Lecture 1 Drugs, Ethics and Medicolegal Issues in Sport Professor Bruce Lynn MSc School of Human Health and Performance

1. Blood doping, erythropoietin and altitude training Lecture 1 Drugs, Ethics and Medicolegal Issues in Sport Professor Bruce Lynn MSc School of Human Health and Performance

2. The basic idea • To raise the oxygen carrying capacity of the blood and so improve performance in events involving aerobic exercise, i.e. any events lasting more than 1-2 minutes. • This can be done by increasing the haemoglobin (Hb) content in the blood • By legal means (altitude training) or • By illegal means • blood doping or “boosting” • use of the hormone erythropoietin.

3. Men Women Hb, g/dl 14-16 12.5-15 Haematocrit 42-50% 38-47% Some facts and figures We have 3.1013 red blood cells, containing a total of 900g of haemoglobin. Red cells last on average 120 days, so we need to replace 1/120 every day, i.e. 2.5.1011 cells, or 3 million every second! This massive production of red cells takes place in the bone marrow. Normal ranges of Hb and haematocrit. Definitions: Hb: Amount of this oxygen-carrying protein within red blood cells. Units g/decilitre blood (decilitre = 100ml). Hematocrit: Proportion of red blood cells to the total blood volume (= packed cell volume or PCV).

4. Blood doping The direct method of increasing blood haemoglobin content. Blood, or more usually concentrated red cells, are transfused via an intravenous catheter. If blood cells from a donor are used this is heterologous transfusion. Alternatively the athletes own blood can be collected, stored, then transfused back – this is autologous transfusion. Either way careful storage of red cells is necessary.

5. Blood doping Blood doping (or boosting) definitely works although estimates of the extent of improvements in performance vary. A recent review concludes that endurance performance is typically improved by 2-3% (Gledhill et al,1999) but other studies have shown maximal oxygen uptake (V02max) up by 13% (Robinson et al, 1982). But remember, even a 2% improvement is massive in competitive endurance events such as athletics. Over 10,000 meters this represents 200 meters, i.e. winning by half a lap!

6. Blood doping • There are a number of difficulties. • Heterologous transfusion • Great care needs to be taken over tissue matching. • There is the risk of getting a blood-borne infection such as AIDS or hepatitis. • Autologous transfusion • Means long periods out of competition as it takes 4-6 weeks to recover from a blood donor session (but see later for a possible fix). • For both methods the effects begin to wane within 3-4 weeks, so then need a repeat transfusion. • Nevertheless there have been plenty of rumours. It would not be surprising if rumours of blood boosting occurred as a significant number of people need to be involved in the process of taking blood, storing it, re-infusing it and checking haematocrits.

7. From the BBC Sport web site, 24 November 2006 Hamilton to ride for Tinkoff team Cyclist Tyler Hamilton, whose two-year suspension for blood doping ended in September, has signed for Italian-based Russian team Tinkoff. Hamilton was sacked by Phonak after his positive test The 35-year-old tested positive for a blood transfusion at the 2004 Tour of Spain, weeks after winning Olympic individual time trial gold. Hamilton was allowed to keep his title because his B test was destroyed by being deep-frozen. "I'm ready to make a comeback," he told the All Sport news agency. 2007 update. Tyler has cycled hardly at all in 2007 and is now beimg connected to Operacion Puerta…see next slide

8. Operaćion Puerta May 2006. Police raids in Madrid and Zaragosa as part of Operation Puerta OP). Found lots of steroids – but also transfusion gear and 100 packets of blood in clinic of Dr Eufemianos Fuertes. Possibly the better testing for EPO is driving people back to transfusions. Can overcome one of the drawbacks of donating your own blood for later use by using EPO to speed the recovery to normal levels apparently the Fuertes method. OP blood packets were labelled with pseudonyms, but a number of top cyclists were easily identified.

9. Operaćion Puerta, cont The authorities are now trying to match blood from bags to cyclists through DNA testing. No cyclists have been successfully prosecuted! But several who initially protested innocence have confessed (e.g. Ivan Basso – says he went to Fuertes but never actually used the blood….). I think the main case against Dr Fuertes has still to go to court – at which point we can expect a lot of finger pointing! But already the authorities have all the Swiss bank account details which must make interesting reading.

