Transfusions and positives

[Race Radio]

The Contador thread is gone off the rails and this topic is related to more then just AC.

I have seen over the years that when an athlete tests positive the theory is floated that they must have gotten it from a transfusion that was from a period of time that they were using the substance.

The first I heard of this theory was back in the 80's when a distance runner was caught for a steroid at the World Championships. The rumor at the time was he used a blood bag from a training period when he was using the steroid for recovery.

It popped up again with Landis. I remember that it was roundly dismissed as being impossible, especially with the multiple positives he had in the IRMS test.

Now it comes up again with AC. Both Bordry and Damsgaard are giving it as a possible explanation.

Anyone know the answer to this? Is it indeed possible? If AC was using Clenbuterol in training how did he dodge the OOC testers? That stuff stays in your body for a long time so he would have to be avoiding tests for a long time.

 

[Anonymous]

Interesting discussion going on over here;

http://forum.cyclingnews.com/showthread.php?t=10342

 

[Balabar]

Anyone know the answer to this? Is it indeed possible? If AC was using Clenbuterol in training how did he dodge the OOC testers? That stuff stays in your body for a long time so he would have to be avoiding tests for a long time.

I'm not an expert, but that never stopped anyone from providing their opinion on the internet.

I'll start with the final question - If Contador was taking clenbuterol during training he may not have been caught because a different lab might have been analyzing his samples. From the recent Cyclingnews article:

http://www.cyclingnews.com/news/contador-food-contamination-or-political-victim

"The Cologne, Germany, laboratory that tested Contador’s July 21 sample is known for being one of the worlds’ most advanced for clenbuterol detection. Had Contador’s sample been tested anywhere else the traces are so minute that the substance would likely have been undetected."

With regards to the first question, a few things need to be determined.
- What concentration of clenbuterol would need to be in Contador's blood to produce the levels detected in his urine.
- What the concentration would need to be in the blood bag. Remember that the transfused blood is greatly diluted when its added.
- How stable clenbuterol is when stored in blood at refrigerated temperatures for an extended period of time.

Once those questions are answered, you can determine what the concentration would likely have been when the blood was drawn. If the resulting theoretical concentration is too high, i.e. at the level that would lead to side-effects or positive drug tests, then the transfusion theory is less plausible.

If the theoretical concentration is still relatively low, the theory becomes more plausible - Contador may have believed that all of the clenbuterol had cleared his system when he would have made the blood draw.

It's unfortunate that these results are from urine samples. If blood samples were taken, the blood values could be closely examined for any evidence of transfusions.

 

[JayZee]

It's unfortunate that these results are from urine samples. If blood samples were taken, the blood values could be closely examined for any evidence of transfusions.

Aren't they always taking blood as well as urine? It would be interesting to see Contador's blood profiles to see if there is any indications of a refill happening on that rest day, which may have contained the PED in trace amounts.

I would also like to know the science behind products contained in transfused blood.

 

[El Pistolero]

I'm not an expert, but that never stopped anyone from providing their opinion on the internet.

I'll start with the final question - If Contador was taking clenbuterol during training he may not have been caught because a different lab might have been analyzing his samples. From the recent Cyclingnews article:

http://www.cyclingnews.com/news/contador-food-contamination-or-political-victim

"The Cologne, Germany, laboratory that tested Contador’s July 21 sample is known for being one of the worlds’ most advanced for clenbuterol detection. Had Contador’s sample been tested anywhere else the traces are so minute that the substance would likely have been undetected."

With regards to the first question, a few things need to be determined.
- What concentration of clenbuterol would need to be in Contador's blood to produce the levels detected in his urine.
- What the concentration would need to be in the blood bag. Remember that the transfused blood is greatly diluted when its added.
- How stable clenbuterol is when stored in blood at refrigerated temperatures for an extended period of time.

Once those questions are answered, you can determine what the concentration would likely have been when the blood was drawn. If the resulting theoretical concentration is too high, i.e. at the level that would lead to side-effects or positive drug tests, then the transfusion theory is less plausible.

