by
Jason Fong
In many endurance specific sports,
such as cycling and cross country skiing, an increase in
maximal aerobic power (VO2max) is desirable. Blood doping
has provided an avenue for athletes to increase VO2max levels
artificially. In recent years, there has been a rise in the
use of recombinant human erythropoietin (rhEPO), which has
many physiological effects, including an increase in oxygen
transport. Because rhEPO can increase athletic performance,
and can possibly have negative side effects, it has been
banned by many competitive sports organizations, such as
the International Olympic Committee (IOC), International
Cycling Union (ICU) and International Skiing Federation (ISF)
(Audran 639). There are problems in directly testing for
rhEPO in blood or urine due to its short half-life between
4 and 13 hours (Magnani 560), and similar structure to endogenous
erythropoietin (EPO) (Wide 1569). Detection of rhEPO may
require an indirect approach that reveals physiological changes
in blood samples. The goal of this paper is to determine
if indirect markers of increased erythropoiesis are accurate
in detecting rhEPO abuse in athletes.
i. Physiological Effects of rhEPO
An increased concentration of red blood
cells translates into more hemoglobin to carry oxygen to
working muscles. This can lead to an increase in VO2max,
the maximum rate of oxygen usage by the body during maximal
work. Those who have a higher VO2max can exercise for longer
periods of time, resulting in an increase of one’s performance
level. By using rhEPO, athletes are able to artificially
stimulate erythropoiesis, the formation of red blood cells
and gain its physiological effects.
When there is low cellular oxygen, the
kidney produces erythropoietin, which travels through the
blood and stimulates erythropoiesis in bone marrow. An increase
in red blood cells results in increased hematocrit levels
(the percentage of blood composed of red blood cells), reticulocytes
(immature erythrocytes), hemoglobin and soluble transferrin
receptors (sTfr), an iron binding protein in the blood. The
amount of sTfr is proportional to erythropoiesis due to its
concentration being dependent on cellular transferrin receptor
(Tfr), or cell mass. These markers may stay at levels above
the baseline (defined as level before rhEPO injection) from
1 to 14 days after the last treatment of rhEPO. In order
to expose the use of rhEPO, one must determine at which levels
these markers show a clear increase in erythropoiesis indicating
rhEPO abuse. This paper will try to determine the amount
of time at which these levels show an increase in erythropoiesis
pointing to rhEPO abuse.
ii. Harmful Side Effects of rhEPO
On the other hand, too much rhEPO can have
detrimental consequences. It is believed that some cyclists
who did not monitor their use of rhEPO died of sudden death.
Apparently, their high concentration levels of red blood
cells led to a significant increase in blood viscosity, and
when coupled to dehydration during intense exercise, blood
clotting occurred. (Bunn 522) Blood clotting can lead to
an increase in heart attacks and strokes. Also, the use of
rhEPO can cause the formation of antierythropoietin antibodies
against rhEPO and endogenous EPO, which leads to red-cell
aplasia (Casadevall 470). In practice, it is common for rhEPO
injections to be accompanied with intravenous injections
of iron. Iron overload may occur and lead to symptoms similar
to those of genetic hemochromatosis. (Cazzola 561) The harmful
side effects of rhEPO along with the physiological changes
that indirectly increase VO2max, have led to the prohibition
of this drug’s use in sports.
iii. Direct Detection of rhEPO
Detection of rhEPO poses many problems.
As already stated earlier, rhEPO has a short half-life and
is similar in structure to endogenous EPO. These two factors
make blood and urine detection difficult since electrophoretic
techniques must be done within a limited timeframe in order
to be able to distinguish between the two forms of erythropoietin.
It is possible to detect rhEPO in urine and blood serum as
was done by Wide. He tested 15 healthy, moderately-trained
men between the ages of 19 to 40 years old. At a fairly low
dosage, 20U/kg three times a week for eight weeks, rhEPO
was accurately detected in blood up to two days after the
last injection; and in urine one day after the last injection.
From the data, sensitivity of the test decreases to fifty
percent in detecting rhEPO in blood or urine after three
days from the last injection. Detection was done by separating
rhEPO from EPO by charge using electrophoresis. (Wide 1574-5)
Endogenous EPO is slightly more acidic than rhEPO (Lasne
635). After one week, this method of detection fails in detecting
any rhEPO. Although this technique is accurate, it is only
accurate for a short period of time. (Wide 1575)
i. General Procedures and Results
In several studies, blood markers were
determined that could possibly be accurate detectors of rhEPO.
Each study, on average, sampled blood once before rhEPO administration,
and twice a week for a month during and after rhEPO administration.
Blood sampling was done in the morning before exercise within
24 hours after each injection. This decreased the likelihood
of hemoconcentration due to dehydration (Parisotto and Gore
570). Most studies had a placebo group, all of which had
results that were similar to baseline values. Baseline values
were determined before the first administration of rhEPO.
