Anti-doping test methods

[Krebs cycle]

I've noticed occasionally there are threads in here about anti-doping methods eg: CO rebreathing for total Hbmass etc but the discussions are sort of scattered all over the place. I am interested in existing and new methods in anti-doping and a central thread to discuss all and sundry seems like it could be a useful thing.

Anyway, I was talking with an anti-doping researcher earlier this week who told me about a new technique being investigated at present that examines oxygen isotopic fractionation in blood. What seems to happen is that the ratio of different oxygen isotopes changes in blood that is stored for a period of time. Thus if you take a blood sample say immediately before the start of a GT and measure the oxygen isotopic fractionation, since the stored blood will have a different fractionation profile, then if you re-infuse stored blood it will alter the profile from the previous sample taken prior to the race.

At present there is large scale testing to examine natural variation in different ethnicities and develop standard reference ranges etc etc.

Hopefully, such an analysis will show up like a sore thumb when stored blood is re-infused and if so, such a test could be a big nail in the coffin of autologous blood doping.



(THE nail in the coffin is of course a massive house cleaning at the UCI, but this thread is about test methods.)

 

[Dear Wiggo]

How soon after infusion would you need to take a blood sample for this test to work?

Or to put it another way: what is the turnover rate of oxygen molecules in the blood supply?

As soon as you infuse blood, your body is using it as an oxygen transporter. The isotopically differentiated oxygen molecules are going to be used as part of the body's natural processes. I am guessing within a day the majority of oxygen molecules would have been replaced by fresh ones breathed in during that day, and the older molecules used in the body's quest for energy production.

Using a very simple guesstimate based on the world record holder's time for static apnea (22:22) and doubling it, I would guesstimate the body's supply of oxygen molecules is turned over / refreshed very hour.

If Iron isotope ratios changed sufficiently in stored blood, that would be helpful - as the RBCs containing the iron last ~120 days (~20 effective days if you factor in the max storage time (~40 days) and a 50% distribution of old:new RBCs in the extracted blood).

 

[nevada]

I've noticed occasionally there are threads in here about anti-doping methods eg: CO rebreathing for total Hbmass etc but the discussions are sort of scattered all over the place. I am interested in existing and new methods in anti-doping and a central thread to discuss all and sundry seems like it could be a useful thing.

Anyway, I was talking with an anti-doping researcher earlier this week who told me about a new technique being investigated at present that examines oxygen isotopic fractionation in blood. What seems to happen is that the ratio of different oxygen isotopes changes in blood that is stored for a period of time. Thus if you take a blood sample say immediately before the start of a GT and measure the oxygen isotopic fractionation, since the stored blood will have a different fractionation profile, then if you re-infuse stored blood it will alter the profile from the previous sample taken prior to the race.

At present there is large scale testing to examine natural variation in different ethnicities and develop standard reference ranges etc etc.

Hopefully, such an analysis will show up like a sore thumb when stored blood is re-infused and if so, such a test could be a big nail in the coffin of autologous blood doping.



(THE nail in the coffin is of course a massive house cleaning at the UCI, but this thread is about test methods.)

Wrong forum mate, the rule of thumb in the forum is

1) a win
2) appearance

 

[Krebs cycle]

How soon after infusion would you need to take a blood sample for this test to work?

Or to put it another way: what is the turnover rate of oxygen molecules in the blood supply?

As soon as you infuse blood, your body is using it as an oxygen transporter. The isotopically differentiated oxygen molecules are going to be used as part of the body's natural processes. I am guessing within a day the majority of oxygen molecules would have been replaced by fresh ones breathed in during that day, and the older molecules used in the body's quest for energy production.

Using a very simple guesstimate based on the world record holder's time for static apnea (22:22) and doubling it, I would guesstimate the body's supply of oxygen molecules is turned over / refreshed very hour.

If Iron isotope ratios changed sufficiently in stored blood, that would be helpful - as the RBCs containing the iron last ~120 days (~20 effective days if you factor in the max storage time (~40 days) and a 50% distribution of old:new RBCs in the extracted blood).

The impression that I got is that the isotopic fractions changed gradually over a period of months ie: the average lifecycle of an individual red blood cell. Therefore, at any given time there should be a more or less stable isotopic profile within an individual (since there is constant turnover of RBCs in the circulation). It would change slightly depending on natural variability due to training (and esp altitude exposure). However since stored blood must always be stored for at least as long as it takes to naturally restore the blood volume which was previously removed, then this would create an abrupt change upon reinfusion. It would be detected immediately and would potentially remain detectable for a couple of weeks. It would be cool if any form of accelerated erythropoiesis were detectable ie: EPO caused an increase in young RBCs in the circulation which would also alter the isotopic ratio, thus revealing blood manipulation.