10. Blood doping – testing Heterologous transfusions The current test looks for 15 different minor antigens and can detect the presence of just one unit (about 500 millilitres) of transfused blood Autologous tranfusions No current test. But could look for low levels of EPO as neg feedback will reduce EPO synthesis following a tranfusion Or can look for unusual RBC:reticulocyte ratio as again transfusion will reduce natural production and so reduce reticulocyte count

11. Erythropoietin (EPO) EPO is a hormone that is released from the kidney in response to tissue hypoxia and travels to the bone marrow.

12. Erythropoietin is a large peptide (or small protein) comprising 165 amino acids with a large number of attached carbohydrate residues (it is 30% glycosylated).

13. Action of EPO In the bone marrow it stimulates production of pronormoblasts (immediate red blood cell (erythrocyte) precursors) from CFU-E cells (committed unipotential colony-forming unit - erythroid). Pronormoblasts in turn develop into normoblasts. The normoblasts start to lose their nucleus and become reticulocytes. The retuculocytes enter the blood system and finally develop into red blood cells with no nuclear material visible at all. Darkened sections of the arrows indicate times of Epo receptor expression and Hb synthesis. BFUe, burst-forming unit erythroid; CFUe, colony-forming unit erythroid. From S. Elliott. British Journal of Pharmacology (2008) 154, 529–541

14. Recombinant human EPO is used to treat anaemia associated with kidney failure, e.g. for those on dialysis, and so is widely available, although pretty expensive. Mean (±SD) level of hematocrit (upper panel) and hemoglobin (lower panel) for the rhEPO treated subjects (filled symbols, N = 10) and control subjects (open symbols, N = 10) for the treatment (days 1–30) and posttreatment period (days P1–P28). Dashed lines show prerace limits of the International Cycling Union (ICU) for hematocrit (hematocrit = 50%) and of the International Skiing Federation (FIS) for hemoglobin (hemoglobin = 18.5 g·L-1).

15. EPO induced increases in blood Hb appear, like blood doping, to increase performance in endurance events (Birkeland et al, 2000). Mean (±SD) level of maximal oxygen uptake (bars) and time to exhaustion (circles) for the rhEPO-treated subjects (hatched/filled symbols, N = 10) and control subjects (open symbols, N = 10) before treatment (day 0) and in the posttreatment period (days P1–P28).

16. It is important not to get Hb too high. Blood viscosity increases with effects on heamodynamics that mean harder work for the heart, and so an increased risk of heart problems. Pathologically elevated red cell counts also lead to a higher risk of clots. But many people who live at altitude have elevated Hb without any circulatory or cardiac problems - but maybe have made compensating adaptations. A spate of unexplained sudden deaths in cyclists in late 1980s was attributed to arrival of EPO on the scene. There have also been problems with side effects in some long- term dialysis patients.

17. Limiting haematocrit Some sports (e.g. cycling) set upper limits on haematocrit (50% for male competitors) Justified as a ban to competing on health grounds (i.e. risks due to increased blood viscosity). High haematocrit was not a doping offence. But 1% of normal population (and 20% of native highlanders) have haematocrits at or above this level. It is also claimed that haematocrit tests are subverted by masking with plasma expanders. If you expand the plasma volume, then the proportion of red cells (the haematocrit) will fall, even though the total number of red cells is still much elevated. Measuring total blood volume is not an easy thing to do, so expanding plasma volume is a straightforward way to avoid detection in a heamatocrit test.

18. Plasma expanders Cyclists apparently get by the haematocrit test by having saline infusions shortly before being tested at the start of events. The short term boost to blood volume may even also aid performance! Others use high molecular weight expanders. But these are easily detected over a long period, as almost the entire Finnish cross-country ski-ing team found to their cost at the world championships in 2001! (Seiler, 2001)

19. Detecting EPO use. It is possible to distinguish recombinant EPO from endogenous EPO, and this can even be done in urine samples. Unfortunately, EPO has a short life in the body and is only detectable for 1-2 days. As blood cells last 120 days, single tests at the time of competition are useless. Mean (±SD) level of serum EPO for the rhEPO-treated subjects (filled symbols, N = 10) before treatment (day 0) and posttreatment period (days P1–P28), as well as the baseline value for the control subjects (open symbols, N = 10).

