But, here’s what I’m saying. The first experiment I run if I want to test if I can detect a given protein in a mixed sample like blood is to spike the protein in. Then, I know it is in there, and I know that I should be able to detect it. If I can, the experiment was a success. Anyone would do this.
So, one guy could have accidentally mixed a spiked tube with an unspiked tube, and suddenly we are damning a guy’s career. I need more evidence than that.
Fiveyearlurker, are you starved for attention here or something? The breakthrough has nothing to do with the medium being used, you fool, the breakthrough is the new antibody being used to latch on to the sugar molecules attached to the EPO protein strands. 3 posts in a row now you’ve swerved by going blah blah blah about 2D gels without once offering anything as to how these new EPO tests are being conducted. Get off your cross already, will ya?
The issue isn’t 2-D gels specifically - rather it’s this… the amino acid sequence of endogenous human erythropoietin (HuEPO) and rHuEPO is identical and consists of 165 amino acids with three N-glycosylation sites and one O-glycosylation site. By extension, the only way to differentiate between the two is to look for the differences in how sugar molecules are attached.
2-D gels are currently the method that the LNDD people are using in their tests, and this is only after the sample urine is concentrated up to 1,000-fold using two ultrafiltration steps with several washes.
The problem is that they’re using a new antibody which binds to rhEPO and native EPO, and they’re depending on the charge differences to know which is which. Unfortunately, that same antibody also non-specifically binds to other urinary proteins too, so instead of getting 2 spots (rh-EPO and native EPO) on the final picture, you get 5-10, some of which might run where the EPO spots are supposed to be. These latter samples fell into the body of 110 “unsure” samples.
Hence, the debate is now whether GC/MS or capillary electrophoresis is faster or more accurate way of doing the process.
loopydude, fiveyearlurker, I don’t question the credentials of either of you in general knowledge of lab procedure, biochem, etc., but just because you’re lab stores frozen samples next to samples which have to be accessed on a daily basis and last week’s Tupperware container with frozen spaghetti and meatballs or marks their samples in messy Crayola magic-marker on masking tape doesn’t mean that one of the preemminent doping labs in the entire world with an annual processing rate in the thousands does it that way too. If some of your growth hormone or enzyme or whatever the hell it is you’re working on goes bad, both of you have a chance to recreate what you’re working on and pick up where you left off. Of course you’re going to show an appropriate level of dilligence in caring for those samples. The lab in question here carries out the dope controls for the most important athletes in the most important events in the world. They probably have hundreds of thousands of samples in storage so there would be no reason to constantly be digging them out of one freezer.
Why does everyone here keep quoting what Dr. Ayotte from the Canadian lab says without bothering to include that she is only expressing suprise at how long the EPO remained stable, not whether the test was valid or not?
All I really have to say in addition to this is that the same lab basically pulled off the exact same study with the 1998 Tour samples and found close to 40 EPO positives. Keep in mind that this was the same year that the Festina scandal broke out. Apparently this science was good enough to be published in Nature. Out of curriosity, have either of you been published in Nature yet?
Uvula, no he never killed any pets. I just thought that in the interest of fighting ignorance and so forth I’d post a follow up to last year’s thread.
All right, if you don’t want to argue the science, that’s fine. But, new antibodies to glycosylation sites are not a breakthrough either. Been done for a long time. They could have done it in 1999 if they had wanted to.
My interest in the tour and Armstrong could be described as remotely aware of it’s existence. What is silly is putting so much faith that you are willing to damn a guy who is taken hundreds of tests by one test that comes up positive that can’t be repeated because the other samples were “destroyed”. If you can’t repeat an experiment, then the results are meaningless and this experiment can’t be repeated.
Again, I’m not in a fantasy land. I think he, and 99% of these guys are cheating. But prove it with better evidence than this before handing down the judgement that the OP started gloating with here.
Not yet, but soon! Hell, I sold out and I’m a company man now. Not likely a Nature paper forthcoming…
They published this in Nature in 2000. So, again, there was no breakthrough recently:
Nature 405, 635 (08 June 2000); doi:10.1038/35015164
Why have these samples not been run before now?
