Biomedical subjects
Publications and source records attributed to Michel Audran.
This study appraised the veracity of claims that athletes can evade doping controls by injecting microdoses of recombinant human erythropoietin (rHuEPO), which rapidly disappear from the circulation. We confirmed that microdosing can reduce the window of detection to as little as 12-18 hours post-injection, suggesting that authorities must adopt appropriate counter measures.
BACKGROUND: Hemoglobin-based oxygen carriers (HBOCs) are being developed as potential substitutes for the oxygen-carrying functions of erythrocytes, but athletes may obtain and experiment with HBOCs as an illicit means of enhancing oxygen transport. An electrophoretic technique has been developed to screen for the presence of HBOCs in blood samples (Lasne et al. Clin Chem 2004;50:410-5). Interest has focused on complementary methods that can provide legally defensible scientific evidence for the presence of HBOCs in blood samples collected for doping control. METHODS: The aim of this research was to develop a size-exclusion SEC-HPLC technique to identify in plasma or serum samples the presence of HBOCs that are currently under development. This method was also used to detect a polymerized bovine hemoglobin (Hemopure) after infusion in 12 healthy males. RESULTS: The chromatograms of all HBOCs tested were clearly separated from the 54-min peak associated with human hemoglobin dimers. It was possible to differentiate between the different HBOC products based solely on their chromatographic profiles, provided they were at high concentrations. Differences were discernible not only based on the presence (or absence) of peaks, but also the separation between respective peaks. The profiles for serum samples collected from the men immediately after infusion of Hemopure showed a distinctive profile. The shape of the chromatographic profile remained consistent for at least 48 h. CONCLUSIONS: Under the analytical conditions reported here, SEC-HPLC was able to separate native hemoglobin from the modified hemoglobin molecules present in each of the HBOC products studied. In tandem with electrophoretic screening, SEC-HPLC provides evidence of the presence of HBOCs and can therefore be regarded as a method that satisfies the criteria for use in an antidoping control setting.
We studied indirect detection models of erythropoietin abuse (EPO) on blood samples collected 48-hr after administration of the drug during 6 weeks of recombinant human erythropoietin (rHuEPO) treatment. Although the efficiency of OFF-models was preserved, we found a loss of sensitivity of ON-models. This study also revealed an increased percentage of stomatocytes in athletes receiving rHuEPO
BACKGROUND AND OBJECTIVES: Enhancement of oxygen delivery to tissues is associated with improved sporting performance. One way of enhancing oxygen delivery is to take recombinant human erythropoietin (rHuEpo), which is an unethical and potentially dangerous practice. However, detection of the use of rHuEpo remains difficult in situations such as: i) several days after the end of treatment ii) when a treatment with low doses is conducted iii) if the rHuEpo effect is increased by other substances. In an attempt to detect rHuEpo abuse, we selected erythroid gene markers from a SAGE library and analyzed the effects of rHuEpo administration on expression of the HBB, FTL and OAZ genes. DESIGN AND METHODS: Ten athletes were assigned to the rHuEpo or placebo group. The rHuEpo group received subcutaneous injections of rHuEpo (50 UI/kg three times a week, 4 weeks; 20 UI/kg three times a week, 2 weeks). HBB, FTL and OAZ gene profiles were monitored by real time-polymerase chain reaction (PCR) quantification during and for 3 weeks after drug administration. RESULTS: The global analysis of these targeted genes detected in whole blood samples showed a characteristic profile of subjects misusing rHuEpo with a increase above the threshold levels. The individual analysis of OAZ mRNA seemed indicative of rHuEpo treatment. INTERPRETATION AND CONCLUSIONS: The performance-enhancing effect of rHuEpo treatment is greater than the duration of hematologic changes associated with rHuEpo misuse. Although direct electrophoretic methods to detect rHuEpo have been developed, recombinant isoforms of rHuEpo are not detectable some days after the last subcutaneous injection. To overcome these limitations indirect OFF models have been developed. Our data suggest that, in the near future, it will be possible to consolidate results achievable with the OFF models by analyzing selected erythroid gene markers as a supplement to indirect methods.
Longitudinal monitoring of athlete's hematologic parameters holds considerable promise as a strategy to detect and thereby deter illicit blood doping. This study documents temporal changes of hemoglobin concentration (Hb) and reticulocyte counts in elite rowers. The 'within subject' variation in rowers was comparable to that of athletes from other sports. Reticulocyte results were dependent on the type of instrument used.
The last Winter Olympics and recent cycling events have once again called attention to the use by endurance sportsmen and women of a variety of approaches designed to increase the blood hemoglobin concentration and oxygen-carrying capacity. An increased hemoglobin concentration and maximal oxygen uptake can be achieved by training at high altitude or by "living high and training low". It can also be procured by "blood boosting" with erythropoietin or darbepoietin, blood infusion, or misuse of hemoglobin-based oxygen carriers. This paper describes methods of increasing oxygen transport, with the accent on blood infusion, which is again relevant today with the development of a test able to detect recombinant human erythropoietins.
