Drugs, Ethics and Medicolegal Issues in Sport

1
Blood doping, erythropoietin and altitude training
Lecture 1 Drugs, Ethics and Medicolegal Issues
in Sport Professor Bruce Lynn MSc School of
Human Health and Performance
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• The basic idea
• To raise the oxygen carrying capacity of the
  blood and so improve performance in events
  involving aerobic exercise, i.e. any events
  lasting more than 1-2 minutes.
• This can be done by increasing the haemoglobin
  (Hb) content in the blood
• By legal means (altitude training) or
• By illegal means
• blood doping or boosting
• use of the hormone erythropoietin.

3
Some facts and figures
We have 3.1013 red blood cells, containing a
total of 900g of haemoglobin. Red cells last on
average 120 days, so we need to replace 1/120
every day, i.e. 2.5.1011 cells, or 3 million
every second! This massive production of red
cells takes place in the bone marrow.
Normal ranges of Hb and haematocrit.
Definitions Hb Amount of this oxygen-carrying
protein within red blood cells. Units
g/decilitre blood (decilitre 100ml). Hematocrit
Proportion of red blood cells to the total blood
volume ( packed cell volume or PCV).
4
Blood doping The direct method of increasing
blood haemoglobin content. Blood, or more
usually concentrated red cells, are transfused
via an intravenous catheter. If blood cells
from a donor are used this is heterologous
transfusion. Alternatively the athletes own
blood can be collected, stored, then transfused
back this is autologous transfusion. Either
way careful storage of red cells is necessary.
5
Blood doping Blood doping (or boosting)
definitely works although estimates of the extent
of improvements in performance vary. A recent
review concludes that endurance performance is
typically improved by 2-3 (Gledhill et al,1999)
but other studies have shown maximal oxygen
uptake (V02max) up by 13 (Robinson et al,
1982). But remember, even a 2 improvement is
massive in competitive endurance events such as
athletics. Over 10,000 meters this represents
200 meters, i.e. winning by half a lap!
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• Blood doping
• There are a number of difficulties.
• Heterologous transfusion
• Great care needs to be taken over tissue
  matching.
• There is the risk of getting a blood-borne
  infection such as AIDS or hepatitis.
• Autologous transfusion
• Means long periods out of competition as it takes
  4-6 weeks to recover from a blood donor session
  (but see later for a possible fix).
• For both methods the effects begin to wane within
  3-4 weeks, so then need a repeat transfusion.
• Nevertheless there have been plenty of rumours.
  It would not be surprising if rumours of blood
  boosting occurred as a significant number of
  people need to be involved in the process of
  taking blood, storing it, re-infusing it and
  checking haematocrits.

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From the BBC Sport web site, 24 November 2006
Hamilton to ride for Tinkoff team
Cyclist Tyler Hamilton, whose two-year suspension
for blood doping ended in September, has signed
for Italian-based Russian team Tinkoff.
Hamilton was sacked by Phonak after his positive
test
The 35-year-old tested positive for a blood
transfusion at the 2004 Tour of Spain, weeks
after winning Olympic individual time trial gold.
Hamilton was allowed to keep his title because
his B test was destroyed by being deep-frozen.
"I'm ready to make a comeback," he told the All
Sport news agency. 2007 update. Tyler has
cycled hardly at all in 2007 and is now beimg
connected to Operacion Puertasee next slide
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Operacion Puerta May 2006. Police raids in Madrid
and Zaragosa as part of Operation Puerta
OP). Found lots of steroids but also
transfusion gear and 100 packets of blood in
clinic of Dr Eufemianos Fuertes. Possibly the
better testing for EPO is driving people back to
transfusions. Can overcome one of the drawbacks
of donating your own blood for later use by using
EPO to speed the recovery to normal levels
apparently the Fuertes method. OP blood packets
were labelled with pseudonyms, but a number of
top cyclists were easily identified.
9
Operacion Puerta, cont The authorities are now
trying to match blood from bags to cyclists
through DNA testing. No cyclists have been
successfully prosecuted! But several who
initially protested innocence have confessed
(e.g. Ivan Basso says he went to Fuertes but
never actually used the blood.). I think the
main case against Dr Fuertes has still to go to
court at which point we can expect a lot of
finger pointing! But already the authorities have
all the Swiss bank account details which must
make interesting reading.
