated
purification and chemical
alteration - reduces the risk for
disease transmission due to
allogeneic transfusion
Basis: cattle blood and human
blood is very similar
Recombinant hemoglobin -comes
from genetically altered hemoglobin
from E. coli
o Processes employed in chemically
altering the Hemoglobin include:
1. Intramolecular cross-linking
- prevents the Hb tetramers
dissociation suppresses renal
filtration of the Hb-based AOC
2. Polymerization increases the
size of the molecule through the
formation of Hb oligomers
prolongs half-life in plasma
3. Conjugation - the covalent bonding
of Hb to a bicompatible polymer
- Also used for size increase
4. Encapsulation- recreates the
natural properties of RBCs without
the antigens
Perfluorocarbon-based AOCs
o Perfluorocarbon (PFC)
Substances which can dissolve
large amounts of oxygen

require emulsification since these

compounds are not readily mixed
in aqueous systems, like blood
plasma
man-made compounds made up
of only carbons and fluorine
no natural sources

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obtained mainly from the

emissions from aluminum
production and into the
environment
2. other sources: manufacture of
electronics (esp. from refrigerants &
semiconductors)
CANNOT bind oxygen, but simply act

as solvents
Its high dissolving capacity (physical

solubility) for gases like oxygen

allow it to load and unload oxygen 2x
as fast as hemoglobin
METHODS OF
ADMINISTRATION/MECHANISM
Intravenous application or INFUSION
slow therapeutic introduction of nonblood fluid into a vein
extremely small; can enter the
microcirculation as emulsion droplets to
be later taken up by reticulo-endothelial
system/macrophages or exhaled for
excretion
A stable 60% emulsion has now been
developed (58% perfluorooctyl bromide
and 2% perfluorodecyl bromide).
released in very small amounts;
excessive amounts have negative
effects on brain, heart, and environment
(potential greenhouse gas)
LIQUID BREATHING
o Phenomenon where PFCs can carry
oxygen so well that humans can
actually survive breathing it in liquid
form (emulsified form usually mixed
with antibiotics, vitamins, salts)!
o discovered in World War II, to
increase chances of marine crew
surviving in case their nuclear
submarines are destroyed

BLOOD DOPING

DISADVANTAGES
o Manifestation of flu-like symptoms
o complement and phagocytic
activation (leading to hypertension)
o immunosuppression upon
administration of high dosages
o bronchospasms
o thrombocytopenia
IDEAL AOCs
o sufficient uptake and delivery at
physiologic oxygen tension, similar to
natural Hb with regard to oxygen and
carbon dioxide transport and delivery
o no major toxic or physiologic effects
o no chemical reaction with oxygen,
activation of complement system, no
increase in WBC count, no reaction
with plasma substitutes or platelets,
weak or no potential to produce
methemoglobin
o metabolized and eliminated by the
body
o does not interfere with capillary
circulation after protein deposition
o sufficient intravascular half-life,
remains in the plasma for 24h
o easy to use and store
o stable at room temp
o universally compatible
o available in large quantities
o low cost
o maintains arterial blood pressure and
pH
o immediately available
o viscosity similar to that of blood
EFFECTS
Beneficial Effects (refer to Table 1 of
appendix)
Harmful Effects
1. Vasoconstriction
-free Hb in the blood in the plasma
(note not in RBC since these Hb are
from the OACs) pass through
endothelium and bind to Nitric
oxides (NO) causes
vasoconstriction in endothelium
may lead to hypertension

ADVANTAGES
o small size
o low cost
o long shelf-life
o lack of vasopressive and pathogenic
effects
o synthetic with minimal risk of
immunologic reactions from
transfusion
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! Hemachromatosis - the disorder wherein too much iron

enters the tissues leading to diabetes mellitus and liver
damage.
4. Neurotoxicity
-Occurs in patients with
dysfunctional blood-brain barrier
-Free Hbs act as neurotoxins
that pass through BBB
damaging nerve tissues
5. Free Radical Production
-result from breakdown products of
free Hb: free heme and iron
causing free radical injury
-Oxidation increase in
concentration of methemoglobin
! Methemoglobin - the form of hemoglobin that cannot
bind to oxygen and thus cannot function in its transport

6. Interference With Laboratory

Studies
- Presence of plasma hemoglobin
and hemoglobinuria interferes with
the diagnosis of transfusion reaction
and other tests
7. Altered Immune Function
- PFCs are at times present in the
reticuloendothelial system (RES),
and prevent its proper functioning
Blood Transfusion
Two forms of blood doping:
o Autologous

Transfusion of ones own blood,

which has been stored (refrigerated
or frozen) until needed.
Homologous
Transfusion of blood that has been
taken from another person with the
same blood type.

! NO regulates the relaxation of smooth muscles by

preventing proendothelin from being converted into
endothelin, a powerful vasoconstrictor
2. Gastrointestinal distress, Malaise,
and Nephrotoxicity
-also caused by the scavenging of
free Hb of NO
3. Hematologic Disturbances
-free Hb in plasma blocks
phagocytosis (e.g. bacterial
ingestion)
-Hbs scavenging of NO may
result to platelet aggregation,
complement activation, coagulation,
iron overload, and hemachromatosis

BLOOD DOPING

MECHANISM
1. Months before competition, a pint of blood is
taken from the athletes body.
2. Blood is gently spun down to separate
serum from RBCs, which are then
refrigerated.
3. The process is repeated after about a
month.
4. This triggers the bodys erythropoietin
mechanism to replenish lost blood.
5. About a week before the event, the two
concentrated units of blood are dripped back
into his system, thereby increasing the RBC
count.
6. This reinfusion causes an induced and
temporary polycythemia or increase in
hemoglobin concentration due to an
increased number of erythrocytes, giving the
body a larger capacity for oxygen.
7. Maximal aerobic power is increased (lasts
only until optimal hematocrit is reached)
SIDE EFFECTS

Allergic reactions
Fever
Rashes
Hives
Repeated blood transfusions over time
dangerous buildup of iron in the body
chronic fatigue, joint pains, abdominal
pains, liver disease
improperly stored blood and improperly
administered transfusions secondary
bacterial infection
High hematocrit less cardiac output
and aerobic power due to steep
increase in blood viscosity risk for
stroke and heart failure
Increased risk of HIV, Hepa B and C
due to methods used (needle
introduction)

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IV. Conclusion

BLOOD DOPING

Figure 2. Mechanism of Blood Transfusion

RBC production is regulated by EPO

levels of RBC = oxygen-carrying capacity
Training at hypoxic conditions like in areas with
high altitudes higher oxygen carrying capacity
(all these can be achieved through blood doping)
EPO, AOCs, Blood Transfusions most common
blood doping techniques
EPO stimulates RBC production
AOCs help increase transport of oxygen without
apparent increase in libro
Blood transfusions blood is obtained, separated
and reinserted in the circulation

V. References
Lecture slides prepared by Group 7 (super daming
references promise sayang space)

VI. Appendix
Figure 1: Hemopoiesis

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BLOOD DOPING

Figure 3. Mechanism of EPO

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