It's early days yet but ya gotta have hope! Maybe it will take a couple of years before it is ready to go, but the important point is that it examines the effect of blood manipulation in a manner that would appear to overcome the limitations of the biopassport.

edit: maybe I totally misinterpreted the explanation and it is in plasma that the isotopic fractions are measured.... remember that when oxygen (O2) gets "used" in aerobic energy metabolism, the bond is split and one oxygen molecule combines with two hydrogens to make water. In this case it would probably take a few days for the isotopic fraction to change in the total body water content. Doubly labelled water equilibrates in less than 24hrs.

 

[Dear Wiggo]

Another thing I have come across when looking into this is the concept of removing the Oxygen entirely and storing it under Helium.

 

[blackcat]

when they take perfluorocarbons dont they have to take pure oxygen in some air tank canister when they mainline it.

did mauro gianetti the principal of Saunier Duval almost die on this $hit, and Museeuw almost lost his leg in that crash

 

[blackcat]

However since stored blood must always be stored for at least as long as it takes to naturally restore the blood volume

but only for the max deposit you wish to get to. then you can just be switching in stored blood every two weeks, and withdrawing fresh blood.

so this point you makes holds, if the person does not wish to start shuffling his/her blood once the maximum bank deposit is reached.

is about 100 days a rule of thumb for RBC?

 

[Alpechraxler]

100/120 days but it depends on the erythrocyte condition. It will get decomposed in the spleen or liver or bone marrow after reaching a certain amount of damage to it's cell surface. For example if you have serious hypertension or a lot of oxidateive stress, sickness going on that can alter the surface it might be faster.

 

[King Boonen]

I've noticed occasionally there are threads in here about anti-doping methods eg: CO rebreathing for total Hbmass etc but the discussions are sort of scattered all over the place. I am interested in existing and new methods in anti-doping and a central thread to discuss all and sundry seems like it could be a useful thing.

Anyway, I was talking with an anti-doping researcher earlier this week who told me about a new technique being investigated at present that examines oxygen isotopic fractionation in blood. What seems to happen is that the ratio of different oxygen isotopes changes in blood that is stored for a period of time. Thus if you take a blood sample say immediately before the start of a GT and measure the oxygen isotopic fractionation, since the stored blood will have a different fractionation profile, then if you re-infuse stored blood it will alter the profile from the previous sample taken prior to the race.

At present there is large scale testing to examine natural variation in different ethnicities and develop standard reference ranges etc etc.

Hopefully, such an analysis will show up like a sore thumb when stored blood is re-infused and if so, such a test could be a big nail in the coffin of autologous blood doping.



(THE nail in the coffin is of course a massive house cleaning at the UCI, but this thread is about test methods.)

Do you have a link to any papers discussing this? It sounds unlikely.

Oxygen exists as stable isotopes in nature, these won't decay over time so any differences would just be due to natural variation in the atmosphere and water supply.

Measuring the difference between 16-O and 18-O in ice cores is a way of charting historic temperatures, which gives an indication of how stable it is.

 

[Dear Wiggo]

Do you have a link to any papers discussing this? It sounds unlikely.

Oxygen exists as stable isotopes in nature, these won't decay over time so any differences would just be due to natural variation in the atmosphere and water supply.

Measuring the difference between 16-O and 18-O in ice cores is a way of charting historic temperatures, which gives an indication of how stable it is.

I was curious about this also - how do the molecules change between O18 and O16 when stored, given they are stable?

 

[Dear Wiggo]

Could it be due to the process of actually preparing the blood for storage? O18 would precipitate more and O16 evaporate more, so you'd get a difference as soon as the blood was finished being prepared for storage?

Clutching at straws somewhat.

 

[King Boonen]

Could it be due to the process of actually preparing the blood for storage? O18 would precipitate more and O16 evaporate more, so you'd get a difference as soon as the blood was finished being prepared for storage?

Clutching at straws somewhat.

It is slightly possible, but I'm very skeptical that it would be a big enough difference to detect, especially when diluted by the blood already in your system.

I would like it to be true though.

 

[Catwhoorg]

I'm more familiar with the principle of C-14 dating, and at first though maybe its simialr.

The C14 is constantly incorporated into living matter. Once the matter 'dies' then the C14 starts to decline due to its radioactivity.

However O18 is stable, so it cannot be analagous to that.

I think there must be something akin to fractionation due to the different molecular weights, but I really wouldn't want to pin my hopes on anything just yet.

Seems like a long shot.

 

[King Boonen]

I'm more familiar with the principle of C-14 dating, and at first though maybe its simialr.

The C14 is constantly incorporated into living matter. Once the matter 'dies' then the C14 starts to decline due to its radioactivity.

However O18 is stable, so it cannot be analagous to that.

I think there must be something akin to fractionation due to the different molecular weights, but I really wouldn't want to pin my hopes on anything just yet.

Seems like a long shot.

There is slight fractionation between the air and rain water but we're taking about prepared blood which I think is spun down, so pretty much no evaporation would occur. The water in the RBC is going to have the same ratio as all water which won't change over time.

It really does seem like a long shot, I'd very much like to read any papers on it though.