10
Blood doping testing Heterologous
transfusions The current test looks for 15
different minor antigens and can detect the
presence of just one unit (about 500 millilitres)
of transfused blood Autologous tranfusions No
current test. But could look for low levels of
EPO as neg feedback will reduce EPO synthesis
following a tranfusion Or can look for unusual
RBCreticulocyte ratio as again transfusion will
reduce natural production and so reduce
reticulocyte count
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Erythropoietin (EPO)   EPO is a hormone that is
released from the kidney in response to tissue
hypoxia and travels to the bone marrow.
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Erythropoietin is a large peptide (or small
protein) comprising 165 amino acids with a large
number of attached carbohydrate residues (it is
30 glycosylated).
13
Action of EPO In the bone marrow it stimulates
production of pronormoblasts (immediate red blood
cell (erythrocyte) precursors) from CFU-E cells
(committed unipotential colony-forming unit -
erythroid). Pronormoblasts in turn develop into
normoblasts. The normoblasts start to lose
their nucleus and become reticulocytes. The
retuculocytes enter the blood system and finally
develop into red blood cells with no nuclear
material visible at all.
Darkened sections of the arrows indicate times of
Epo receptor expression and Hb synthesis. BFUe,
burst-forming unit erythroid CFUe,
colony-forming unit erythroid.
From S. Elliott. British Journal of Pharmacology
(2008) 154, 529541
14
Recombinant human EPO is used to treat anaemia
associated with kidney failure, e.g. for those on
dialysis, and so is widely available, although
pretty expensive.
Mean (SD) level of hematocrit (upper panel) and
hemoglobin (lower panel) for the rhEPO treated
subjects (filled symbols, N 10) and control
subjects (open symbols, N 10) for the treatment
(days 130) and posttreatment period (days
P1P28). Dashed lines show prerace limits of the
International Cycling Union (ICU) for hematocrit
(hematocrit 50) and of the International
Skiing Federation (FIS) for hemoglobin
(hemoglobin 18.5 gL-1).
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EPO induced increases in blood Hb appear, like
blood doping, to increase performance in
endurance events (Birkeland et al, 2000).
Mean (SD) level of maximal oxygen uptake (bars)
and time to exhaustion (circles) for the
rhEPO-treated subjects (hatched/filled symbols, N
10) and control subjects (open symbols, N 10)
before treatment (day 0) and in the posttreatment
period (days P1P28).
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It is important not to get Hb too high. Blood
viscosity increases with effects on heamodynamics
that mean harder work for the heart, and so an
increased risk of heart problems.
Pathologically elevated red cell counts also
lead to a higher risk of clots. But many people
who live at altitude have elevated Hb without any
circulatory or cardiac problems - but maybe have
made compensating adaptations. A spate of
unexplained sudden deaths in cyclists in late
1980s was attributed to arrival of EPO on the
scene. There have also been problems with side
effects in some long- term dialysis patients.
17
Limiting haematocrit Some sports (e.g. cycling)
set upper limits on haematocrit (50 for male
competitors) Justified as a ban to competing on
health grounds (i.e. risks due to increased blood
viscosity). High haematocrit was not a doping
offence. But 1 of normal population (and 20 of
native highlanders) have haematocrits at or above
this level. It is also claimed that
haematocrit tests are subverted by masking with
plasma expanders. If you expand the plasma
volume, then the proportion of red cells (the
haematocrit) will fall, even though the total
number of red cells is still much elevated.
Measuring total blood volume is not an easy
thing to do, so expanding plasma volume is a
straightforward way to avoid detection in a
heamatocrit test.
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Plasma expanders Cyclists apparently get by the
haematocrit test by having saline infusions
shortly before being tested at the start of
events. The short term boost to blood volume may
even also aid performance! Others use high
molecular weight expanders. But these are
easily detected over a long period, as almost the
entire Finnish cross-country ski-ing team found
to their cost at the world championships in 2001!
(Seiler, 2001)
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Detecting EPO use. It is possible to distinguish
recombinant EPO from endogenous EPO, and this can
even be done in urine samples. Unfortunately,
EPO has a short life in the body and is only
detectable for 1-2 days. As blood cells last 120
days, single tests at the time of competition are
useless.
Mean (SD) level of serum EPO for the
rhEPO-treated subjects (filled symbols, N 10)
before treatment (day 0) and posttreatment period
(days P1P28), as well as the baseline value for
the control subjects (open symbols, N 10).