BACKGROUND: Hemoglobin-based oxygen carriers (HBOCs) have recently been included in the International Olympic Committee and World Anti-Doping Agency lists of substances and methods prohibited in sports. To enforce this rule and deter abuse of HBOCs in elite sports, it is necessary to develop HBOC-specific screening and confirmation tests that are the usual steps in antidoping control analysis. METHODS: We developed a screening method based on electrophoresis of serum samples cleared of haptoglobin (Hp). Four successive steps (immunoprecipitation of Hp, electrophoresis of the cleared serum, Western blotting of the separated proteins, and detection of hemoglobin-related molecules based on the peroxidase properties of the heme moiety), provided electropherograms that could be easily interpreted in terms of the presence of HBOCs. This method was tested with serum samples enriched with various types of HBOCs: polymerized, conjugated, and cross-linked hemoglobins. It was also applied to blood samples collected from 12 healthy volunteers who had been infused with either 30 or 45 g of Hemopure, a glutaraldehyde-polymerized bovine hemoglobin. RESULTS: The method clearly detected the presence in serum of the various types of HBOCs tested and demonstrated no possible confusion with endogenous hemoglobin that may be present in cases of hemolysis. The test was able to detect Hemopure for 4-5 days after administration of 45 g to healthy individuals. CONCLUSIONS: The electrophoretic method is a simple, fast, and sensitive procedure that appears to fulfill the criteria of a screening test for the presence of HBOCs in antidoping control samples.
Blood oxygenation is a fundamental factor in optimising muscular activity. Enhancement of oxygen delivery to tissues is associated with a substantial improvement in athletic performance, particularly in endurance sports. Progress in medical research has led to the identification of new chemicals for the treatment of severe anaemia. Effective and promising molecules have been created and sometimes used for doping purposes. The aim of this review is to present methods, and drugs, known to be (or that might be) used by athletes to increase oxygen transport in an attempt to improve endurance capacity. These methods and drugs include: (i) blood transfusion; (ii) endogenous stimulation of red blood cell production at altitude, or using hypoxic rooms, erythropoietins (EPOs), EPO gene therapy or EPO mimetics; (iii) allosteric effectors of haemoglobin; and (iv) blood substitutes such as modified haemoglobin solutions and perfluorochemicals. Often, new chemicals are used before safety tests have been completed and athletes are taking great health risks. Such new chemicals have also created the need for new instrumental strategies in doping control laboratories, but not all of these chemicals are detectable. Further progress in analytical research is necessary.
The purpose of this paper is: (i) to compare recombinant human erythropoietin (rHuEPO) pharmacokinetics in athletes and healthy individuals; and (ii) to report pharmacokinetic/pharmacodynamic (PK/PD) studies performed in athletes. Effect parameters in PK/PD studies included: (i) red blood cell variables (haematocrit, reticulocyte count); and (ii) markers of iron metabolism (serum soluble transferrin receptors [sTfR], ferritin [fr] and sTfR : fr ratio). To understand the choice of these markers, we first performed a brief review of the pharmacological effects of rHuEPO. Few studies have been conducted in healthy individuals and there are minimal references concerning pharmacokinetics in athletes. A 'flip-flop' phenomenon was noted after subcutaneous administration. The pharmacokinetics appeared linear from 50-1000 U/kg, but this linearity was not observed at the lowest dose of 10 U/kg. A negative-feedback loop of endogenous erythropoietin production occurred at the end of treatment. The half-life of the terminal part of the curves seemed to be slightly higher in athletes (36-42 vs 32 hours) than in untrained individuals and total clearance tended to be greater (17.5 vs 6.5 mL/h/kg). In conclusion, more investigations are needed to better understand the relationship between rHuEPO administration and changes in haematological and iron-metabolism parameters in athletes, particularly after chronic low-dose administration of rHuEPO.
Maximal oxygen uptake is one of the major performance limiting factor in endurance sports. Sophisticated training methods have been developed to increase this variable. On the other hand, attempts have been made to improve maximal oxygen uptake by artificial means: blood doping, administration of human recombinant erythropoietin and, probably, by the use of a new class of therapeutic agents: the oxygen carriers. All these substances and methods are prohibited by the International Olympic Committee. But, until now, the detection of the misuse of these compounds is a problem: there is no detection method for blood doping, no screening method for oxygen carriers and the current test method for rhu-Epo lacks sensitivity if a urine sample is collected more than 3-4 days after the last injection of the protein. An indirect method for detecting rhu-Epo could be an alternative solution. Our researches in this field highlight the difficulties to detect doping from biological parameters.
A direct detection method for anti-doping control of recombinant human erythropoietin (rHuEPO) abuse in racehorses is proposed. This method involves screening of plasma (or serum) by an enzyme-linked immunosorbent assay specific for human EPO and confirmation in urine samples by characterization of the urinary EPO isoelectric profile. This method was tested on horses that were administered epoetin alpha (rHuEPO) and the hyper-glycosylated form of this drug (darbepoetin alpha).