If the theoretical concentration is still relatively low, the theory becomes more plausible - Contador may have believed that all of the clenbuterol had cleared his system when he would have made the blood draw.

It's unfortunate that these results are from urine samples. If blood samples were taken, the blood values could be closely examined for any evidence of transfusions.

Only unfortunate if you have already declared him guilty of blood doping. Blood samples were taken.

 

[Tyler'sTwin]

They should test his TdF blood samples for traces of plastic.

 

[Balabar]

Only unfortunate if you have already declared him guilty of blood doping. Blood samples were taken.

Maybe a poor choice of words. Any time you are conducting an investigation, the more data available, the better the conclusions will be. An evaluation of Contador's blood values could help exonerate him or be used to support the clenbuterol positive.

I don't doubt that blood samples were taken. In fact it would be absurd to not take a blood sample from the yellow jersey on the rest day before the queen stage. But it's likely that not as many blood samples were taken as urine samples. According to Contador's expert, Dr. Douwe de Boer, urine samples were taken every day between July 19 and 24. It's unlikely that blood samples were taken each of these days as well. Blood samples are more expensive to handle and analyze and repetitive blood sampling is intrusive to riders.

A close examination of his blood profile would still be useful.

 

[Cobblestones]

The question about the concentration:

If you take 1 pint with Clen-contaminated blood (the restday refill) and pump it into a rider with about 10 pints of uncontaminated blood, the concentration you'll measure is about 1/10 of the concentration which was in the refill bag. Now that assumes that Clen is only dissolved in the bloodstream. If it migrates out of the blood then the refill bag concentration had to be higher. Now the bag concentration at the time of the refill might have been lower than at the time the blood was drawn. This is because Clen might have been metabolized in the bag (even at low temperatures). I don't know whether there's data on that.

 

[Merckx index]

With regards to the first question, a few things need to be determined.
- What concentration of clenbuterol would need to be in Contador's blood to produce the levels detected in his urine.
- What the concentration would need to be in the blood bag. Remember that the transfused blood is greatly diluted when its added.
- How stable clenbuterol is when stored in blood at refrigerated temperatures for an extended period of time.

1) The media reports are giving the test results in terms of amounts, e.g., 50 picograms. I assume that the values were actually determined per ml. of urine??? If so, the total amount in urine would be one hundred or more times higher, say, 5000-10,000 picograms, or 5-10 nanograms. The urine is formed over a period of several hours, which means it would take a fairly small fraction of the clenbuterol actually present in the blood. As a very rough guess, let's say 50-100 nanograms in the circulation.
2) Depends on how much blood was to be transfused, but roughly, the amount transfused would be 5-10% of the total blood volume in his system, so concentration would have to be 10-20x greater. IOW, when the blood was withdrawn, his total circulation contained 500-1000 nanograms, or 0.5-1.0 microgram.
3) As I discussed briefly on another thread, clenbuterol is rapidly metabolized in blood in the body, because in various animal experiments, more than half of it is converted into other substances by the time it appears in the urine. This is a period of a few days. In refrigerated blood, the process would be much slower, of course, but there would also be much greater periods of time involved. As a very wild-*** guess, I will say that in samples refrigerated for several weeks, 90% or more of the clenbuterol would be metabolized.

To summarize, if he had 50 pg/ml. of urine, maybe 10 nanograms in the total urine sample. Thus maybe 100 nanograms in his circulation. And one to two micrograms in his circulation when he withdrew blood for transfusion.

Needless to say, these are extremely rough estimates, but may serve as a framework in which to approach this possibility.

 

[roundabout]

Ok, this may sound extremely stupid, but can a positive by clenbuterol be related to actovegin use?

 

[luckyboy]

They should test his TdF blood samples for traces of plastic.

What's this stuff about plastic now? Just read a few posts in the main AC thread, but I'm not delving back through it all.