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Detecting EPO use. In practice, the boost from
EPO does not last 120 days. As can be seen from
the earlier figure, it is well down by 20 days
(presumably why the Festina cycling team was
toting EPO around in the 1998 Tour de France, an
event that lasts 3 weeks). And urine testing
gets more sensitive all the time. There is also
a blood test that looks at the proportion of
immature red cells that has the ability to detect
EPO use up to 14 days after stopping
injections. So the window of opportunity is
narrowing
21
Urine testing for EPO
Anti-doping urine analysis demo-nstrating the
presence of recom-binant human erythropoietin
(rHuEPO) in urine (see lane 4). Lane 1 rHuEPO
standard 2 positive urine (control) 3
negative urine (control) 4 sample declared
positive 5 darbepoetin alfa (Aranesp standard).

Test based on isoelectric focusing patterning and
a double blotting protocol. Works because the
exogenous isoforms of rHuEPO are less acidic than
the endogenous EPO Lasne F, de Ceaurriz J. Nature
(2000) 405635.
22
NESP Interestingly, a longer lasting version of
EPO is now available for clinical use,
darbepoetin or Aranesp (nesp in the peleton!).
This is effective for longer, so patients (or
athletes) need fewer injections and this is more
convenient and cheaper. It does make it easier
to detect, however, and this version of EPO was
in fact successfully detected in competitors in
the 2002 Winter Olympics. DYNEPO rHuEPO made in
a human cell line. Has sialate etc residue
profile very similar to natural human
EPO. French anti-doping lab claims it can detect
it, and found it in samples from 10 riders in the
2007 Tour de France. But test not yet approved
by WADA, so these do not count as positives
23
CERA Continuous erythropoietin receptor
activator. Another longer lasting version of EPO
available for clinical use. This is effective
for longer, so patients (or athletes) need fewer
injections and this is more convenient and
cheaper. This molecule is EPO plus a large
methoxy-polyethylene glycol polymer, doubling the
molecular weight to 60kDa. Binds with low
affintiy to EPO receptor but not internalised, so
keeps acting. Large size and unique structure
makes detection easy. But test not out until
this summer but CERA in trials, and so available
before that. ADAs now want to back test old
samples. NB there were hits for CERA in Tour de
France in Summer 2008. EMPs Small molecule
EPO-mimetic peptides. Typically 20 amino acid
residues. One EMP, Hematide, is now in trials.
No sequence homology to EPO. Could imagine a
rich sponsor getting an effective EMP synthesised
without ever publishing the sequence.
24
Iron metabolism, ferritin, transferrin It is
also possible to look at iron metabolism. Hb
contains iron and the body has well developed
transport and storage systems for iron so that
enough is available to produce the red cells we
need. If EPO stimulates red cell production
then the levels of the iron storage protein
ferritin fall. At same time the levels of
soluble transferrin receptor (needed to get iron
into developing red cells) rise in response to
the increased demand. So by looking at the
ferritintransferrin ratio you get quite a good
indication of red cell production rates.
Unfortunately you can mask these changes by
increasing iron and folic acid intake in diet.
It may be possible to track transferrin receptor
mRNA levels - these rise up to 40 times with EPO
use, but no-one has a test based on this yet.
25
Mean (SD) level of soluble transferrin receptor
concentration (sTfR upper panel), ferritin
concentration (middle panel), and sTfR/ferritin
ratio (x100 lower panel) for the rhEPO treated
subjects (filled symbols, N10) and control
subjects (open symbols, N10) for the treatment
(days 130) and posttreatment period (days
P1-P28). Dashed line in upper panel shows the
mean 2 SD (95 CI) for all subjects at baseline
( 4.6 mg/L).
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With better testing, the problem of EPO abuse may
now wane. Given the dynamics (about 30 days
useful boost, but possible detection up to 14
days), a strategy of random tests in the run up
to major competitions looks like the way to go.
It will be interesting to see how many sports
in how many countries develop such a
programme! In the UK most Olympic sports now
have at least some out-of-competition
testing. Cycling, after the fiasco of last years
Tour de France and the loss of major sponsors, is
also getting out-of-competitions testing
organised. And some team managers claim they are
going to test their own riders.
27
Time line of EPO abuse

• 1984 rh-EPO licensed
• EPO abuse reported
• Festina team car full of EPO in tour de France
• First reliable test for EPO
• Chinese rowers withdraw from Sydney Olympics
• No confirmed EPO ves at Sydney
• Yegereva tests ve, gets off on technicality,
  goes on to win world 5000m
• Winter Olympics. 3 cross-country skiers banned
  for Darbepoetin use.
