Mohammad, Yaser A. (2010) Detection of blood doping in athletes (masking substances and methods). MSc(R) thesis, University of Glasgow.Full text available as:
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The evidence in the detection of blood doping among endurance athletes is conflicting especially when these athletes are undergoing autologous blood transfusions and abusing recombinant human erythropoietin (rHuEPO). Continuous monitoring all year round and creating haematological profiles, known as athlete’s haematological or blood passport with cut-off values, are being practiced by some international sport federations to ban athletes from participating or apply sanctions and punishments when the tested athletes are proven to be doped. However, athletes who use prohibited substances or methods that increase the concentration of red blood cells and haemoglobin (Hb) are also using masking agents and other methods to manipulate the samples. The use of plasma expanders are prohibited as they induce haemodilution and can potentially result in false negatives of athletes’ blood samples. Other substances to date, specifically the ingestion of Creatine (Cr) and Glycerol (Gly), can cause plasma expansion when taken together by their novel synergistic effect on the human body and particularly on total body water (TBW). However, Gly has recently been added to the WADA 2010 prohibited substances list (following the completion and publication of some of the results from the experiments presented here). In addition, the posture of the sampled athlete during blood collection may also influence the concentration of blood parameters and potentially plasma volume. The main objectives of the studies presented in this thesis are to test the hypothesis that the use of hyperhydration agents, particularly Cr and Gly, in conjunction with postural changes will dilute blood samples to the extent that this manipulation could potentially influence the results of a blood doping test. In the first experiment (EXP1), eight subjects volunteered to take part in Cr and Gly fluid loading for 7 days. Urine samples and body mass were obtained before subjects lay in a supine (0°) postural position for 30 min. During that period, body water compartments were estimated and urine and blood samples were obtained from the subjects. This hyperhydration strategy and posture resulted in a significant increase in body mass by 0.82 ± 0.22 kg, TBW by 0.67 ± 0.17 L, intracellular water (ICW) by 0.35 ± 0.09 L, extracellular water (ECW) by 0.37 ± 0.15 L and urine osmolality by 161 ± 115 mOsmol.kg-1 (P<0.05) with significant decreases in Haematocrit (Hct) values at 10 min, 20 min and 30 min blood samples by 1.26% ± 0.65%, 1.3% ± 0.96% , 1.35% ± 0.73%, respectively, (P<0.05) and an insignificant decease in Hb concentration (P=0.07). These results showed that the fluid loading protocol and posture used induced a degree of hyperhydration and haemodilution that could potentially influence the outcome of a drug test. The second experiment (EXP2) focused on the changes in Hb and Hct with different postural positions of ten euhydrated subjects. The extensive research done to detect blood doping by indirect markers and equations used the supine postural position for 5-10 min prior to obtaining blood samples. However, WADA’s only approved postural position is to have the selected athlete, undergoing a doping test, be seated for 10 min before venipuncture. However, most of WADA’s anti-doping research relating to this application have been based on tests developed with subjects tested in a supine position. In this experiment, three postural positions were applied: Supine (0°), sitting and standing. As subjects changed their postures from supine (0°), sitting and ending with standing, there was an increased haemoconcentration as reflected by an increase in Hb and Hct values (P<0.05). Hb concentration and Hct values increased by 0.55 ± 0.06 g.L-1 and 0.87% ± 0.12%, respectively, when subjects changed their posture from supine to sitting and increased by 0.71 ± 0.14 g.L-1 and 1.70% ± 0.08%, respectively, when subjects changed their posture from sitting to standing. This experiment showed that the posture in which the blood samples were obtained from human subjects has a direct effect on the concentration of blood parameters such as Hb and Hct. While not a new finding, this observation confirmed the idea that posture requires standardisation for meaningful research outcomes aimed at developing valid doping tests to be developed (currently not the case). In the third experiment (EXP3), seven healthy trained subjects volunteered to take part in Cr and Gly hyperhydration followed by 2 running bouts at 60% of their maximum oxygen uptake (VO2 max) in an environmental chamber. This experiment employed a different Cr and Gly hyperhydration strategy to the one used in EXP1. Specifically, Cr was loaded for 7 days and Gly was only ingested on the day of experiment and precisely 5 hours prior to the first blood sample. Each exercise bout consisted of 30 min running bouts but at two different climatic temperatures 10°C and 35°C, respectively with 70% relative humidity. Body mass and urine samples were taken before the start of the test. Blood samples were obtained after the subjects were comfortably seated for 10 min and just prior to the start of the test as well as after each running bout. This protocol resulted in no significant changes in Hb, Hct, Reticulocytes (Retics%) and OFF-hr scores and values. In general this revised hyperhydration regiment did not induce the same degree of haemodilution and plasma expansion as the previous approach and could therefore account for the negative findings on blood profiles. The hyperhydration protocol of EXP1 using Cr and Gly for 7 days succeeded in significantly manipulating and increasing body mass, TBW, ICW, ECW and urine osmolality (P<0.05) and decreasing Hct (P<0.05) values but not Hb concentration (P=0.07) when blood was withdrawn in supine (0°) posture. In EXP2, the change of posture from supine (0°), to sitting and ending with standing resulted in haemoconcentration and increases in Hct and Hb values. With a slight change of hyperhydration protocol in EXP3 of Cr for 7 days and Gly on the seventh day only, it resulted in no significant changes in Hct, Hb, %Retics and OFF-hr scores and therefore no haemodilution effects when compared to pre-supplementation tests at rest and following submaximal exercise bouts in cool and warm environments. In all EXP1, EXP2 and EXP3, Hct among other blood parameters was the most affected by hyperhydration, posture and time at which blood samples were obtained. The hyperhydration protocol of EXP1 was more successful in decreasing Hct and Hb values than EXP3. More research should be focused on determining whether the extent of blood dilution induced by hyperhydration strategies such as those used in the present series of experiments can alter the outcome of a doping test. Other hyperhydration/fluid loading protocols should be assessed including plasma expanding substances and methods in view of these substances being considered by WADA to be included in the prohibited substances and methods list (if proven effective). It is recommended that the emphasis of WADA-related research is also shifted to the identification of other physiological/molecular markers that are not significantly affected by haemodilution such genetic signatures in response to drug perturbations (e.g. autologous blood transfusions and rHuEPO injections).
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