 

[cyclanalyst]

A couple of things to be aware of:

1) the number 50pg/mL refers to a concentration, not an amount. We don't have any data - there isn't any - to suggest how much urine AC passed in 24 h, so it's inappropriate to guess at the amount in his body. Concentration is a surrogate measure of drug blood/tissue levels. For clenbuterol there is no threshold, so no drug level is tolerated.
2) I'm unable to uncover (in a rapid literature search) any data on stability of clenbuterol in human blood or plasma, and if there were any it would be from experiments done at 37C (body temp). Lots of data from rat biofluids on pharmacokinetics and metabolism, but it's complicated - some of the liver metabolites are converted back to clenbuterol in urine.

A couple of points about the CN article on De Boer's statement:

1) Food poisoning from clen contaminated meat is uncommon in Europe since the 1990s, and the Spanish incidents date to 1990. There are recent reports only in Asia & SE Asia. That doesn't mean that cattle aren't being given clen, but if they are they likely aren't being given high doses as in the past.
2) If you give a bodybuilder level dose of clen to AC in off season for training, and he gets an out of competition test, the levels will likely be detectable for at least 10-20 days following the last dose (extrapolation from horse data, where the half-life is more like 9 hours). Doesn't seem like a risk worth taking, so I think the blood bag scenario is unlikely.
3) Typo near bottom - 0.1ng/mL should be ng/kg to make any sense. Usual dose is 20,000-120,000 ng for bodybuilding, and AC weighs what, 60 kg? It's worth careful attention to units and measures and orders of magnitude, since numbers are usually used to confuse rather than to illuminate.

My guess: he ate some meat with clenbuterol in it. Only a guess, though. Still, the damage to the sport could be huge.

 

[Tyler'sTwin]

What's this stuff about plastic now? Just read a few posts in the main AC thread, but I'm not delving back through it all.

http://www.velonation.com/News/ID/5...ays-rider-may-have-received-transfusions.aspx

 

[Merckx index]

A couple of things to be aware of:

1) the number 50pg/mL refers to a concentration, not an amount. We don't have any data - there isn't any - to suggest how much urine AC passed in 24 h, so it's inappropriate to guess at the amount in his body. Concentration is a surrogate measure of drug blood/tissue levels. For clenbuterol there is no threshold, so no drug level is tolerated.
2) I'm unable to uncover (in a rapid literature search) any data on stability of clenbuterol in human blood or plasma, and if there were any it would be from experiments done at 37C (body temp). Lots of data from rat biofluids on pharmacokinetics and metabolism, but it's complicated - some of the liver metabolites are converted back to clenbuterol in urine.

A couple of points about the CN article on De Boer's statement:

1) Food poisoning from clen contaminated meat is uncommon in Europe since the 1990s, and the Spanish incidents date to 1990. There are recent reports only in Asia & SE Asia. That doesn't mean that cattle aren't being given clen, but if they are they likely aren't being given high doses as in the past.
2) If you give a bodybuilder level dose of clen to AC in off season for training, and he gets an out of competition test, the levels will likely be detectable for at least 10-20 days following the last dose (extrapolation from horse data, where the half-life is more like 9 hours). Doesn't seem like a risk worth taking, so I think the blood bag scenario is unlikely.
3) Typo near bottom - 0.1ng/mL should be ng/kg to make any sense. Usual dose is 20,000-120,000 ng for bodybuilding, and AC weighs what, 60 kg? It's worth careful attention to units and measures and orders of magnitude, since numbers are usually used to confuse rather than to illuminate.

My guess: he ate some meat with clenbuterol in it. Only a guess, though. Still, the damage to the sport could be huge.

Did you read my post? I provided a link that discusses metabolism of clenbuterol in the body. From that, I made my rough estimate of how much would be metabolized in cold storage. Likewise ROUGH estimates can be made about the amount of urine, and from that how much must have been in blood. Order of magnitude at best, but still useful.

Wrt contaminated food, see the very interesting study posted by BroDeal in the other thread on clenbuterol. This study shows that volunteers who ate contaminated meat had much higher clenbuterol concentrations in the urine that AC, up to 2 days after the meal. So food contamination is very possible, if contaminated meat is actually around.