• 2003 David Miller, darling of British cycling,
  caught using EPO
• Iban Mayo banned from Tour de France for EPO
  ve
• Riccardo Ricco, Moises Duenas, Manuel Beltran,
  all ve for CERA in TdF

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Other actions of EPO EPO is actually a cytokine,
and like many cytokine-like hormones, is turning
out to have many actions They mostly involve
hypoxia or ischaemia For example the respiratory
response to hypoxia is boosted by EPO, partly by
an action on chemoreception in the carotid
body. EPO is also produced in the brain and
affects hypoxic responses centrally. (Note EPO
does not cross the blood-brain barrier) Finally,
EPO is tissue protective against stroke and
cardiac infarct!
29
Altitude acclimatisation Living at altitudes
above about 2000m leads to enough tissue anoxia
to stimulate EPO release and red cell production.
For example, a group who spent 30 days at the
top of Pikes Peak (4300m) in Colorado had average
increases in Hb from 13.7 on arrival to 16.2 at
departure, with parallel increases in haematocrit
(from 43-48). These are similar to the
increases seen with blood doping or EPO use.
30
Altitude acclimatisation However, on return to
sea level, little if any increase in endurance
performance is found. The reasons are complex.
Firstly, it is not possible to train at maximum
intensity at altitude just because the
atmospheric oxygen level is lower. Secondly,
the adjustments of the circulation to altitude
involve more than just an increased Hb.
Hyperventilation, a normal response to the
lowered oxygen level, leads to increased carbon
dioxide excretion and eventually to a reduction
in the buffering power of the blood. This may
reduce performance levels as lactic acid produced
during high intensity exercise will not be so
well neutralised. There may also be reductions in
blood volume and shifts in the Hb dissociation
curve, changes that may impair performance.
Note, however, that for competitions held at
altitude, suitable acclimatisation is essential.
31
Live high, train low To get round some of these
problems you live and sleep up the mountain, but
travel down to sea level to train. This option
is now available to those of us who do not live
conveniently close to a suitable mountain the
nitrogen tent or house. Athletes live in
nitrogen tents with the oxygen level reduced to
15-16 (equivalent to being at 2500m altitude).
These strategies allow you to train in the
normal way, but should provoke a useful boost in
blood Hb due to the time spent at simulated
altitude. There will still be problems with
other, disadvantageous, circulatory adaptations.
Results have been contradictory some showing
improvements, others not. There is even dispute
as to how far HiLo actually stimulates increased
Hb. See articles by Wilbur and by Gore in Med Sci
Sports Medicine (2007) 39(9)
32
The coaches and athletes certainly believe
altitude training important Here is Steve Cram
writing in the Guardian Tuesday October 2, 2007,
after the Great North Run this year For most
this still means training in the mountains but
increasingly it involves hypoxic tents, where the
pressure can be adjusted to mimic any altitude
and it is where many sleeping hours are spent.
Salazar likes to combine the two. In preparation
for the world championships in Osaka his group
trained at Park City in Utah at 7,000-8,000ft. In
addition he advises sleeping in the tents at a
setting of anything up to 13,000ft.
http//sport.guardian.co.uk/athletics/comment/0,
,2181691,00.html
Kara Goucher, coached by Salazar, beating Paula
Radcliffe. 2007 Great North Run
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Nitrogen tent
http//altitudetraining.com/wrap/images/four30_lar
ge
34
Percent change in 5000-m time from baseline
performance (at 6 weeks) in three training
groups.  
Recalculated and redrawn from data of Levine
Stray-Gundersen, 1997. The bar labelled likely
range of true change is the author of this
reviews estimate of the 95 confidence interval
for points other than the baseline. 
http//www.sportsci.org/traintech/altitude/wgh.htm
l
35
Report posted 7 Oct 2008 Bellamy sleeping in
altitude tent Wales captain Craig Bellamy is
sleeping in an altitude tent in a bid to save
his career. The injury-prone West Ham striker is
to make his first start for a year when Wales
host Liechtenstein on Saturday Oct 11, 2008.
"I have got my own altitude tent and I sleep in
it every night because it speeds up recovery and
improves overall fitness," Bellamy admits. His
tent simulates high altitude by maintaining a
lower oxygen concentration. One of the benefits
is to increase red blood cells and enzymes to
speed recovery. "My rehabilitation has been
quite extraordinary," Bellamy told BBC Sport.
"And I want to really attack the last years of
my career in a positive manner. "The altitude
tent improves endurance, helps speed and makes
you a fitter athlete. "I'm ready to play 90
minutes and play two games in five days because I
feel good and I've done all of the groundwork."
