The case for/against transfusion. For science geeks.

[Merckx index]

WADA has said they will appeal the RFEC decision, apparently with or without UCI. An important part of the case Bert is trying to make in his defense is the improbability that he could have tested positive for clenbuterol (CB) as a result of a blood transfusion containing this substance. His arguments will hinge on pharmacokinetics, that is, the rate at which CB is cleared from the body. Here are three studies of CB I found helpful and relevant to this issue (I haven‘t provided links, but given enough information to access the article or abstract quickly; the first one I believe is not at PubMed, but they're all available at Google Scholar):

Yamamoto et al. (1985) - single dose of 20, 40 or 80 ug given to human volunteers. Plasma levels reached maximum of 100, 200 and 350 pg/ml. in 2.5 hours and lasted for 6 hours. When administered orally twice daily, maximum of 200-300 (20 ug dose) and 500-600 (40 ug dose) reached in four days. After 72 hours, 20% of single dose found in urine.

Comment: So when a single oral dose of 20 ug was taken, about 4 ug total was passed in the urine in 72 hours. For a typical individual, about 4 l. of urine are passed in this time period, so the average concentration of CB in urine is about 1 ng/ml., below some detection limits. Of course, this is only an average. It is much higher shortly after dosing. I will get to this in a moment. It would also be higher if the CB is taken regularly. Thus when subjects were given CB twice daily, after four days plasma levels were 2-3 times higher than after a single dose. Presumably, urine levels would be proportionately higher as well.

Meyer and Rinke (1991) - 5 ug/kg. given twice a day for 3 weeks to calves. Urine taken at multiple periods after dosing stopped. Highest concentration was 112 ng/ml., but this is probably outlier. Most concentrations up to 1 day withdrawal fell between 10-40 ng/ml, decreasing after that.

Comment: The dose of 5 ug/kg used in this study is referred to as an anabolic dose, compared to a lower therapeutic dose (for asthma, e.g.). I believe, however, when CB is used by athletes for weight loss or to increase muscle/fat ratio, doses in the therapeutic range (20-100 ug) are used. Someone who has used CB for this purpose, or knows others who have, can maybe correct and/or enlighten me further on this issue.

Anyway, my assumption is that a rider would take about 10% of this dose, e.g., 30 ug for a 60 kg rider would be 0.5 ug/kg. Extrapolating from this study of a different species, urine levels would mostly be in the range of 1-4 ng/ml, IOW, sometimes below the sensitivity of some labs to detect. And after one day, they would generally drop below 1 ng/ml. So unless a rider was being tested on very sensitive equipment, clearing to the point of a negative test could take place within as little as 24 hours, and most often within 2-3 days.

Smith and Paulson (1997) - 3mg/kg single oral dose given to calves. Pooled urine taken at 6 or 12 hour intervals. 36-47% of original CB administered found in urine after 48 hours. Individual pools contained 5-13%.

This study of a single dose found a larger % of CB ending up in the urine than the Yamamoto study. This could be a species difference, humans vs. calves. Extrapolating to a 20 ug. dose in humans, about 8 ug would collect in the urine in 48 hours, and about 1-2.5 ug per 6 or 12 hour pool, as they were described in this study. Assuming a pool contains 500 ml., this corresponds to 2-5 ng/ml.

So from these data, even a single relatively low dose of CB would be fairly detectable in urine up to 48 hours. However, the study noted that most of the original dose had been accounted for by this period, so presumably after 48 hours, a subject would test negative except possibly for the most sensitive equipment.

Conclusions:
1) a rider taking CB at therapeutic but I believe still performance-enhancing levels, say, less than 100 ug per dose, could probably pass all but the most sensitive tests within three days of terminating the doping. So if the program were used for a single week, there would be a ten day period of vulnerability. Moreover, if he took a dose in the low range, say 20-30 ug, it's conceivable that, depending on the sensitivity of the test, he could pass all the tests even during the doping period.
2) I don’t know if CB would be very useful if taken as a single dose, but if a rider did do this, it would very likely go undetected unless he was tested within 48 or even 24 hours of taking the substance. IOW, a very low risk move.
3) If micro dosing is possible and effective--say, taking a dose of less than 10 ug.daily over a period of time--a rider could very conceivably pass all but the most sensitive tests even if they were administered during the period he was doping.
4) These conclusions address the issue of whether testing during June, when speculation suggests any blood withdrawal most likely would have occurred, could have detected the use of CB that then ended up in the blood being transfused. However, even if it can be established that Bert could have gotten away with taking CB during this period, there is an additional question: how much CB would he have to take for it to show up as a 50 pg positive following transfusion?