20. Detecting EPO use. In practice, the boost from EPO does not last 120 days. As can be seen from the earlier figure, it is well down by 20 days (presumably why the Festina cycling team was toting EPO around in the 1998 Tour de France, an event that lasts 3 weeks). And urine testing gets more sensitive all the time. There is also a blood test that looks at the proportion of immature red cells that has the ability to detect EPO use up to 14 days after stopping injections. So the window of opportunity is narrowing

21. Urine testing for EPO Anti-doping urine analysis demo-nstrating the presence of recom-binant human erythropoietin (rHuEPO) in urine (see lane 4). Lane 1: rHuEPO standard; 2: positive urine (control); 3: negative urine (control); 4: sample declared positive; 5: darbepoetin alfa (Aranesp standard). Test based on isoelectric focusing patterning and a double blotting protocol. Works because the exogenous isoforms of rHuEPO are less acidic than the endogenous EPO Lasne F, de Ceaurriz J. Nature (2000); 405:635.

22. NESP Interestingly, a longer lasting version of EPO is now available for clinical use, darbepoetin or Aranesp (nesp in the peleton!). This is effective for longer, so patients (or athletes) need fewer injections and this is more convenient and cheaper. It does make it easier to detect, however, and this version of EPO was in fact successfully detected in competitors in the 2002 Winter Olympics. DYNEPO rHuEPO made in a human cell line. Has sialate etc residue profile very similar to natural human EPO. French anti-doping lab claims it can detect it, and found it in samples from 10 riders in the 2007 Tour de France. But test not yet approved by WADA, so these do not count as “positives”

23. CERA Continuous erythropoietin receptor activator. Another longer lasting version of EPO available for clinical use. This is effective for longer, so patients (or athletes) need fewer injections and this is more convenient and cheaper. This molecule is EPO plus a large methoxy-polyethylene glycol polymer, doubling the molecular weight to 60kDa. Binds with low affintiy to EPO receptor but not internalised, so keeps acting. Large size and unique structure makes detection easy. But test not out until this summer but CERA in trials, and so available before that. ADAs now want to back test old samples…. NB there were hits for CERA in Tour de France in Summer 2008. EMPs Small molecule EPO-mimetic peptides. Typically 20 amino acid residues. One EMP, Hematide, is now in trials. No sequence homology to EPO. Could imagine a rich sponsor getting an effective EMP synthesised without ever publishing the sequence….

24. Iron metabolism, ferritin, transferrin It is also possible to look at iron metabolism. Hb contains iron and the body has well developed transport and storage systems for iron so that enough is available to produce the red cells we need. If EPO stimulates red cell production then the levels of the iron storage protein ferritin fall. At same time the levels of soluble transferrin receptor (needed to get iron into developing red cells) rise in response to the increased demand. So by looking at the ferritin:transferrin ratio you get quite a good indication of red cell production rates. Unfortunately you can mask these changes by increasing iron and folic acid intake in diet. It may be possible to track transferrin receptor mRNA levels - these rise up to 40 times with EPO use, but no-one has a test based on this yet.

25. Mean (±SD) level of soluble transferrin receptor concentration (sTfR; upper panel), ferritin concentration (middle panel), and sTfR/ferritin ratio (x100; lower panel) for the rhEPO treated subjects (filled symbols, N=10) and control subjects (open symbols, N=10) for the treatment (days 1–30) and posttreatment period (days P1-P28). Dashed line in upper panel shows the mean +2 SD (95% CI) for all subjects at baseline (= 4.6 mg/L).

26. With better testing, the problem of EPO abuse may now wane. Given the dynamics (about 30 days useful boost, but possible detection up to 14 days), a strategy of random tests in the run up to major competitions looks like the way to go. It will be interesting to see how many sports in how many countries develop such a programme! In the UK most Olympic sports now have at least some out-of-competition testing. Cycling, after the fiasco of last years Tour de France and the loss of major sponsors, is also getting out-of-competitions testing organised. And some team managers claim they are going to test their own riders.

27. Time line of EPO abuse • 1984 rh-EPO licensed • EPO abuse reported • Festina team car full of EPO in tour de France • First reliable test for EPO • Chinese rowers withdraw from Sydney Olympics • No confirmed EPO +ves at Sydney • Yegereva tests +ve, gets off on technicality, goes on to win world 5000m • Winter Olympics. 3 cross-country skiers banned for Darbepoetin use. • 2003 David Miller, darling of British cycling, caught using EPO • Iban Mayo banned from Tour de France for EPO +ve • Riccardo Ricco, Moises Duenas, Manuel Beltran, all +ve for CERA in TdF

28. Other actions of EPO EPO is actually a cytokine, and like many cytokine-like hormones, is turning out to have many actions They mostly involve hypoxia or ischaemia For example the respiratory response to hypoxia is boosted by EPO, partly by an action on chemoreception in the carotid body. EPO is also produced in the brain and affects hypoxic responses centrally. (Note EPO does not cross the blood-brain barrier) Finally, EPO is tissue protective against stroke and cardiac infarct!