I don’t disagree with you on this. I think it’s extremely unlikely that someone who went through as much chemotherapy as Armstrong did would not ALSO have been administered EPREX (the rHuEPO of choice in 1997-1998) as a valid medicinal therepeutic drug. I’ll lay London to a Brick that it was EPO in particular which allowed him to resurrect his career as quickly as he did. But I’ll also add that in 1999 there was no valid EPO test on file, hence, a truckload of other riders were also on the gear too.
I also agree that there is no valid evidence in these latest LNDD findings which are sufficiently overwhelming for me to accuse Armstrong of being a definitive drug cheat. The methods involved are way too open to debate regarding their validity - however, they obviously represent a fantastic step in the right direction.
Still, given the stakes and financial magnitude of all these accusations, you’d think that WADA would have (by now) a twenty-million dollar machine that goes ‘bing’ when it detects rh-EPO…
Actually, for an amateur, I’d like to think I did a fucking good job of arguing the science. However, that’s just my conceit talking. Nonetheless, if you reckon you can do a better job at retrospective testing, fucking go for it. There’s obviously a bucket of money to be made. Certainly, it’s out of my level of expertise.
Ahhh… thank you for reminding me. I’ve been trying to comment on this for ages but I kept digressing.
The thinking behind doing the 1999 tests in 2004 was as follows… firstly, all of the 1998 tests had been used - so there were none that they had on file. However, the LNDD labs apparently have a batch of 1998 Vuelta Doping Control tests wihch they intend to process shortly.
Anyways, the theory is that the 1999 Tour tests were from a year when it was still widely known that no EPO test existed - hence, by extension, there was also no need for a class of masking agents to also be present in urine to “hide” the presence of EPO.
Apparently, by 2000 the word was starting to get out that an EPO test was going to be available and the assumption is that masking agents would also started to have appeared on the scene by that stage.
That’s what I’ve heard at least…
I’ve done some 2D electrophoresis myself (though I’m nowhere near an expert in that technique - admittedly I’d be hard pressed to do it today without help or good instructions it’s been so long), and their ability to properly distiguish alternate glycoforms by charge (whether the carbohydrate chains are N or O linked, I guess) is certainly going to be highly dependent on having sufficient unmodified EPO in the sample, because 2D electrophoresis isn’t infinitely sensitive, despite its resolving power. What happens in urine, over many years, where polysaccharides are just floating around in a solution of potentialy reactive pH filled with nitrogenous compounds, I haven’t the foggiest idea, but it’s not something I’m willing to take for granted is a straightforward problem, or a non-issue. It’s known that ammonia and aminated sugar buildup (through normal cell metabolism) in CHO cell cultures (CHO is a kind of cell line from which the major manufactureres produce secreted recombinant human EPO in huge quantities, and which does indeed yield a differentially-glycosylated form of EPO) can cause greater heterogeneity of secreted EPO glycoforms. As the protein is in urine, which, even in cold storage can be expected to have some ammonia in it, I think it’s not unreasonable to worry about what’s happened to old samples.
I will concede that I did not know they screened so many samples, and it is interesting that only a subset of those were picked up in the assay with a high degree of confidence. Would they expect such an unequivocable answer in this assay, in an old sample, would come from samples that were negative in older assays?
And what is the rate of false positives in this test under the best of conditions? How is that affected over time? Have such variables been dealt with? We all have EPO in our pee. That’s where it first was isolated from for clinical applications, after all. But obviously, not many of us have CHO-derived EPO in there. What does sitting around in a chemical witches brew like urine do to either variety? Why should there be a 90 day limit on the samples for regulatory purposes if this was a non-issue? What happens after 90 days? A year? Five years?
I think it’s reasonable to want those questions answered, as they’re not absurd nitpicks.
Wow. Had to go away from the computer for a few minutes, and should have used preview before posting. Hope I didn’t make a redundant post.
Why not now? They wanted to add these samples to their basic understanding regarding the reliability and length the test worked over. Obviously, research like this needed to be done, because outside of the whole Armstrong connection to this, researchers in other labs didn’t even belive that this test was possible to use after such a period.