The values in this paper also demonstrate something else that had not occurred to me. AC could have eaten contaminated meat not at the Tour but at some earlier time, then withdrawn blood, and re-infused it at the Tour. There could have been enough clenbuterol from the contaminated meat to show up in his urine at the reported 50 pg/ml, after transfusion.

So contaminated food and transfusion are not necessarily exclusive explanations.

 

[Squares]

1) In refrigerated blood, the process would be much slower, of course, but there would also be much greater periods of time involved. As a very wild-*** guess, I will say that in samples refrigerated for several weeks, 90% or more of the clenbuterol would be metabolized.

That all depends on how the clenbuterol is metabolized.

Most xenobiotics such as clenbuterol are metabolized in the liver by cytochrome P450 oxidases. If the metabolites are formed by liver metabolism, there could be effectively NO metabolism in the blood bag at 4 C when the blood was stored. It does appear that oxidative metabolism is the predominant breakdown, which would be liver based.

This all boils down to the half life of clenbuterol in ex vivo whole blood.

The data is probably somewhere in the files at the pharmaceutical companies who make the stuff.

 

[hektoren]

The plasticizer DEHP is a dead end, as it might just as well stem from certain makes of plastic cling film, absorbed by fatty foods and entering his body. There's no 1:1 relationship between finding DEHP and having had a transfusion. DEHP is used in a wide range of soft PVC products ranging from lifesaving medical devices such as medical tubing and blood bags, to footwear, electrical cables, packaging, tarpaulins for lorries, flooring, stationery and roofing.

 

[cyclanalyst]

Did you read my post? I provided a link that discusses metabolism of clenbuterol in the body. From that, I made my rough estimate of how much would be metabolized in cold storage. Likewise ROUGH estimates can be made about the amount of urine, and from that how much must have been in blood. Order of magnitude at best, but still useful.

Wrt contaminated food, see the very interesting study posted by BroDeal in the other thread on clenbuterol. This study shows that volunteers who ate contaminated meat had much higher clenbuterol concentrations in the urine that AC, up to 2 days after the meal. So food contamination is very possible, if contaminated meat is actually around.

The values in this paper also demonstrate something else that had not occurred to me. AC could have eaten contaminated meat not at the Tour but at some earlier time, then withdrawn blood, and re-infused it at the Tour. There could have been enough clenbuterol from the contaminated meat to show up in his urine at the reported 50 pg/ml, after transfusion.

So contaminated food and transfusion are not necessarily exclusive explanations.

I did indeed read your post. I was unable to find data on plasma or blood stability, I didn't say there wasn't data on metabolism and PK in vivo. Won't be the same at all in a bag.

Agreed that we can conclude that if the urine concentration is low, the plasma level is low as well. That's why I think the meat is the likely source, since we're all agreed these are sub-therapeutic levels. Other animal data indicates the concentration in urine is much higher than plasma at all time points, so this supports your argument even more. I still think it needs to be said, maybe for the non-scientist readers of the forum, that a urine concentration is not the same as a drug level, and it's risky to guess at the original dose.

Regarding your "eat meat & give blood" hypothesis, it seems less likely but plausible. I do agree that if AC (or any of the well-funded top ranked cadre) were doping he'd likely be as undetectable as LA.

 

[bigloco]

The plasticizer DEHP is a dead end, as it might just as well stem from certain makes of plastic cling film, absorbed by fatty foods and entering his body. There's no 1:1 relationship between finding DEHP and having had a transfusion. DEHP is used in a wide range of soft PVC products ranging from lifesaving medical devices such as medical tubing and blood bags, to footwear, electrical cables, packaging, tarpaulins for lorries, flooring, stationery and roofing.

And yet didn't I read that they had used an "undisclosed" procedure for testing blood in hopes of proving blood bag storage?

It may be all bs, but I'd definitely like to hear an official release of that data.

I don't know any specifics of the test or even if it really exists, but one would think they could differentiate DEHP leaching from a storage bag and that existing from ingestion.

 

[cyclanalyst]

That all depends on how the clenbuterol is metabolized.