With each study, the results showed that there was an increase
in concentrations of reticulocytes, hemoglobin, EPO, and
sTfr during treatment; increases were also seen in the hematocrit
levels and the sTfr/serum protein ratios during treatment.
The following five studies are summarized in Figure 1.
ii. Study 1: Measurement of Blood and Physiological
Markers
In a study done by Audran, nine well-trained
athletes (seven males, two females) received a 50U/kg dosage
of rhEPO daily for 26 days. Tested were four triathletes,
two cyclists, one rower, one swimmer, and one handball player,
averaging an age of 24 years old and weight of 73kg. During
treatment, significant increases in reticulocyte, EPO and
sTfr concentrations and sTfr/serum protein ratios were seen
by day ten, whereas hemoglobin and hematocrit levels did
not clearly increase until day 14. From the results after
the last rhEPO injection, reticulocyte, hemoglobin and sTfr
concentrations remained above baseline values for seven days;
and hematocrit levels remained above baseline up to 14 days;
and EPO levels stayed above baseline for two days, as was
expected due to its short half-life. Physiological tests
were also done to measure the effect of rhEPO. On average,
VO2max increased by 5ml/min/kg, and maximum heart rate lowered
by 9 beats/min after the treatment period. Audran also analyzed
sTfr/serum protein ratio data of athletes who lived in high
and low altitudes and who did not receive rhEPO, and compared
them to those who had received rhEPO in his study. Using
the average ratio of those that lived at high altitudes as
the threshold limit, the athletes in his study had an average
that was higher than the athletes that lived at high altitudes
up to seven days post-treatment. (Audran 641-2)
iii. Study 2: Effect of Iron Supplementation
with rhEPO Injections
In a similar study, Parisotto and Gore
gave 27 well-trained athletes (22 males, 5 females) 50U/kg
of rhEPO three times a week for 25 days. The average age
of the subjects was 25 years old and average weight was 70kg,
and their sports were not specified. In conjunction with
rhEPO injections, one group received oral iron supplements
daily and another group received iron injections once a week,
both containing 100mg of iron. During rhEPO treatment, reticulocyte
and sTfr levels had increased by day ten. Hematocrit and
hemoglobin levels did not show a clear increase until the
third week. In post-treatment tests, reticulocyte concentration
stayed above baseline up to one week, hematocrit and hemoglobin
concentrations remained elevated for three weeks, EPO concentrations
decreased to baseline values within five days, and sTfr concentrations
remained above baseline for two weeks. VO2max increased by
6.5 ml/min/kg after the last treatment and returned to baseline
within four weeks. Those taking the oral supplements had,
on average, higher concentrations of each blood marker compared
to those with iron injections. (Parisotto and Gore 567) The
overall results from Parisotto and Gore are very similar
to Audran’s study, and confirm the validity of both
investigations.
iv. Study 3: Differences between Ethnic
Groups and Sampling Times
In a study by Parisotto and Wu, the
above results were reproduced. Two groups were studied, one
resident of Sydney (49 athletes- 33 males, 16 females) and
the other resident of Beijing (24 athletes- 12 males, 12
females). For the Sydney group, average age and height were
28 years old and 77kg. For the Beijing group, average age
and height were 21 years old and 62kg. In the Beijing group,
blood sampling was taken 24 and 48 hours after each injection
as opposed to 24 hours for the Sydney group. The objective
was to verify if the tests could identify rhEPO users after
a passing of time since EPO levels tend to decrease due to
its short half-life. The results of the of the Sydney and
Beijing group were similar to one another, and similar to
the study done by Parisotto and Gore. In addition, Parisotto
and Wu derived equations to detect rhEPO users. The equation
for detecting current users of rhEPO combined hematocrit
level, reticulocyte concentration, EPO concentration, sTfr
concentration and macrocyte percentage. The equation for
detecting recent users of rhEPO (two weeks after the final
injection) combined hematocrit level, reticulocyte concentration
and EPO concentration. rhEPO users had higher values based
on each equation that distinguished them from the nonusers.
Both equations had a very high sensitivity and high specificity
in detecting those that were rhEPO users and those that were
not. Parisotto and Wu demonstrated that rhEPO users could
be distinguished accurately based on model equations with
threshold values for detection. (Parisotto and Wu 129-34)
v. Study 4: Effect of Differing Dosages
In a study done by Magnani, one group
(A) received 200U/kg rhEPO twice a week for 10 days; a second
group (B) received 200U/kg of rhEPO along with 25mg of iron,
25mg of folic acid and 2500mcg of Vitamin B12 twice a week
for 10 days; a third group (C) received 30U/kg of rhEPO along
with the same amount of supplements as the group B twice
a week for four weeks. Group C did not have a post-treatment
period. The study included 18 male athletes ranging from
19 to 28 years old; their ages, weights and sports were not
identified. Group A and B displayed a clear increase in sTfr
concentrations by day four of treatment, and were well above
baseline after one week post-treatment. Group C increased
above baseline by day four, but never showed a great increase
like group A and B. Reticulocyte concentration increased
by day four for each group. Four days after the last injection,
group A and B began to decline in reticulocyte concentration,
and group C began to decline by day 18. Hematocrit levels
remained well above baseline up to two weeks post-treatment
for group A and B, and until day 24 for group C. This study
also used beta-globin mRNA as a marker for detecting rhEPO.