 

[sideshadow]

I don't have time at the moment to look through this thoroughly, but thought some might find it useful.

http://www.bloodtransfusion.it/articolo.aspx?idart=002350&idriv=75

 

[Catwhoorg]

Thanks.

I have downloaded the text and will peruse over the weekend.

 

[biokemguy]

Kinetic Isotope effect

I think there must be something akin to fractionation due to the different molecular weights, but I really wouldn't want to pin my hopes on anything just yet.

Seems like a long shot.

I think you're on to something. It's called the kinetic isotope effect. My guess would be that the isotope ratio of oxygen bound hemoglobin differs depending on the partial pressure of O2 which would change for stored blood vs. circulating blood.

Since O2 transport from oxyhemoglobin to myoglobin in muscles isn't 100%, there should be a small reservoir of O2 from the reinfused blood with a different isotope profile that has a lifetime similar to RBCs.

Personally I think the methods that look for changes in surface proteins of RBCs in storage are more promising and likely easier to detect.

 

[Dear Wiggo]

Since O2 transport from oxyhemoglobin to myoglobin in muscles isn't 100%, there should be a small reservoir of O2 from the reinfused blood with a different isotope profile that has a lifetime similar to RBCs.

Can you explain this further? 4 x O molecules can bind to a Hgb molecule, right?

You're saying that after X number of days in storage, the ratio of O18:O16 will be different for stored blood, and when reinfused, not all the oxygen bound to the Hgb in the reinfused blood is divested, so some proportion of the reinfused blood will continue to circulate in the bloodstream, somehow managing to maintain the differentiating O18:O16 ratio. In a best case for the doper scenario, they have 10l of blood and reinfuse 0.5l. So 5% of their blood, max, has a different ratio of O18:O16.

Where do the new Oxygen molecules come from whilst the blood is in storage?

 

[Dear Wiggo]

Another link - I am too tired to parse this properly.

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032137
The homeostasis of 18O and 2H of blood plasma resists the fluctuation of 18O and 2H levels of water sources

 

[King Boonen]

Another link - I am too tired to parse this properly.

On my phone so can't read it, but I'll take a guess at it saying something along the lines of, 18O levels in blood are similar to that in air, rather than the lower levels found in water. At least, that's what seems obvious to me. Will click the link when I get chance.

 

[Dear Wiggo]

On my phone so can't read it, but I'll take a guess at it saying something along the lines of, 18O levels in blood are similar to that in air, rather than the lower levels found in water. At least, that's what seems obvious to me. Will click the link when I get chance.

They are actually discussing blood vs rain water ratios - it was the study in its entirety that I can't parse properly right now. 3am or something.

Would laugh if the guys racing at altitude managed to alter their ratio by riding in the rain (the study says you won't, but still).

 

[biokemguy]

It's le chatelier's fault

Where do the new Oxygen molecules come from whilst the blood is in storage?

The change in isotope ratio could occur because the O2 that is in solution (gas can dissolve in liquid) but not necessarily bound to Hb can exchange at different rates based on isotope and partial O2 pressure. What changes in stored blood is partial O2 pressure.

I haven't put a lot of thought into this, so I'm just guessing as to the actual mechanism behind isotope ratio change in stored blood.

 

[Dear Wiggo]

The change in isotope ratio could occur because the O2 that is in solution (gas can dissolve in liquid) but not necessarily bound to Hb can exchange at different rates based on isotope and partial O2 pressure. What changes in stored blood is partial O2 pressure.

(Only just saw the Chatelier principle reference when replying - thank you that explains it succinctly).

Thanks, that makes far more sense than the scenario I was visualising where oxygen in the blood was only present when bound to Hgb.

Theoretically, you could design a molecule with good oxygen affinity (but not as good as Hgb oops!) and inject it and increase your blood oxygen levels, yeah?

So your Hgb could pick up oxygen directly from the blood stream more easily / reiably, rather than having to make the return journey all the way back to the lungs.

There's more oxygen in your blood, so you can train harder. Not as hard as a EPO-fuelled Hgb mass increase, but certainly harder than a normally aspirated rider.

And because it's not Hgb, your Hgb and Hct would not show increases. In fact, they would decrease due to the additional non-RBC material in the blood.

So you could carefully microdose EPO to bring your Hct/Hgb up to par.

Or does the affinity vs Hgb not even matter - the molecule is transported to the mitochondria anyway, and can give up its payload at the site just like Hgb would?

 

[biokemguy]

New EPO test?

So we know Di Luca & Santambrogio have tested positive for EPO and also rumours of Nibali, does this mean that WADA have a new test that can pick up even micro-dosing or are they just all being careless?

Amid the speculation in the Nibali thread there's some question as to whether a new EPO test has been developed.

This looks promising
http://www.ncbi.nlm.nih.gov/pubmed/23720238


Perhaps it can take the suspicious samples and definitively show they're positive.

 

[Dear Wiggo]

This also looks promising. MAIIA test: http://www.ncbi.nlm.nih.gov/pubmed/23347069

Morkeberg and Ashenden, et al.