Most xenobiotics such as clenbuterol are metabolized in the liver by cytochrome P450 oxidases. If the metabolites are formed by liver metabolism, there could be effectively NO metabolism in the blood bag at 4 C when the blood was stored. It does appear that oxidative metabolism is the predominant breakdown, which would be liver based.

This all boils down to the half life of clenbuterol in ex vivo whole blood.

The data is probably somewhere in the files at the pharmaceutical companies who make the stuff.

Agree. I'm not used to seeing any stability data in cold whole blood in the literature. I'd expect that if blood was bagged and stored during a clenbuterol dosing cycle there would be plenty of drug left after 6 months to give a urine positive. But from everything I read about sophistication in doping at the top levels, this seems like it would be a rookie mistake for a rider in AC's class.

Interesting discussion- thanks!

 

[bigloco]

Agreed that we can conclude that if the urine concentration is low, the plasma level is low as well. That's why I think the meat is the likely source, since we're all agreed these are sub-therapeutic levels. Other animal data indicates the concentration in urine is much higher than plasma at all time points, so this supports your argument even more. I still think it needs to be said, maybe for the non-scientist readers of the forum, that a urine concentration is not the same as a drug level, and it's risky to guess at the original dose.

Regarding your "eat meat & give blood" hypothesis, it seems less likely but plausible. I do agree that if AC (or any of the well-funded top ranked cadre) were doping he'd likely be as undetectable as LA.

Re: low level indicating meat as a likely source...

explain this...to me, I don't see the logic.

To me there will be a point where, having blood drawn at such a time after using clembuterol, factoring in its metabolism by the body and the in vitro degradation that will occur, that a urine test would give the results shown in the positive test.

Maybe the levels shown would make his usage impossible, maybe not...I just dont think anyone here has done the math on it

 

[Tyler'sTwin]

The plasticizer DEHP is a dead end, as it might just as well stem from certain makes of plastic cling film, absorbed by fatty foods and entering his body. There's no 1:1 relationship between finding DEHP and having had a transfusion. DEHP is used in a wide range of soft PVC products ranging from lifesaving medical devices such as medical tubing and blood bags, to footwear, electrical cables, packaging, tarpaulins for lorries, flooring, stationery and roofing.

It's not just the presence of DEHP, it's having "ten times over the higher value" of the stuff in his blood. That's surely highly suspicious whether it's grounds for a suspension or not (obviously not).

 

[131313]

Now it comes up again with AC. Both Bordry and Damsgaard are giving it as a possible explanation.

Anyone know the answer to this? Is it indeed possible? If AC was using Clenbuterol in training how did he dodge the OOC testers? That stuff stays in your body for a long time so he would have to be avoiding tests for a long time.

"Possible"? Anything is possible. Likely? No. My opinion it's highly unlikely, and to be honest I was disappointed in Damsgaard's response because it seems high on opinion and short on science, and he's a guy I generally respect.

I don't know what Bertie's testing schedule is like, but given the amount detected, here are the two possible scenarios:

1) he was using Clenbuterol as a stimulant and as a bronchodilator. This is certainly "possible", but it's highly unlikely since I'm almost certain he has a TUE for a more effective bronchodilator and caffeine is a more effective stimulant. Still, if that were the case, he'd have had to have had about 4 weeks between blood tests

2) he was using it to build muscle mass and lose weight. Again, HIGHLY unlikely, since it's now known that in the dosage required for this effect, it has significant deleterious effects on the cardiovascular system (this was less well-known until the last decade, which is why some earlier cyclists probably used it. Now, it's in the domain of the body nazis and maybe some shot putters or sprinters). But let's say he got some bad advice and was using it for this purpose. He'd probably have to go between 4-6 months between drug tests.

These are back of napkin calculations, but they're in the ballpark. Someone can check my maths if they're so inclined. My opinion, it's just not plausible.