29. Altitude acclimatisation Living at altitudes above about 2000m leads to enough tissue anoxia to stimulate EPO release and red cell production. For example, a group who spent 30 days at the top of Pikes Peak (4300m) in Colorado had average increases in Hb from 13.7 on arrival to 16.2 at departure, with parallel increases in haematocrit (from 43-48%). These are similar to the increases seen with blood doping or EPO use.

30. Altitude acclimatisation However, on return to sea level, little if any increase in endurance performance is found. The reasons are complex. Firstly, it is not possible to train at maximum intensity at altitude just because the atmospheric oxygen level is lower. Secondly, the adjustments of the circulation to altitude involve more than just an increased Hb. Hyperventilation, a normal response to the lowered oxygen level, leads to increased carbon dioxide excretion and eventually to a reduction in the buffering power of the blood. This may reduce performance levels as lactic acid produced during high intensity exercise will not be so well neutralised. There may also be reductions in blood volume and shifts in the Hb dissociation curve, changes that may impair performance. Note, however, that for competitions held at altitude, suitable acclimatisation is essential.

31. Live high, train low To get round some of these problems you live and sleep up the mountain, but travel down to sea level to train. This option is now available to those of us who do not live conveniently close to a suitable mountain – the nitrogen tent or house. Athletes live in nitrogen tents with the oxygen level reduced to 15-16% (equivalent to being at 2500m altitude). These strategies allow you to train in the normal way, but should provoke a useful boost in blood Hb due to the time spent at simulated altitude. There will still be problems with other, disadvantageous, circulatory adaptations. Results have been contradictory – some showing improvements, others not. There is even dispute as to how far HiLo actually stimulates increased Hb. See articles by Wilbur and by Gore in Med Sci Sports Medicine (2007) 39(9)

32. The coaches and athletes certainly believe altitude training important Here is Steve Cram writing in the Guardian Tuesday October 2, 2007, after the Great North Run this year: “For most this still means training in the mountains but increasingly it involves hypoxic tents, where the pressure can be adjusted to mimic any altitude and it is where many sleeping hours are spent. Salazar likes to combine the two. In preparation for the world championships in Osaka his group trained at Park City in Utah at 7,000-8,000ft. In addition he advises sleeping in the tents at a setting of anything up to 13,000ft. “ http://sport.guardian.co.uk/athletics/comment/0,,2181691,00.html Kara Goucher, coached by Salazar, beating Paula Radcliffe. 2007 Great North Run

33. Nitrogen tent http://altitudetraining.com/wrap/images/four30_large

34. Percent change in 5000-m time from baseline performance (at 6 weeks) in three training groups.  Recalculated and redrawn from data of Levine & Stray-Gundersen, 1997. The bar labelled likely range of true change is the author of this review’s estimate of the 95% confidence interval for points other than the baseline.  http://www.sportsci.org/traintech/altitude/wgh.html

35. Report posted 7 Oct 2008 Bellamy sleeping in altitude tent Wales captain Craig Bellamy is sleeping in an altitude tent in a bid to save his career. The injury-prone West Ham striker is to make his first start for a year when Wales host Liechtenstein on Saturday [Oct 11, 2008]. "I have got my own altitude tent and I sleep in it every night because it speeds up recovery and improves overall fitness," Bellamy admits. His tent simulates high altitude by maintaining a lower oxygen concentration. One of the benefits is to increase red blood cells and enzymes to speed recovery. "My rehabilitation has been quite extraordinary," Bellamy told BBC Sport. "And I want to really attack the last years of my career in a positive manner. "The altitude tent improves endurance, helps speed and makes you a fitter athlete. "I'm ready to play 90 minutes and play two games in five days because I feel good and I've done all of the groundwork."