As I have discussed in the Contador acquitted thread, the amount of blood transfused is typically 5-10% of total blood volume. So had he hypothetically been tested at the time he was withdrawing blood, he would have had, at a minimum, 10-20 times higher urine levels, i.e., 500-1000 pg/ml. This range, while still below the sensitivity of some labs, is actually an underestimate (see the other thread). The actual value would have to be somewhat higher, probably 2 ng/ml or more. As we have seen, this is consistent with using therapeutic doses of CB. So the level Bert actually tested for is, in this respect, quite consistent with resulting from a transfusion.

However, to reach this level originally, before withdrawal, he would most likely have taken CB regularly, at moderate to high therapeutic doses, for at least several days, extending his period of vulnerability to testing. This is where data on when he was tested become very critical to his case, IMO. If he was tested during this period, Bert needs to make the case that the test would not have allowed him to get away with taking doses large enough and often enough to provide the levels obtained after transfusion.

If it turns out he was not tested at all during this period, then I think his case that transfusion would not explain his positive collapses. If he was tested at some point, then the fun begins.

 

[bhilden]

I think what I take away from your query is that given all the science mumbo-jumbo you cite it is clear to me why WADA has a zero tolerance policy on some substances like Clenbuterol.

The drug testers can't be everywhere all the time so unless a rider can positively prove contamination by providing a sample of the contaminated product they took, it is all just speculation on what they took, how much they took and when they took a particular banned substance.

This can clearly create unfortunate situations where innocent riders get a suspension, but the current policy is zero tolerance. Someone please tell me the difference between Contador's situation and Tom Zirbel's situation. Neither can prove they unknowingly took a banned substance and only one is serving a suspension.

 

[BroDeal]

Someone please tell me the difference between Contador's situation and Tom Zirbel's situation.

Contador has clean tests from the days before he tested positive for miniscule amounts of a banned substance. Zirbel does not.

Here is a question. I assume the studies that show how quickly Clen is eliminated are done with regular people who drink regular amounts of liquid. How does that compare to someone who drinks six or seven liters of liquid during a day at the TdF?

 

[JA.Tri]

muscle concentration of CB in calf ...and AC

What dosage of CB would need to be administered to calf/cow to generate muscle CB levels sufficient to deliver sufficient CB to AC?

As an alternative what level of CB would have been present in AC's muscle tissue at the time he delivered the questioned urine sample?

I guess it would be at least 1 order of magnitude less than urine but this truly is a guess.

I ask these questions simply because they seem to be an important part of the total picture.

If previous posts have addressed please pardon (and PM the link).

 

[Dallas_]

Must admit I had to really concentrate hard, but credit where credit is due Merckx index, a top post.

To me, this is the most relevant post in the Clinic, regarding Clen and AC's case. I hope WADA do appeal, as I have never believed even 1% of the BS beef theory.

Berto needs to read this for a reality check -->> http://www.skynews.com.au/health/article.aspx?id=582203&vId=

cheers

 

[python]

i could not resist but say that drunk monkeys come to mind (as was the recent case with a currently folk hero/doper) when complex human pharmacokinetics are derived from bovines.

not to metion a totally unique species - professional cyclists. well, may be cav qualifies for the sudy.

 

[peterst6906]

i could not resist but say that drunk monkeys come to mind (as was the recent case with a currently folk hero/doper) when complex human pharmacokinetics are derived from bovines.

+1

Any attempt to look at this scientifically is good, but there are large risks in extrapolating data and making assumptions/presumations about dosing and effects.

Taking a conservative approach, the level of error that should be allowed for in this analysis is magnitudes larger than the listed values.