While loopydude continues to talk out of his ass on this one, I wonder why he’s so sure that EPO is necessarily unstable after this time period. I frankly doubt that he has specific knowledge of how long EPO can remain stable in a urea rich environment at -20 C; he’s just throwing in his own guesses from his research with entirely separate proteins.
lurker, I think the most important question here is, what exactly would you propose could cause a false positive on this test, outside of cases such as the Belgian triathlete where he always produced basic isoforms in his urine. That there was some vast conspiracy to plant EPO in the blinded test tubes? That someone managed to track down Armstrong’s sample numbers, sneak into the lab, unfreeze the samples, place EPO in them, add EPO to six other random samples, and then add trace amounts to a bunch of others so that they would come back “weak positives” and never get reported? Really, I understand how it’s possible, but what’s more likely? That Armstrong was doped with a shit-load of EPO and that EPO will survive for a few years at -20 C or the above conspiracy.
The next question I have to ask is, like personal blood doping versus external blood doping, what are the chances that riders are already saving every single bit of their urine to have their own endogynous EPO get recycled? What sort of labs do you need to do such a thing?
Sure, valid questions that we’re all willing to discuss, but again, which explanation is more reasonable? Do you actually propose that normal human EPO morphs into something that looks like recombinant EPO just from sitting in urine? The lab which has been testing tons and tons of samples for just such issues has found that it apparently is still possible to test for synthetic or natural EPO after the said time period at the relavant conditions.
To see the substance, you have to get your head out of there.
Look: I worked for years supporting a project (when I wasn’t slaving on my own, which was going nowhere) where part of what I did was generate mutants of secreted glycoproteins to be expressed in a CHO cell system, which I purified on IMAC columns and by HPLC, and ultimately analysed for their relative abilities to dimerize members of the cytokine receptor family (as well as some receptor tyrosine kinases)…I am NOT going to discuss what those proteins were, but it was my business to know something about members of the family of growth factors that includes things like EPO and HGH. I had to do a lot of work to characterize the mutants I was working on, and in so doing gained a fair amount of technical biochemistry, even though that’s not ostensibly my area of expertise. I’d like to think I’m not at all talking out of my ass on the subject of EPO storage. I can certainly say without feeling uncomfortable that I’ve worked with the commercial product, and know how to handle it in the lab. Iffy samples with potentially poor integrity were of no use to me. So I learned how to properly handle and store them, and leaving EPO to sit around in urine in a regular freezer does not impress me. Getting good data out of such sensitive assays from a sample like that would impress the hell out of me if it is indeed reliable.
I believe LA. Not just because he seems sincere because we all know from recent events that some athletes are most excellent liars.
Here’s why I believe him. He won [i[this* year, when he was under a great deal of scrutiny. And he obviously tested clean. Absolutely, positively, certifiably clean.
Now, if a dude can win the TdF this year without PED’s, why is it so damn implausible that he could have won it 7 years ago without PED’s, when he had fitness, training, AND age on his side.
Unless someone comes up with proof, I’ll give him the benefit of the doubt.
Threemae, unless you were in France a few weeks ago racing around on your tricycle, you don’t have a rooster in this cockfight. So why the hostility?
I already know there can be heterogeneity of glycoforms generated just by sitting in old culture medium. I don’t know what the labs’ gels look like, so I can’t say how likely it is that something that looks like recombinant EPO in that assay could be generated. If, under regulatory conditions, the samples must be no older than 90 days, I have to wonder what the problem with older samples is.
I’d still like to know if the stuff could have been there residually from Armstrong’s cancer treatments.
If it was naturally occurring EPO, wouldn’t he have continued to test positive for it after 1999?
It would be easier for individual riders to build their own hypobaric chambers than extract their own epo in their basement, I’m guessing. You would need affinity columns or some sort of reasonably sophisticated chromatography setup, concetrators of some sort (maybe AMICON centrifuge filters - and, uh, a centrifuge), you’d need a way to assay lots for activity so you don’t either screw the pooch or give yourself erythrocytopenia, keep everything sterile and preserved, etc. While I don’t doubt the riders could be trained to do the work in a few years, it would be tough to manage, and the costs in supplies would be huge. Not to mention the fact that a home EPO-extraction lab would probably be difficult to conceal for long, much less explain.
Again, no. The assay is only valid for 72 hours, max, and certainly not from his cancer treatment.
Impossible. Lance was being treated in '96, I think. The protein is lost through excretion and breakdown fairly quickly, and is undetectable in just a few days after the last injection. The effects on your blood last much longer, as the lifespan of red blood cells can be measured in months. You’d think if Lance was doping that much, he’d have a suspicious hematocrit, but maybe he did for all I know. It’s not a proof of much by itself, especially if you’re carefully monitored by your cheating sports doc.