Beta-globin mRNA expression increased during administration
of rhEPO for all three groups. For group A and B, beta-globin
increased until day 14 and then experienced a great decrease.
Group C reached its highest level on day 18 and then experienced
a slow decline. Magnani formed an equation to detect rhEPO
users based on hematocrit level, reticulocyte concentration,
sTfr concentration, Tfr mRNA concentration and beta-globin
mRNA concentration. All three groups were detectable from
days 4 to 18 accurately. Times before and after those specified
showed less than a 20% accuracy in detecting rhEPO users.
(Magnani 560-7)
vi. Study 5: Measurement of Blood and Physiological
Markers
In a study done by Birkeland, 20 healthy,
well-trained male athletes received 200U/kg of rhEPO three
times a week for 30 days. All subjects were given 270mg of
oral iron supplementation. These athletes had specialties
in cycling, orienteering, running, triathlon, swimming and
cross-country skiing. Average age and weight were 23 years
old and 73kg. sTfr levels increased above baseline by day
four of treatment and lasted one week post-treatment. Hemoglobin
concentrations showed significant increase by day ten of
treatment and remained above baseline for three weeks post-treatment.
Hematocrit levels had increased by day seven of treatment
and remained well above baseline ten days post-treatment.
VO2max increased by five ml/min/kg after the last treatment
and remained above baseline for over 25 days. (Birkeland
1240-1)
From the results, one can see that
there are indirect methods that can test for the presence
of rhEPO use. The short half-life of rhEPO makes it difficult
to use direct methods of blood and urine testing for detection.
However, after discontinuing the constant use of rhEPO, its
physiological effects may be prolonged to a certain degree.
These effects can prove useful in the detection of rhEPO
even if it is not present in the blood. On average, hematocrit
levels remain detectable 14 days post-treatment, and concentrations
of reticulocytes, hemoglobin, and sTfr remain detectable
seven days post-treatment. Also, different doses of rhEPO
can produce different physiological effects. In general,
greater dosages of rhEPO induce a quicker response of increased
erythropoiesis than lower dosages. In effect, less time is
required until the effects of rhEPO are apparent in blood
sampling. Iron supplementation, whether oral or injection,
increases the effect of rhEPO in terms of concentration levels.
However, it does not affect the time of onset of the physiological
effects. From the data, it is possible to conclude that iron
intake increases the effects of rhEPO induced erythropoiesis
after the administration period, but further tests must be
done to verify this.
For the most part, the results of each
test confirm one another, and show that there are predictable
markers that can account for the usage of rhEPO. However,
indirect markers of rhEPO cannot be used individually. There
is considerable variation in hematocrit level, and concentrations
of reticulocytes, sTfr and hemoglobin among people in the
population (Parisotto and Wu 134). Also, hematocrit levels
tend to flux by tilting one’s head or drinking a saline
solution due to changes in hydrostatic pressure within the
body (Schmidt 135). Parisotto and Magnani proposed two different
equations combining several of these parameters that can
accurately predict rhEPO use within a certain timeframe.
More studies must be done in order to determine this window
of testing. In Magnani’s study, he also used beta-globin
mRNA and Tfr mRNA concentrations as parameters to detect
for rhEPO use. These markers increased the accuracy of detection,
but again, further studies must be done to confirm this.
In order to gain the physiological effects
of rhEPO, athletes need to continue its use until a late
stage of preparation for an event. A test for increased erythropoiesis
in the two to six weeks before competition would have a high
likelihood of detecting rhEPO abuse (Parisotto and Gore 569).
Detection of rhEPO in urine should be performed to confirm
results of blood sampling for indirect markers. However,
it should not be made a requirement because urine testing
suffers from many restraints. Blood sampling should be done
in the morning before exercise and daily intake of fluids,
as these factors can alter hematocrit levels in the body.
In conclusion, it is possible to detect
usage of rhEPO through indirect blood markers, such as hematocrit
levels, sTfr concentrations and reticulocyte concentrations.
rhEPO can have physiological effects that last beyond its
use, and these effects are what one measures. These parameters
must be factored in together and not individually to have
a more accurate detection. rhEPO abuse can be detected most
accurately using indirect erythroid markers that expose artificially
altered erythropoiesis.
—copyright © 2002, Jason Fong
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