As a result, it's still not possible from this analysis to sort fact from pure conjecture.

 

[pnwrider]

I believe Clenbuterol is generally sold in 20 ug pills.

One question on this theory--I thought when blood doping, the plasma was spun off, and just the packed red cells were infused. Wouldn't that eliminate most of the anything he could test positive for?

 

[dbrower]

I believe Clenbuterol is generally sold in 20 ug pills.

One question on this theory--I thought when blood doping, the plasma was spun off, and just the packed red cells were infused. Wouldn't that eliminate most of the anything he could test positive for?

There seems to be a lot of simple, whole-blood re-infusion going on. Spinning requires a lot more effort, and infrastructure.

-dB

 

[Merckx index]

Here is a question. I assume the studies that show how quickly Clen is eliminated are done with regular people who drink regular amounts of liquid. How does that compare to someone who drinks six or seven liters of liquid during a day at the TdF?

Sure. Actually the key questions here revolve around elimination before the Tour, when speculation is that Bert took CB and transfused. If he was training hard, then yes, he was drinking a lot of water. But remember, we drink a lot of water during intense physical exercise because the water is used by the body and because a lot is sweated off. Don’t **** all of that water out.

A more important factor than water per se is increased metabolic rate. This could result in faster clearance of the drug. But remember, most of the vulnerability to testing window is probably taken up by period in which CB is used, not the withdrawal period. Faster clearance might reduce the window from ten days to nine, not a huge deal.

What dosage of CB would need to be administered to calf/cow to generate muscle CB levels sufficient to deliver sufficient CB to AC?

This question was apparently addressed in detail in the WADA files used in the RFEC decision. WADA claims that Bert’s 50 pg/ml level is indicative of a CB levels in calf of at least 3x the standard of detection, and possibly as high as 100 times. Here's the relevant passage:

According to AMA, evidence reports have established contact with one of the clenbuterol manufacturing companies, and with the help of previous studies they were able to estimate the amount of clenbuterol which should have been present in the meat ingested (Appendices 3,4,5). Taking into account various scenarios and assumptions, the one most favorable to the athlete shows the contamination [of the meat] to be about 312 ng/kg., at least three times higher than the minimum detection level [Note: the detection level is 100 ng/kg] set by the EU. Other scenarios are up to 11,000 ng/kg, based on excretion studies developed by the Cologne laboratory, i.e., 110 times the minimum level of detection within the EU (these pharmacokinetic calculations were confirmed by one of the clenbuterol manufacturers). In all cases, mean high clenbuterol concentrations would be fully detectable by health authorities.

But this could be, and apparently was (and will be again) challenged by Bert’s team. Depends on a lot of assumptions. I might dig into this at some point.

i could not resist but say that drunk monkeys come to mind (as was the recent case with a currently folk hero/doper) when complex human pharmacokinetics are derived from bovines.

There is a reason a lot of pharmacokinetic studies are done on bovines (and other mammals), and it’s not just because of fear of CB-tainted meat. They are good model systems for humans in many respects. Of course there are differences--I noted a possible one--and these have to be taken into account. I discussed these two bovine studies because it’s clear from the data presented in those articles that their CB clearance rates are broadly similar to humans--several days, not hours, not weeks--with more than half of the initial dose ending up in tissues, not urine.

For example, in the Yamamoto study cited, following doses of 20, 40 and 80 ug., plasma levels in humans reached maxima of 100, 200 and 350 pg/ml. after 2.5 hours. The 40 and 80 ug doses can be assumed to be roughly 0.5 and 1.0 ug/kg. When rabbits were given a dose of 0.5 and 2.0 ug/kg, plasma maxima of 200 and 800 pg/ml. were reached within 2 hours, so the absorption into blood is very similar for these two species. The half life of CB in rabbits was only 9 hours, as compared to 35 hours in humans, and 30 hours in rats. The Meyer and Rinke study found a half-life of CB in plasma of calves of 18 hours, so humans generally have a somewhat slower rate of elimination. This is further complicated by a biphasic elimination that is found in dogs, humans and rats as well as calves. After a day or two, clearance is slower.

