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17% Of urine samples don't contain EPO. At all

Jul 28, 2010
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Just read an interesting article (in Dutch). Martial Saugy, a Swiss doping expert, has said between 2003 and 2006 of 3050 samples he tested 17% contained no EPO at all. So no natural EPO as well. Specifically tests done after races often contained no EPO. Which is strange because the body produces more during high stress.
This points to the use of EPO erasing substances being widely used.
http://www.ncbi.nlm.nih.gov/pubmed/17692836

This can be done by adding a small about of Proteases. (For example even putting the stuff in the body/bladder with a catheter to cheat control).
http://www.ncbi.nlm.nih.gov/pubmed/20355180 There are tests for high concentration of the stuff, but already a small amount wipes the urine clean.
A protease (also termed peptidase or proteinase) breaks down proteins. A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein.

German article already mentioning the stuff in 2006
http://ergogenics.org/434.html#poeder

Dutch article about the topic
http://ergogenics.org/anabolica253.html

This makes it even more possible to cheat a doping test with a little advance warning (or time to 'shower').

Did anyone hear any recent news about this, current amounts of samples being EPO free? Or is there a non-doping explanation for some being EPO free even?
 
Jun 22, 2010
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brul12 said:
Did anyone hear any recent news about this, current amounts of samples being EPO free?
Tampering with your samples qualifies as a "prohobited method".
In cycling nobody is making use of prohibited methods, so you won´t be hearing a lot about this.
 
Aug 9, 2010
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brul12 said:
This points to the use of EPO erasing substances being widely used.
I presume you mean masking agents?

Is it too obvious to ask if masking agents (eg Probenicid a la Mr Delgado) are tested for as well as the headline PEDs?
 
Sep 25, 2009
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probenecid has long been tested for but it has NOTHING to do with masking epo or proteases. it was used to mask aa steroids.

masking agents fall under wada regulations for test adulteration and or interference. same punishment. use of proteases (known as 'fuentes red powder', laundry detergent etc) arguably has decreased with more sophisticated epo microdosing - the idea was to have the rEPO min-microdose naturally broken down by the body overnight thus not necessitating a risky adulteration. flandis told us of iv microdosing as opposed subcutaneous that speeds up epo 'digestion'
 
Aug 9, 2010
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python said:
probenecid has long been tested for but it has NOTHING to do with masking epo or proteases. it was used to mask aa steroids.
I did know that, I was just citing Probenicid as an example of a masking agent that was tested for.

I'd heard of detergent being used, presumably administered by the athlete at the point of test? No wonder some riders have a whiter than white record when it comes to passing dope tests...
 
Aug 13, 2009
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After USPS was found dumping bags of drugs and syringes in the 2000 Tour there samples from the Tour were retested. They were found to be "Too Clean" with no EPO at all in them.
 
Sep 25, 2009
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Chuffy said:
I did know that, I was just citing Probenicid as an example of a masking agent that was tested for.
got you now.

I'd heard of detergent being used, presumably administered by the athlete at the point of test? ..
yes, as the op noted, the cheaters use
either
- catheter
- a grain stuffed under a nail
- or a grain pre-inserted in the urethra

the effect is wiping out both the natural epo and the synthetic one. the latest famous case is leogrande's.

since the 'classic' epo test depends on the comparison of basic bands in rEPO to the natural EPO (of course also concurrently using references - positive and negative samples), after electrophoresis induced separation, the urine sample is rendered useless.
 
Jul 22, 2009
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Jesús Manzano talked extensively about cyclist's sample-destruction techniques in his Cómo se dopa un ciclista exposé in Diario As. In it, amongst many other things, the Spanish cyclist talks about this little reddish little pill, "the size of a grain of rice", which they (at Kelme) were trained to use (by inserting in the penus) right before a urine test during the "training phase" or right after being given something by the teal doctor. Apparently this little pill destroys the sample altogether. However, destroying a sample nowadays will land you on the "suspect" list. And once you're put under the microscope it's just a matter of time before you are busted doing something.
 
While protease use has been going on a long time, it should also be noted that quite frequently urine samples contain no detectable EPO. It doesn't necessarily mean that riders are degrading EPO. In fact, the 17% figure sounds low to me.
 
May 5, 2009
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the substance to entirely remove EPO from urin was also found at Fuentes' during operacion puerto. at that time when the article came out, quite a few tests of very famous athletes were said to be entirely EPO free...
 
Apr 8, 2010
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I always thought that the reason that Heras got busted was that he was, for some reason, unable to get the proteinase into the sample.
 
Jul 22, 2009
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Square-pedaller said:
I always thought that the reason that Heras got busted was that he was, for some reason, unable to get the proteinase into the sample.
Honestly? I think the Heras positive was a set up. Not because I like the guy or saying that he didn't dope (he did) mind you. From what I've read, AKA IMO, the pre-Puerto investigations weren't going anywhere precisely because the cyclists were simply 1) not talking and 2) not testing positive, meaning the authorities were one step behind the dopers.

