- Jul 27, 2010
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The report that Contador’s TDF samples had high levels of metabolites of the plasticizer DEHP seems to provide strong evidence that he transfused blood, which would also account for the small amount of clenbuterol (CB) in his samples. Much of the evidence supporting DEHP metabolites as markers for blood transfusion comes from a paper by Segura and others comparing values of these metabolites in urine from patients who had transfused blood with 48 hours with controls who had not undergone a transfusion (http://onlinelibrary.wiley.com/doi/10.1111...09.02352.x/full
), though the German lab that actually tested AC's samples apparently used a slightly different protocol. More than half the transfusion patients exhibited metabolite levels of greater than 200 ng/ml. In contrast, none of the controls (n = 30) had values anywhere close to this. Another, much larger study, involving more than 2500 subjects, likewise reported that the vast majority of non-transfused patients (>95%) had levels of DEHP metabolites below 100 ng/ml. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2920922/?tool=pubmed)
). Since Bert’s reported values were 480 and 210 ng/ml, the conclusion that he blood doped seems very strong.
However, there are other studies of DEHP that suggest more variability of its urine metabolite levels than that reported by Segura. Two studies of a relatively small number of non-transfused subjects (n = 25, n = 45) both reported a highest MEHHP level of about 250 ng/ml. (http://data.healthis.org/pv/201004/a03.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929921/
). MEHHP is the metabolite of DEHP generally found in urine in the highest concentration, and the 480 ng/ml value reported for Bert probably is for this metabolite. In any case, each of these studies found a highest control value for this metabolite that was considerably higher than the highest value found for controls in the Segura study. In the study of 2500 subjects mentioned earlier, the highest value for this metabolite was more than 3000 ng/ml, a whopping seven times higher than Contador’s highest reported value. The highest value for another metabolite studied by Segura, MEHP, was more than 700 ng/ml, and for a third, MEOHP, about 2000 ng/ml.
Of course, such a large study would be expected to find higher outlier values, but even taking into account the number of subjects tested, these values are far larger than would be expected on the basis of a normal distribution. In fact, both this study and the two other studies I mentioned reveal a distribution of DEHP metabolite values that is highly skewed--there are a few people with extremely high values. This poses a serious problem for any WADA test, which has to minimize the possibility of false positives.
Even more damning to the WADA test than the magnitude of some control values, though, is their variability within a single individual. In addition to producing high values of DEHP, a blood transfusion is thought to result in a spike of such values. They go way up shortly after the transfusion, as large amounts of DEHP enter the bloodstream, then rapidly decline as the substance is metabolized. Contador’s values allegedly showed just such a spike, with values declining from several hundred ng/ml to 50 or less the following day, and this is considered further evidence of transfusion.
However, a very recent study reports that enormous variation in MEHHP levels can occur within non-transfused individuals, from day to day or even hour to hour (http://www.ncbi.nlm.nih.gov/pubmed/20797930
). One subject in this study, for example, showed an increase in MEHHP levels from about 100 ng/ml to 1000 ng/ml in a few hours. Another subject showed an increase from 10 ng/ml to 1000 ng/ml in a period of a few days. This study thus not only demonstrates that very high levels of DEHP can be detected in some non-transfused subjects, but that levels can show sudden spikes over time.
A key factor in observing such spikes is collection and analysis of spot urine samples; that is, every sample produced by a subject over a period of several days or more is examined. In the Segura study, urine samples obtained over a 24 period were pooled, then tested as a single sample; this would tend to average out such variations. In the other studies I cited above, spot samples were obtained, but only a single one was analyzed for each subject. Using this approach, one would expect to detect some relatively high values--and indeed, as I discussed earlier, these studies did indeed find some values higher than any reported by Segura for control subjects. But of course, variability within individuals could not be determined.
Could Contador be the victim of such natural variation? It should be easy enough to see if this is the case. If he really is innocent, as he claims, he ought to submit to spot testing of urine samples for DEHP metabolites over a period of several days. If the values recorded for his TDF samples were the result of normal variation, that variation should be evident in such a re-test. At least a few of his samples ought to show values in the range of those reported for his TDF samples, while others would be much lower. In other words, the spike reported in his TDF samples would be a natural phenomenon, as shown in other individuals. If this is indeed the case, he will have a very strong argument that the DEHP results do not establish blood doping, which in turn, will strengthen his claim that his CB positive resulted from contaminated meat.
On the other hand, if his samples show no evidence of variation that could account for high DEHP values--if they remain consistently low over a period of several days--then a conclusion of blood doping becomes much stronger. The only alternative explanation would be that he was exposed to some other source of DEHP. But this is unlikely, not only because other sources that can account for such high levels are rare, but because the exposure was temporary. It’s difficult to imagine what he could have done during a TDF--when he spent most of his time either racing or in a hotel room--that would have brought him into contact with high levels of DEHP, and only on one particular day.
Regardless of the outcome of the Contador case, I think there are some serious problems with the proposed DEHP test for blood doping. Others have pointed out that athletes could avoid excessive exposure by storing blood in containers that have much lower levels of DEHP than commonly-used blood bags. But even if all transfusions took place with DEHP-containing blood bags, interpreting results may be very difficult, given the variability of metabolite levels. Possibly, a clear-cut difference between transfuses and non-transfuses can be defined by taking 24 hour samples, averaging out variations over time, as in Segura’s study. But it’s very difficult to obtain such samples from athletes; normally, a spot sample is taken. And of course, all samples currently stored from earlier events are of this kind.
