People have been asking me about Riccardo Ricco, the Italian cyclist who was thrown out of the Tour de France for testing positive for the hormone erythropoietin (Epo), so I want to do a little Q&A about Epo detection and abuse.
What is Epo?
Epo is a hormone released mostly by the kidney that regulates red blood cell (RBC) number. Cells in the kidney respond to hypoxia by releasing Epo into the blood stream. Epo then goes to stem cells in your bone marrow that produce RBCs and increases their production. The appeal to use Epo from the point of view of a cyclist is that in raising your RBC number you are increasing the oxygen carrying capacity in your blood. This is useful if you are in an endurance sport like cycling.
What is it used for medically?
As a drug Epo is used under a couple of circumstances. One of the most common is to supplement the lack of endogenous production in renal failure. Renal failure patients can run a pretty bad anemia, and synthetic forms of Epo were created to prevent the need for transfusions. Also, Epo is used for cancer patients who have undergone radiation or chemotherapy and may also have anemia. Finally, anemia can be the side-effect of some HIV medications, so Epo is sometimes used to treat it in these patients. (Technical description of Epo drugs and their uses here.)
Does Epo abuse have any side effects?
The concern with Epo abuse is that the individual will get a dangerously high hematocrit. Hematocrit is the percentage of blood volume that is taken up by blood cells. It is also related to the blood viscosity. If your blood gets too viscous, you run the risk of stroke, blood clot, or heart attack.
How would we test for Epo abuse?
There are two general approaches to test for Epo abuse. First, you could test for the amount of blood cells in someone's blood and judge that way. If they were using Epo you would expect them to have a very high hematocrit -- the percentage of blood volume that is taken up by RBCs. Your average male has about 46% and your average female has about 38%. You could set an arbitrary standard and say that if you have this high hematocrit, you are not eligible to compete.
The unfortunate part though is that hematocrit varies very widely among individuals in response to disease, genetics, and environment. Below is a table from a survey of Italian adults with and without hypertension (you can ignore the ones with hypertension). (Click to enlarge.)
The table is from Crillo et al. 1992.
You can see that the values for hematocrit vary widely even in normal individuals. Some common known causes for high hematocrit are obstructive lung disease such as COPD, a myeloproliferative disorder (a disease where you make too much of one type of blood cells...basically a type of cancer) called polycythemia vera, and even living at high altitude. The International Cycling Union (UCI) sets a limit of 50% for men and 47% for women, but in a survey of athletes, a small percentage had higher than these limits even though they were not using Epo. So setting an arbitrary maximum for hematocrit poses problems. (Ricco claims that he has a naturally high hematocrit, and that claim may not be false. Unfortunately, he also failed the other type of test.)
The alternative strategy -- and the strategy used by the World Anti-Doping Agency (WADA) -- is to try and detect synthetic Epo in blood or urine. This is possible but also difficult. Epo is a protein hormone, so it is possible to synthesize it with an identical protein sequence -- either through peptide synthesis in the lab or through some sort of cell expression system. However, while Epo produced in this way would have an identical protein sequence, it would not be exactly the same as Epo in your body because proteins in your body go through post-translational modification. Sugars are added to the protein through a process called glycosylation. This glycosylation would not be present in a peptide synthesize in the lab. In cell expression systems, the resulting glycosylated protein would be different than the human version unless it was produced in a human-derived cell line because different species have different genes to do this. It turns out that the commonly available forms of synthetic Epo are produced in cell lines derived from hamsters (CHO and BHK cells for the initiated). Hamsters -- being mammals -- have very similar but not exactly the same glycosylation profiles as humans (they lack some of the genes). Anyway, the take home message is that it is possible to detect synthetic Epo because of differences in glycosylation.
The technique that they use to do this is called isoelectric focusing. Briefly, isoelectric focusing separates proteins by their pH. Isoelectric focusing is combined with electrophoresis -- which separates the proteins by size -- to produce a 2-dimensional blot that has all of the proteins in a sample separated by pH and size. (The combined technique is called 2-D PAGE.) Each dot on this blot represents a different protein, and you can identify whether a protein is present or not by its position on the blot. Synthetic Epo would appear in a different position that the Epo from your body because of small changes in its pH and size due to the different glycosylation. So basically, you take a sample of blood or urine, put it through this regimen, and in theory you could detect synthetic Epo. (A technical description of this technique is available here.)
I say in theory because this technique has been heavily criticized for either being prone to false positives or false negatives. Recently, a scientist from Denmark named Carsten Lundby decided to test the accuracy of various WADA-certified laboratories with respect to this test. They treated several test subjects with Epo and then sent urine samples to two WADA labs for testing. One lab failed to identify the positive samples altogether, and another lab failed to identify all of the samples as positive (granted this included some samples taken a couple weeks after the subjects stopped using the Epo).
Also, even if accurate testing could be assured, Epo itself has a relatively short half-life in the body (a couple days) whereas the lifetime of a RBC is about 2 months. If testing is only done during competition, it would be very easy for an athlete to take the drug to boost their hematocrit and then stop taking it during competition to evade detection.
Basically, the take-home is that while tests have been developed to look for Epo abuse, this is a lot more difficult that you would think.
What test did Ricco fail? What drug was he abusing?
Ricco (and two others) was caught for using another form of synthetic Epo called CERA (short for Continuous Erythropoiesis Receptor Activator). CERA is a pegylated form of Epo that was created to give the drug a longer half-life in patients. While I assume that CERA is tested for in a very similar manner as regular synthetic Epo, WADA has raised a bunch of eyebrows with this because the technical details of their test for it have not been released. Considering the issues with other Epo tests, the test details need to be verified by independent parties before it can be deemed in WADA's words "valid and reliable." Particularly because the longer half-life allows you to use smaller doses, some scientists are skeptical that the CERA test is accurate.