The Heras positive was the precursor to the entire operation. The authorities needed to crack the case open and needed a high profile cyclist in one of the "suspect" teams (Liberty Seguros, Kelme, et cetera) to test positive. And Heras getting caught opened the door veeeeeeery wiiiiiiiiide. Too suspiciously wide if you ask me.
 
Jan 19, 2010
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When people here talk about 17% of samples having NO EPO, can someone clarify something for me. Are they suggesting that there is absolutely NO EPO in the samples or is it that the level of EPO is below the limits of detection for the assay they are using?

Those things are as different as the sun and the moon. If they are saying there is absolutely NO epo in the samples it leads to certain conclusions.

If they are saying there was no EPO above their limit of detection, that leads to entirely different conclusions.

It is pretty easy to test for protein in the urine and if there is no protein of any type, it would substantiate the protease theory as the protease itself would be the only protein left.
 
Jul 20, 2010
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enzymes like those proteases are not perfect and will fall apart fairly quickly... because natural proteases in the body only need to work at specific sites in the body... proteases are made to eventually fall apart. But using them sound ridicoulous to me having no proteïns in your urine makes your sample very very suspicious to doping agents... ( those people arent exactly what u call stupid )
 
Squares said:
When people here talk about 17% of samples having NO EPO, can someone clarify something for me. Are they suggesting that there is absolutely NO EPO in the samples or is it that the level of EPO is below the limits of detection for the assay they are using?

Those things are as different as the sun and the moon. If they are saying there is absolutely NO epo in the samples it leads to certain conclusions.

If they are saying there was no EPO above their limit of detection, that leads to entirely different conclusions.

It is pretty easy to test for protein in the urine and if there is no protein of any type, it would substantiate the protease theory as the protease itself would be the only protein left.
All any scientific/medical procedure can ever say is that the substance was below the level of detection. The samples are not tested for protein in general. Even if you treated urine with a protease, some proteins would remain undegraded, particularly if the treatment were of the low tech variety available to someone trying to avoid an EPO positive.

Btw, proteases frequently attack themselves! But again, 100% of the proteins are not going to be degraded.
 
Jan 19, 2010
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Merckx index said:
All any scientific/medical procedure can ever say is that the substance was below the level of detection. The samples are not tested for protein in general. Even if you treated urine with a protease, some proteins would remain undegraded, particularly if the treatment were of the low tech variety available to someone trying to avoid an EPO positive.

Btw, proteases frequently attack themselves! But again, 100% of the proteins are not going to be degraded.
I agree and was, I guess, asking a rhetorical question of those who claimed that the was no EPO and said that was a sign of doping, but they didn't answer.

Furthermore, addition of proteases wouldn't eliminate EPO, but would make the EPO bands run smaller than the expected mass, which would give a very conspicuous pattern on the gel. The same would occur if someone added amylase or glycosidases that would remove the sugars from both the natural EPO and the synthetic EPO, which is where the real difference occurs in the two.
 
Squares said:
I agree and was, I guess, asking a rhetorical question of those who claimed that the was no EPO and said that was a sign of doping, but they didn't answer.

Furthermore, addition of proteases wouldn't eliminate EPO, but would make the EPO bands run smaller than the expected mass, which would give a very conspicuous pattern on the gel. The same would occur if someone added amylase or glycosidases that would remove the sugars from both the natural EPO and the synthetic EPO, which is where the real difference occurs in the two.
Keep in mind that in the EPO test, the proteins are visualized not by nonspecific staining, but by antibodies. So if digestion is very substantial, probably whatever is left won't react with the antibodies, so you won't see anything. And though they may be doing it differently now (I'm not sure), the original test was based purely on electrophoretic mobility, not on size (1D not 2D), so it would be changes in charge only, not in size, that would be detected.

Glycosidase treatment might or might not affect antibody binding; but even if it didn't, it would probably convert the EPO into a form different from both natural and synthetic, which should also raise suspicions.
 
Apr 13, 2010
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Merckx index said:
While protease use has been going on a long time, it should also be noted that quite frequently urine samples contain no detectable EPO. It doesn't necessarily mean that riders are degrading EPO. In fact, the 17% figure sounds low to me.
Can you elaborate a bit? For example, what would you expect a "natural" proportion of tests with no EPO in to be? And how does it happen?

Are you basically saying that (more than) 17% of samples are useless even without tampering?
 
Jan 19, 2010
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JPM London said:
Can you elaborate a bit? For example, what would you expect a "natural" proportion of tests with no EPO in to be? And how does it happen?

Are you basically saying that (more than) 17% of samples are useless even without tampering?
I'll take a stab at it.

For every medical test, there is a limit to the ability to detect what you are looking for. Think of it like the speedometer on your bike.