So yes, there are somewhat different values for clearance times for different species, but the general kinetics are very similar. There are other factors, such as individual differences, and dose differences, so a rough range is all we can ever estimate when discussing clearance rates. These rates are critical to determining the window of testing vulnerability, but asusming he would have been taking CB for at least a week or so, changes of a day or so in estimates of clearance don't much affect this window.

On the other hand, the peak values in urine are not very dependent on clearance rates, and so can be estimated more accurately, even using animal data. These are most relevant to conclusions about whether Bert could have passed CB tests, depending on the dosage he was using.

One question on this theory--I thought when blood doping, the plasma was spun off, and just the packed red cells were infused. Wouldn't that eliminate most of the anything he could test positive for?

Discussed upstream. This may well be the case. Certainly the case for long-term storage when RBC are stored frozen. But even in that case, the rider might choose to store the plasma and reconstitute the cells with that rather than saline. And in short-term storage, as dB notes, not separating is of course easier. That’s what Ricco did, though I’m sure not going to use him as an example of how the rest of the peloton dopes.

 

[lean mean &green]

there are plenty of assumptions that need to be made when tackling a problem such as this and merckx index is upfront about those uncertainties, he acknowledges where the research is incomplete, and he's made it clear in his writing that there's a lot of guess work involved. he isn't submitting his findings for peer review or testifying in front of CAS, he's only presenting them as "back of the napkin" calculations to gain perspective. his contribution here is commendable in a number of ways.

So the level Bert actually tested for is, in this respect, quite consistent with resulting from a transfusion.

this is the take home message. once you've gained a better understanding of what the values actually mean and you start creating an expectant range you no longer trivialize a concentration of 50 pg/mL. in fact, contador's values are more consistent with transfusion than they are with contamination. sadly, i've dug deep and crunched a lot of numbers myself and that's about the only conclusion i can stand behind. it's not much but it's a few steps forward at least.

hopefully the complexity of establishing a threshold for this and similar substances is coming into clearer focus as well. therapeutic (and very effective) doses are usually less than 100 micrograms. that's very little of the drug by weight and that results in very low concentrations in blood/urine. those already low concentrations can be further diluted by factors of 100 or more when transfused. even setting a threshold of just 100 pg/mL could be regrettable.

 

[Merckx index]

there are plenty of assumptions that need to be made when tackling a problem such as this and merckx index is upfront about those uncertainties, he acknowledges where the research is incomplete, and he's made it clear in his writing that there's a lot of guess work involved. he isn't submitting his findings for peer review or testifying in front of CAS, he's only presenting them as "back of the napkin" calculations to gain perspective. his contribution here is commendable in a number of ways.

I should have made clear at the outset that because I'm a cheapskate who doesn't like to pay for online articles--and because I'm out of town and can't access a med library--I haven't been able to read a complete paper on human CB kinetics. This is why I was relying on calf data to some extent, where I can see the complete article. WADA, for sure, will have access to data that I don't.

But the differences between humans and cattle can work both ways. For example, cattle apparently clear CB from plasma faster than humans, which would suggest that humans will have quantities of CB in their urine longer than cattle. But it also suggests that these quantities will be lower, since the longer the period of clearing, the less cleared at any particular time. This has relevance to speculation about the dose a rider could take and avoid detection even during or very soon after the period of ingestion.

this is the take home message. once you've gained a better understanding of what the values actually mean and you start creating an expectant range you no longer trivialize a concentration of 50 pg/mL. in fact, contador's values are more consistent with transfusion than they are with contamination. sadly, i've dug deep and crunched a lot of numbers myself and that's about the only conclusion i can stand behind. it's not much but it's a few steps forward at least.

One of the posters asked about how much CB a calf would have to be given for Bert to test positive. I should have made clear that the real issue, which is touched on briefly in the RFEC report, is how much contamination--i.e., how much CB in meat--would be necessary for a positive. I quoted a passage from the RFEC decision concerning that.