 

[Guttercat]

A couple of things to be aware of:

1) the number 50pg/mL refers to a concentration, not an amount. We don't have any data - there isn't any - to suggest how much urine AC passed in 24 h, so it's inappropriate to guess at the amount in his body. Concentration is a surrogate measure of drug blood/tissue levels. For clenbuterol there is no threshold, so no drug level is tolerated.
2) I'm unable to uncover (in a rapid literature search) any data on stability of clenbuterol in human blood or plasma, and if there were any it would be from experiments done at 37C (body temp). Lots of data from rat biofluids on pharmacokinetics and metabolism, but it's complicated - some of the liver metabolites are converted back to clenbuterol in urine.

A couple of points about the CN article on De Boer's statement:

1) Food poisoning from clen contaminated meat is uncommon in Europe since the 1990s, and the Spanish incidents date to 1990. There are recent reports only in Asia & SE Asia. That doesn't mean that cattle aren't being given clen, but if they are they likely aren't being given high doses as in the past.
2) If you give a bodybuilder level dose of clen to AC in off season for training, and he gets an out of competition test, the levels will likely be detectable for at least 10-20 days following the last dose (extrapolation from horse data, where the half-life is more like 9 hours). Doesn't seem like a risk worth taking, so I think the blood bag scenario is unlikely.
3) Typo near bottom - 0.1ng/mL should be ng/kg to make any sense. Usual dose is 20,000-120,000 ng for bodybuilding, and AC weighs what, 60 kg? It's worth careful attention to units and measures and orders of magnitude, since numbers are usually used to confuse rather than to illuminate.

My guess: he ate some meat with clenbuterol in it. Only a guess, though. Still, the damage to the sport could be huge.

Just want to say thanks for your posts.

 

[BroDeal]

Re: low level indicating meat as a likely source...

explain this...to me, I don't see the logic.

To me there will be a point where, having blood drawn at such a time after using clembuterol, factoring in its metabolism by the body and the in vitro degradation that will occur, that a urine test would give the results shown in the positive test.

Maybe the levels shown would make his usage impossible, maybe not...I just dont think anyone here has done the math on it

We are never going to find the definitive evidence one way or another. Even if Contador's legal team tracks down some contaminated meat, we still won't know if it is an excuse. All we know is that the previous days' testing shows that the low level detected was not the result of declining values from a dose taken on a previous day. The positive definitely could have come from food contamination. It may be possible that it came from a transfusion.

I think what we can clearly say about this is that it shows the stupidity of not having a threshold for a positive. A quick bit of googling shows Clenbuterol contamination issues all over the world, everywhere from China to Turkey to Portugal. I don't know enough to say that the level that the level found for Contador should be too low to constitute a positive, but there surely exists a level that is too low. Anyone who handles money could be found positive for illegal drugs if the testing was sensitive enough.

 

[gobuck]

clen info

This is what Willy Voet had to say about clenbuterol.

Banned from the market in France, Clenbuterol is one of the most powerful hormones when it comes to developing muscular mass. Beef rearers are well aware of its properties: the more meat they can sell, the more money they make. It can give spectacular muscle growth. To work out its effects precisely, we needed a guinea-pig, but it couldn't be one of the riders. They are so happy to be given something new that they tend to lose all restraint and the whole pelelon knows exactly what's happening over the next few weeks. We found the right man soon enough: me. Before the Dauphine Libere in 1996 I took ten pills over seven days, then urinated conscientiously into a jar from days five to eight after taking the final pill. The whole works was then sent to a laboratory in Ghent. The Clenbuterol had been eliminated from my system by day eight. For a cyclist, who will get rid of chemicals far more quickly than someone sedentary like me, the period was still shorter.

And the effects were felt almost immediately. Three hours after I took the first pill, I began shivering. I had the impression that my lungs were swelling, that I had a new battery somewhere in the system. I felt confident, full of energy, strong as a bull - on hormones. The effects lasted for more than a month, effects which we used with good results in the big Tours after that.

some quotes from Victor Conte talking about this drug

Quote:
“It has faster clearance than any of the anabolic steroids, and it's an anabolic substance,”

“It will accelerate healing and tissue. It could also be used as a stimulant but would more likely be used between events to enhance recovery.”


http://www.bikeradar.com/forum/viewtopic.php?p=16508093