Go out, stand next to your bike, turn on the Speedometer and take 1 small step. Take another small step about 10 seconds later and another every 20 seconds. You are moving, but your speedometer will be showing a reading of 0 kph, your movement is just below the limit of detection for your bike speedometer.

The same thing applies to drug tests.

Say you need 10,000 molecules of EPO in a 1 mililiter sample of urine for the test to work and give you a signal (the non-zero reading on your speedometer). If for some reason because of drinking a lot of water you have diluted your urine so that the natural levels of EPO are at 5,000 molecules per mililiter, the test will show no signal and give a reading of 0. It could also be 9,100 molecules per mL and still show 0. As you can see, there is EPO there but it is just below the limit of detection.

For this reason, it is hard to hear people say that 17% of urine samples contain no EPO and jump to the conclusion that people are doping.

Perhaps it really means that the EPO test needs to be improved so that it has a lower limit of detection that is low enough to pick up EPO in all samples. Then again, people seem to like the idea of a conspiracy better.
 
Jul 29, 2010
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Squares said:
I'll take a stab at it.
Are you sure that's correct? My understanding was there is natural EPO produced by the body, but by throwing a little detergent in the pee sample, you can wipe out ALL Epo (both naturally-occuring, and artificial).

This is not new, as I think previously this allegation was leveled against JUllrich as well, by the german anti-doping guy, Werner Franke (?).

Anyways, I can buy the argument that the test for synthetic EPO needs a base reading (10,000 in your example), or else it registers as a "zero". But I think that is not what is being described here -- no EPO at all, not even naturally-occurring that you expect to find in healthy human being. If so, it is not merely "conspiracy theory" conjecture...
 
JPM London said:
Can you elaborate a bit? For example, what would you expect a "natural" proportion of tests with no EPO in to be? And how does it happen?

Are you basically saying that (more than) 17% of samples are useless even without tampering?
It’s well documented that urine samples do not always contain EPO. For example, this reference reported that EPO was detected in the urine of just 64% of anonymous Olympic athletes tested:

Berglund, B., & Wide, L. (2002). Erythropoietin concentrations and isoforms in urine of anonymous Olympic athletes during the Nagano Olympic Games. Scandinavian Journal of Medicine and Science in Sports, 12, 354-357

Another study looked at some of the factors that might result in indetectable levels of EPO in urine, with the aim of distinguishing natural factors from the use of proteases:


Séverine Lamona, Neil Robinsona, Pierre-Edouard Sottasa, Hugues Henryb, Matthias Kamberc, Patrice Mangina and Martial Saugya Possible origins of undetectable EPO in urine samples. Clinica Chimica Acta 85, 61-66

They concluded that:

"Statistical analyses indicated that undetectable EPO profiles were clearly related to urine properties such as low EPO concentrations or extreme specific gravities. The addition of very small quantities of protease was shown to remove all traces of EPOs in urine. This finding led to the development of a simple, specific and sensitive test that reveals proteasic activity based on albumin digestion."
 
To reiterate some points I made earlier, addition of protease to urine by an athlete (as opposed to incubating urine with protease in a laboratory setting) is very unlikely to degrade all proteins. But since EPO is detected by a specific antibody, all that's necessary is to degrade the portion of the EPO molecule that the antibody recognizes. This is presumably why addition of protease by a tested athlete can remove all traces of EPO.
 
Aug 9, 2010
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A lot of the science is going straight over my head.

To reduce all this to it's simplest - absence of EPO in a urine sample is not uncommon and is not necessarily proof of tampering. But it might possibly be. Therefore, absence of EPO doesn't actually tell us a great deal. Correct?

Thanks for the replies Science Guys. I joined this Forum hoping to learn more. Please be gentle while I try and make sense of it all!
 
Chuffy said:
A lot of the science is going straight over my head.

To reduce all this to it's simplest - absence of EPO in a urine sample is not uncommon and is not necessarily proof of tampering. But it might possibly be. Therefore, absence of EPO doesn't actually tell us a great deal. Correct?

Thanks for the replies Science Guys. I joined this Forum hoping to learn more. Please be gentle while I try and make sense of it all!
You're mostly correct. Except that there are ways of analyzing urine samples that have no EPO to determine whether they might have been tampered with. Basically, if an athlete treated the urine with protease to degrade the EPO, this would also have an effect on other proteins in the urine, and this effect can be detected. If, on the other hand, the absence of EPO was due to natural factors, no effects on other proteins will be observed.

The scientists in the second article I mentioned above are claiming that if they have a bunch of urine samples with no EPO, they can determine which ones resulted from tampering with protease, and which ones didn't.
 
Mar 10, 2009
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Lets just look at this in the simplest terms. No EPO = tampering with the sample or masking. Evidence of cheating. The absence of Synthetic EPO is not the consideration but if the naturally occurring EPO is missing too then the athlete is sick or tampering with his samples. Seems simple.
 

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