36
Exercise efficiency improves with altitude
training From   GORE Med Sci Sports Exerc,
Volume 39(9).September 2007.1600-1609
Percent decrease in oxygen consumption during
submaximal exercise after various forms of
altitude acclimatization. mean SD. 1) Three
hours per day for 2 wk of intermittent exposure
to normobaric hypoxia (12.3 FIO2). 2) 20 nights
of about 8 h per night of sleep at a simulated
altitude between 2000 and 3100 m, and training
near sea level (600 m). 3) 23 nights of about 10
h per night of exposure to a 3000-m simulated
altitude. 4) 29 days of about 12 hd-1 of
exposure to simulated LHTL at 3000 m. 6) Exposure
to intermittent hypobaria of 4500 m, 3 90
minwk-1, for 3 wk. 7) Five to six nights at 2500
m followed by 8 to 12 nights at 3000-3500 m,
sleeping at simulated altitude and all training
at 1200 m. 7) Mountain climbing for 3 wk to 6194
m. 8 and 9) Long-term residence at 3500-4500 m.
37

• Summary Blood doping, erythropoietin and
  altitude training
• Increasing blood haemoglobin levels to the very
  top end of the normal range increases aerobic
  exercise capacity significantly.
• Such effects are obtained with direct blood
  transfusions or with EPO injections.
• Both procedures are banned. The current tests
  look adequate to eliminate EPO abuse if applied
  out of competition.
• Altitude (real or simulated) acclimatization is
  not the same as blood boosting. The overall
  changes to blood and circulation are more
  complex, and deliver smaller increases in terms
  of sea level performance. There are also
  interesting improvements in non-haematological
  aspects of performance.

38
Methods to enhance oxygen transport include
stimulation of endogenous Epo (eEpo) synthesis,
stimulation of erythropoiesis (e.g., through
administration of erythropoiesis-stimulating
agents (ESAs) such as recombinant human Epo
(rHuEpo)) or through direct increases in delivery
of oxygen (e.g., transfusion, administration of
artificial oxygen carriers or through enhanced
oxygen unloading). 2,3-DPG, 2,3-diphospho
glycerate HBOC, Hb-based oxygen carrier.
From S. Elliott. British Journal of Pharmacology
(2008) 154, 529541
39
References Overviews Drugs in sport blood
doping, erythropoeitin and altitude training. A.
Narvani et al, Key Topics in Sports Medicine,
pp78-82, 2006. Blood boosting and sport.
Armstrong, DJ. and Reilly, T. Chapt 7 in Drugs in
sport, 3rd edn, ed DR Mottram, 2003. S Elliott
(2008) Erythropoiesis-stimulating agents and
other methods to enhance oxygen transport.
British Journal of Pharmacology (2008) 154,
529541 Papers cited Birkeland, KI,
Stray-Gundersen, J et al (2000) Effect of rhEPO
administration on serum levels of sTfR and
cycling performance. Med-Sci-Sports-Exerc. 32(7)
1238-43 Gore, C. J., et al. (2003).
Second-generation blood tests to detect
erythropoietin abuse by athletes. Haematologica
88, 333-344. Gledhill, N, Warburton, D and
Jamnik, V. (1999) Can J Appl Physiol, 24,
54-65 Hahn, A. G. Gore, C. J. (2001). The
effect of altitude on cycling performance a
challenge to traditional concepts. Sports Med.
31, 533-557. Robinson, RJ et al, (1982) The
effect of induced erythrocythemia on hypoxia
tolerance during exercise. J Appl Physiol. 53,
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Avois, P Mangin, M Saugy, Erythropoietin and
blood doping, Br J Sports Med 200640(Suppl
I)i30i34. Seiler, S (2001) Doping Disaster for
Finnish Ski Team a Turning Point for Drug
Testing? Sportscience 5(1), sportsci.org/jour/0101
/ss.html Stray-Gundersen J, Videman T, Penttilä
I, Lereim I., Abnormal hematologic profiles in
elite cross-country skiers blood doping or?
Clin J Sport Med. 2003 May13(3)132-7.Links GORE,
CHRISTOPHER JOHN, CLARK, SALLY A., SAUNDERS,
PHILO U. Nonhematological Mechanisms of Improved
Sea-Level Performance after Hypoxic Exposure.
Medicine Science in Sports Exercise.
39(9)1600-1609, September 2007. WILBER, RANDALL
L., STRAY-GUNDERSEN, JAMES, LEVINE, BENJAMIN D.
Effect of Hypoxic "Dose" on Physiological
Responses and Sea-Level Performance. Medicine
Science in Sports Exercise. 39(9)1590-1599