36. Exercise efficiency improves with altitude training From:   GORE: Med Sci Sports Exerc, Volume 39(9).September 2007.1600-1609 Percent decrease in oxygen consumption during submaximal exercise after various forms of altitude acclimatization. mean ± SD. 1) Three hours per day for 2 wk of intermittent exposure to normobaric hypoxia (12.3% FIO2). 2) 20 nights of about 8 h per night of sleep at a simulated altitude between 2000 and 3100 m, and training near sea level (600 m). 3) 23 nights of about 10 h per night of exposure to a 3000-m simulated altitude. 4) 29 days of about 12 h·d-1 of exposure to simulated LHTL at 3000 m. 6) Exposure to intermittent hypobaria of 4500 m, 3× 90 min·wk-1, for 3 wk. 7) Five to six nights at 2500 m followed by 8 to 12 nights at 3000-3500 m, sleeping at simulated altitude and all training at 1200 m. 7) Mountain climbing for 3 wk to 6194 m. 8 and 9) Long-term residence at 3500-4500 m.

37. Summary – Blood doping, erythropoietin and altitude training • Increasing blood haemoglobin levels to the very top end of the normal range increases aerobic exercise capacity significantly. • Such effects are obtained with direct blood transfusions or with EPO injections. • Both procedures are banned. The current tests look adequate to eliminate EPO abuse if applied out of competition. • Altitude (real or simulated) acclimatization is not the same as blood boosting. The overall changes to blood and circulation are more complex, and deliver smaller increases in terms of sea level performance. There are also interesting improvements in non-haematological aspects of performance.

38. Methods to enhance oxygen transport include stimulation of endogenous Epo (eEpo) synthesis, stimulation of erythropoiesis (e.g., through administration of erythropoiesis-stimulating agents (ESAs) such as recombinant human Epo (rHuEpo)) or through direct increases in delivery of oxygen (e.g., transfusion, administration of artificial oxygen carriers or through enhanced oxygen unloading). 2,3-DPG, 2,3-diphospho glycerate; HBOC, Hb-based oxygen carrier. From S. Elliott. British Journal of Pharmacology (2008) 154, 529–541

39. References Overviews Drugs in sport – blood doping, erythropoeitin and altitude training. A. Narvani et al, Key Topics in Sports Medicine, pp78-82, 2006. Blood boosting and sport. Armstrong, DJ. and Reilly, T. Chapt 7 in Drugs in sport, 3rd edn, ed DR Mottram, 2003. S Elliott (2008) Erythropoiesis-stimulating agents and other methods to enhance oxygen transport. British Journal of Pharmacology (2008) 154, 529–541 Papers cited Birkeland, KI, Stray-Gundersen, J et al (2000) Effect of rhEPO administration on serum levels of sTfR and cycling performance. Med-Sci-Sports-Exerc. 32(7): 1238-43 Gore, C. J., et al. (2003). Second-generation blood tests to detect erythropoietin abuse by athletes. Haematologica88, 333-344. Gledhill, N, Warburton, D and Jamnik, V. (1999) Can J Appl Physiol, 24, 54-65 Hahn, A. G. & Gore, C. J. (2001). The effect of altitude on cycling performance: a challenge to traditional concepts. Sports Med.31, 533-557. Robinson, RJ et al, (1982) The effect of induced erythrocythemia on hypoxia tolerance during exercise. J Appl Physiol. 53, 490. N Robinson, S Giraud, C Saudan, N Baume, L Avois, P Mangin, M Saugy, Erythropoietin and blood doping, Br J Sports Med 2006;40(Suppl I):i30–i34. Seiler, S (2001) Doping Disaster for Finnish Ski Team: a Turning Point for Drug Testing? Sportscience 5(1), sportsci.org/jour/0101/ss.html Stray-Gundersen J, Videman T, Penttilä I, Lereim I., Abnormal hematologic profiles in elite cross-country skiers: blood doping or? Clin J Sport Med. 2003 May;13(3):132-7.Links GORE, CHRISTOPHER JOHN, CLARK, SALLY A., SAUNDERS, PHILO U. Nonhematological Mechanisms of Improved Sea-Level Performance after Hypoxic Exposure. Medicine & Science in Sports & Exercise. 39(9):1600-1609, September 2007. WILBER, RANDALL L., STRAY-GUNDERSEN, JAMES, LEVINE, BENJAMIN D. Effect of Hypoxic "Dose" on Physiological Responses and Sea-Level Performance. Medicine & Science in Sports & Exercise. 39(9):1590-1599