This is not to suggest that Mr. Ricco did or did not use CERA. He may have. But we need to be certain that these tests work.
Are there any better methods available to detect blood doping?
A better method than trying to detect synthetic Epo would be to develop a baseline for an athlete's traits and report any deviation from this baseline. This is the "biological passport" method being touted by among others the Garmin-Chipotle team. How this program works is that you have regular random testing of the athletes both in and out of season. From this background of testing, you can know what their average hematocrit is, so you would notice if it went up by 8%. Pilot studies using this technique have been successful. I mentioned this option before in reference to testosterone abuse. The test used to detect testosterone (measuring something called the testosterone/epitestosterone ratio) can be confounded by individuals who have genetically low T/E ratios. For these individuals, creating a baseline for the T/E would be the only way to detect the abuse.
Anyway, that is about all I have to say about Epo abuse. Relative to testosterone abuse, I think that WADA is going to have a much more difficult time controlling Epo. I hope that this pushes them towards a "biological passport"-like program because it looks like such a program is the only one that will be effective.
If you would like to know more about this story, the guys at Trust but Verify have it (as usual) well-covered.
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The reason WADA (well actually the French national testing group and WADA) seems to be keeping their test under wraps is that they supposedly worked with Roche (the developer of CERA) to put a marker in the drug that has a unique molecular signature. Thus easier detection. This seems to be a future trend.
Evidently at the Giro d'Italia this year they detected some odd chemical in Ricco & co. i.e. a new spot on the grid, but didn't know what it was and asked the French. So there was suspicion going into the tour.
WADA has always ground on me because of their arrogant statements of no false positives. The problem is that there is (hopefully that is turning into was) a high percentage of true positives which we know from police actions and such, so there isn't as much of a prosecutors effect and the odds are that the positives really are. I am sure that some riders are going to get falsely accused.
I think the passports are the savior also - look at blood characteristics in general for changes and anomalies rather than specific markers.
There's an interesting blurb at Pharmalot that claims:
I'm not sure what to make of that, though. They make it sound like something was added for the sole purpose of detecting doping, but it's hard to believe FDA or EMEA would go along with that. Maybe they're just taking advantage of the fact that the Roche product includes a molecule of polyethylene glycol covalently attached to the protein, to prolong its half-life?
Picking a couple of nits - isoelectric focusing separates proteins by pI (isoelectric point), not pH. Also, I don't think peptide synthesis would be a viable way to make epo. Epo consists of 165 amino acids, which I think is too large for that approach, even if you only wanted enough for one person.
The sheer difficultly of detecting this sort of thing makes it harder for me to see it as worth bothering. If you can't distinguish between people with high hematocrit and people with high hematocrit because of drugs, except on the basis of frequent and subtle analysis of metabolites and such, what sporting purpose is served by trying? It seems to come down to little more than a fuzzy, emotional feeling that it is more honest to have high hematocrit because your ancestors have lived at 15,000 feet for N generations, rather than because you are using Epo.
I understand that there are health concerns for the athletes using the stuff, and I respect the right of the organizations in question to have whatever club rules they want; but the harder doping gets to detect, the more trying to detect it feels like dubious hair-splitting to me.
When drugs are so hard to detect are they really having an effect on performance?
re: splitting hairs; tell that to the almost 100 families of riders in the 90s who died of heart attacks, the result of too thick blood
re: really having an effect on performance;
....a one day classics rider magically transform from into a 7 year tour winner? (please dont quote any PR material from his camp - lies)
....a current team manager who won the tour and then confessed for political reasons, his nickname was mr. 60 as in 60% crit level.
if only 5% of insider trading is found, why bother trying to stop it?
this isnt really about EPO. a rider can spend a lot of money on a 'full program' (transfusions with own blood), win a lot, and never be caught. this is about cheating.
Jake,
thank you for the informative article. As a middle ager, who is genetically prone to be thin, and suffer from a poor memory, I'm curious about intellectual performance enhancing medicines. In my case the performance is associated with the my reduced ability to sponge and synthesize new information while reading or during a conversation. My mother claimed to have a terrible memory, and I have a body type very similar to hers. And now I suffer from all of the ailments she began to suffer from in her 40s; lactose intolerance, gluten intolerance, hypothyroid. As I age past 44 I realize that my charm, wit and good humor is slipping. Excercise does help and I often ride my bike to work 9 miles each way and even do 1 to 5 hour rides one or two weekends a month. An occasional run, and I am constantly nervously moving. Excercise is often touted as the saving grace to stay sharp and witty into the second half of life. However, it seems to me that if ordinary people can get a face lift, tummy tuck, botox enhancements, there ought to be a way ( in addition to excercise ) where one can maintain or even improve reaction times, memory, wit, recall, problem solving, etc. i.e. generally improve ones intellect, and ability to learn new things. I'm also aware that attitude, close relationships and emotins associated with relationships play dramatic roles in the intellectual pursuit, along with good diet and sleep habits. With all of this and more in mind, I'm curious as to what a young medical student knows about designer chemicals to improve neural networks? There must be ways that a middle ager who is not participating in a competitive sport, but who is required to think on her feet, and who must actively use her problem solving skills and engage others, can enhance intellectual performance.
Highest regards, and thankyou again for this informative post.