But I can say a little more. A simple approach to this question is possible. As I pointed out on another thread, an ingestion of 135 ng of CB--a value LMG estimated that might result from blood transfusion--could result in a urine concentration of the 50 pg/ml found in Bert. My estimate was that 6.5 - 17 pg would appear in a urine sample taken within a day of the transfusion, so for a 200 ml sample, the range would be about 30-85 pg/ml, with the range moving higher or lower if the urine sample were smaller or larger. There is thus a large variability in this estimate, maybe as much as 10-200 pg/ml., but clearly 50 pg is well within the expected range, which is all the prosecution should have to establish.

The EU detection standard is supposed to be 100 ng/kg of meat, so to ingest 135 ng of CB from meat that passed inspection, one would have to eat more than a kg., about three pounds of it, minimum. I’m pretty sure Bert doesn’t eat meat in these quantities. In fact, I believe he has already stated about how much he ate?? If he has, more precise calculations are obviously possible concerning how much CB/kg would have to be in the meat.

Now consider some of the qualifiers. First, there is a range, depending particularly on the exact kinetics. So one could lower substantially the amount of meat that would need to be eaten. WADA, as I said before, has concluded that the most favorable possibility for Bert is still off by a factor of three. I assume they know about how much meat Bert ate to draw this conclusion. These rough calculations I have provided here may help in understanding that.

Second, this estimate was based on calf data, since I haven‘t been able to access all the human studies. Since calves clear CB somewhat faster than humans, the estimate might have to be lowered; that is, if humans clear more slowly, less CB would probably appear in urine at any given time from a given initial dose. But this works against Bert, because it means that, if anything, one would have to ingest more CB in meat to reach that 50 pg. value.

These two qualifiers, I should add, also apply to transfusion. More CB might have to be present in blood to get the 50 pg value. But in this case, I think more leeway is easily possible, since there is a pretty large range of CB doses that a rider could take. The main thing that increasing the amount of CB in blood does for these arguments is increase the possibility of detection. So the question of whether Bert was tested during this period of possible blood withdrawal becomes important.

Another issue is the dose. I very much doubt that CB kinetics have ever been carried out on such a low dose, so one could argue that peak urine values, as % of dose, are much higher than those determined from the usual dose. I think this is unlikely. More likely, they would be lower, because CB probably binds to proteins and other factors in the blood, and the smaller the dose, normally, if anything, the greater the % of the dose that is bound (this gets into receptor binding theory). So less would be passed into the urine, at least in the short-term.

However, one could always make this argument, that very low doses pass more quickly into the urine. One could speculate about any number of factors to support such a claim. This would lower the amount of contaminated meat necessary. I imagine the kinetic data/arguments WADA referred to involve this, among other things.

But one can see from the three pound figure that it would have to be lowered quite a bit, 5-10 fold I should think. And at the end of the day, all one has is the unproven possibility that a relatively small amount of meat below the detection limit could result in the positive. Obviously, this doesn’t come close to rising to the standard that Bert needs.

hopefully the complexity of establishing a threshold for this and similar substances is coming into clearer focus as well. therapeutic (and very effective) doses are usually less than 100 micrograms. that's very little of the drug by weight and that results in very low concentrations in blood/urine. those already low concentrations can be further diluted by factors of 100 or more when transfused. even setting a threshold of just 100 pg/mL could be regrettable.

I argued in an earlier post that the fact that Bert's urine value actually went up from 7 to 17 pg/ml from July 24 to July 25 argued that values below 20 pg/ml are uncertain, and therefore a threshold might be called for. Another scientist has advised me that while there might be considerable uncertainty in this range, the presence of CB itself is not in doubt.

Another factor that may ultimately mitigate for a threshold, though, is environmental contamination. The RFEC report acknowledges that some EU farmers may well be using CB. The argument is that they let it clear from the cattle before slaughter, to prevent testing positive. This does not affect Bert's case--cattle treated in this way continue to fly under the EU detection radar--but if they are giving CB to cattle at any time, it is getting into soil and groundwater through manure, much as synthetic hormones, which can be legally given to cattle in America, are. So at some point down the road, we all may have traces of CB, just as we all apparently now have significant amounts of DEHP in our bodies. Welcome to the brave new world.