), though the German lab that actually tested AC's samples apparently used a slightly different protocol. More than half the transfusion patients exhibited metabolite levels of greater than 200 ng/ml. In contrast, none of the controls (n = 30) had values anywhere close to this. Another, much larger study, involving more than 2500 subjects, likewise reported that the vast majority of non-transfused patients (>95%) had levels of DEHP metabolites below 100 ng/ml. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2920922/?tool=pubmed)
). Since Bert’s reported values were 480 and 210 ng/ml, the conclusion that he blood doped seems very strong.
However, there are other studies of DEHP that suggest more variability of its urine metabolite levels than that reported by Segura. Two studies of a relatively small number of non-transfused subjects (n = 25, n = 45) both reported a highest MEHHP level of about 250 ng/ml. (http://data.healthis.org/pv/201004/a03.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929921/
). MEHHP is the metabolite of DEHP generally found in urine in the highest concentration, and the 480 ng/ml value reported for Bert probably is for this metabolite. In any case, each of these studies found a highest control value for this metabolite that was considerably higher than the highest value found for controls in the Segura study. In the study of 2500 subjects mentioned earlier, the highest value for this metabolite was more than 3000 ng/ml, a whopping seven times higher than Contador’s highest reported value. The highest value for another metabolite studied by Segura, MEHP, was more than 700 ng/ml, and for a third, MEOHP, about 2000 ng/ml.
Of course, such a large study would be expected to find higher outlier values, but even taking into account the number of subjects tested, these values are far larger than would be expected on the basis of a normal distribution. In fact, both this study and the two other studies I mentioned reveal a distribution of DEHP metabolite values that is highly skewed--there are a few people with extremely high values. This poses a serious problem for any WADA test, which has to minimize the possibility of false positives.
Even more damning to the WADA test than the magnitude of some control values, though, is their variability within a single individual. In addition to producing high values of DEHP, a blood transfusion is thought to result in a spike of such values. They go way up shortly after the transfusion, as large amounts of DEHP enter the bloodstream, then rapidly decline as the substance is metabolized. Contador’s values allegedly showed just such a spike, with values declining from several hundred ng/ml to 50 or less the following day, and this is considered further evidence of transfusion.
However, a very recent study reports that enormous variation in MEHHP levels can occur within non-transfused individuals, from day to day or even hour to hour (http://www.ncbi.nlm.nih.gov/pubmed/20797930
). One subject in this study, for example, showed an increase in MEHHP levels from about 100 ng/ml to 1000 ng/ml in a few hours. Another subject showed an increase from 10 ng/ml to 1000 ng/ml in a period of a few days. This study thus not only demonstrates that very high levels of DEHP can be detected in some non-transfused subjects, but that levels can show sudden spikes over time.
A key factor in observing such spikes is collection and analysis of spot urine samples; that is, every sample produced by a subject over a period of several days or more is examined. In the Segura study, urine samples obtained over a 24 period were pooled, then tested as a single sample; this would tend to average out such variations. In the other studies I cited above, spot samples were obtained, but only a single one was analyzed for each subject. Using this approach, one would expect to detect some relatively high values--and indeed, as I discussed earlier, these studies did indeed find some values higher than any reported by Segura for control subjects. But of course, variability within individuals could not be determined.
Could Contador be the victim of such natural variation? It should be easy enough to see if this is the case. If he really is innocent, as he claims, he ought to submit to spot testing of urine samples for DEHP metabolites over a period of several days. If the values recorded for his TDF samples were the result of normal variation, that variation should be evident in such a re-test. At least a few of his samples ought to show values in the range of those reported for his TDF samples, while others would be much lower. In other words, the spike reported in his TDF samples would be a natural phenomenon, as shown in other individuals. If this is indeed the case, he will have a very strong argument that the DEHP results do not establish blood doping, which in turn, will strengthen his claim that his CB positive resulted from contaminated meat.
On the other hand, if his samples show no evidence of variation that could account for high DEHP values--if they remain consistently low over a period of several days--then a conclusion of blood doping becomes much stronger. The only alternative explanation would be that he was exposed to some other source of DEHP. But this is unlikely, not only because other sources that can account for such high levels are rare, but because the exposure was temporary. It’s difficult to imagine what he could have done during a TDF--when he spent most of his time either racing or in a hotel room--that would have brought him into contact with high levels of DEHP, and only on one particular day.
Regardless of the outcome of the Contador case, I think there are some serious problems with the proposed DEHP test for blood doping. Others have pointed out that athletes could avoid excessive exposure by storing blood in containers that have much lower levels of DEHP than commonly-used blood bags. But even if all transfusions took place with DEHP-containing blood bags, interpreting results may be very difficult, given the variability of metabolite levels. Possibly, a clear-cut difference between transfuses and non-transfuses can be defined by taking 24 hour samples, averaging out variations over time, as in Segura’s study. But it’s very difficult to obtain such samples from athletes; normally, a spot sample is taken. And of course, all samples currently stored from earlier events are of this kind.
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