Biomedical subjects
Publications and source records attributed to Emmanuelle Varlet-Marie.
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.
The metabolic syndrome which is at high risk for diabetes and atherothrombosis is associated with hemorheologic abnormalities. Initially, insulin resistance was considered as the core of the syndrome. However, it becomes clear that the syndrome is a cluster in which the combined effects of obesity, insulin resistance, and hyperinsulinemia can be inconstantly associated, contributing to a various extent to a global impairment of blood rheology. We previously reported in 157 nondiabetic subjects that both obesity and insulin resistance increase red cell rigidity (Dintenfass's Tk) and plasma viscosity (eta p), and that whole blood viscosity at high shear rate (eta b 1000 s(-1)) reflects rather obesity than insulin resistance. In this study we aimed at defining the specific hemorheologic profile of insulin resistance and hyperinsulinemia by separating a sample of 81 subjects into 4 subgroups according to quartiles of insulin sensitivity (SI) (measured with the minimal model of an intravenous glucose tolerance test) and baseline insulin. Results show that (1) values of SI within the upper quartile are associated with low eta b due to low eta p; (2) low SI regardless insulinemia is associated with increased aggregation indexes; (3) when low SI is associated with hyperinsulinemia (insulin the upper quartile and SI in the lower) there is a further increase in eta b due to an increase in eta p; (4) neither SI nor insulinemia modify Hct. Thus hyperinsulinemia and insulin resistance induce hyperviscosity syndromes which are somewhat different, although they are associated most of the time. Low SI increases RBC aggregation while hyperinsulinemia increases eta p.
The metabolic syndrome, which is associated with an high risk for diabetes and atherothrombosis, is associated with hemorheologic abnormalities. These abnormalities seem more and more to be explained by its various symptoms than by insulin resistance which represents theoretically the core of the syndrome. In this study we aimed at defining the specific hemorheologic profile of insulin resistance and hyperinsulinemia by separating a sample of 90 subjects into 4 subgroups according to the clinical score "NCEP-ATPIII" which is the best recognized standardized definition of the syndrome. Results show no significant changes of blood rheology across classes of NCEP score despite a borderline rank correlation between RBC aggregability "M1" and the score. Whole blood viscosity was mostly correlated to HDL-cholesterol (r = -0.353, p = 0.007) and triglycerides (r = 0.574, p = 0.0001). Plasma viscosity was correlated with total cholesterol (r = 0.3359, p = 0.02) and with LDL-cholesterol (r = 0.357, p = 0.03). Red blood cell rigidity "Tk" was negatively correlated to HDL-cholesterol (r = -0.430, p = 0.007). Aggregability "M" was correlated to total cholesterol (r = 0.356, p = 0.01) and "M1" to HDL-cholesterol (r = -0.406, p = 0.006). Thus, despite previously described correlations with glucose disposal parameters, the hyperviscosity syndrome of the metabolic syndrome is not proportional to its clinical scoring and is strongly dependent upon the lipid profile.
Aging impairs blood rheology while various training protocols improve it. The purpose of this study was to delineate the respective role of aging and endurance training on blood rheology. Thirty-two subjects [16 middle-aged men: 8 cyclists (MAcy) and 8 sedentary men (MAsed) and 16 young men: 8 cyclists (Ycy) and 8 sedentary men (Ysed)] were compared in this study. Results showed higher red blood cell (RBC) rigidity and aggregability (AFFIBIO), lower RBC disaggregability (AFFIBIO) at middle age than at 25 yr, regardless of training status. However there was no age-related difference in whole blood viscosity at either native or corrected hematocrit, plasma viscosity, hematocrit, and Myrenne aggregation indexes M and M1. Training was associated with a reduced hematocrit in middle age subjects but not in 25 yr old ones. We evidenced no effect of training on red cell rigidity (Dintenfass's Tk index), in whole blood viscosity at either native or corrected hematocrit, and plasma viscosity. Thus, regular cycling at middle age maintains a low hematocrit but does not prevent aging-related increase in red cell rigidity and aggregability. Specific effects of cycling among other sports may explain this specific pattern.
There is a lack of consensus about the biological diagnosis of the overtraining syndrome (OTS). Recently, efforts have been made to standardize its clinical diagnosis (e.g., standardized questionnaires like that of the French consensus group on overtraining of the Société Française de Médecine du Sport-SFMS). We previously reported that the early signs of overtraining (= "overreaching") in elite sportsmen are associated with a hemorheologic pattern (raised hematocrit and plasma viscosity etap) that suggests some degree of reversal of the "autohemodilution" which characterizes fitness, and that the feeling of heavy legs in overtrained athletes is related to higher etap and higher red cell aggregation. We thus investigated on a sample of 48 athletes (age 24 +/- 1 yr), referred for possible diagnosis of overtraining to what extent plasma viscosity is a predictor of OTS. From those 48 athletes 10 had a value of etap in the highest quartile (etap > 1.44 pPa.s) and 8 of them had a diagnosis of overreaching, while in the 38 whose etap was < 1.44 mPa.s there were 20 cases of overreachings. Overt cases of OTS were found in 1 subject of the highest quintile and two in the lowest. Thus the predictive value of etap for early stages (overreaching) or chronicized stages (overtraining syndrome) is as follows: (a) prediction of overreaching: sensitivity 28.57%; specificity 90%; positive predictive value 80%; negative predictive value 47.37%; (b) prediction of chronicized overtraining: sensitivity 2.70%; specificity 18.18%; positive predictive value; 10.00%; negative predictive value 5.26%. These results show that etap is a rather specific, although poorly sensitive predictor of overreaching but has no interest in the diagnosis of the overtraining syndrome itself.
Hemorheologic effects of exercise training ("hemorheologic fitness") are very different according to the mode and the intensity of this training. We previously reported that low intensity endurance training in sedentary patients suffering from the metabolic syndrome sumultaneously improved blood rheology, body composition and lipid oxidation at exercise. We aimed at analyzing the link among these improvements in 24 patients submitted to a 2 months targeted training designed for increasing exercise lipid oxidation. Variations of whole blood viscosity at high shear rate (etab 1000 s(-1)) were explained here by two statistically independent determinants: hematocrit and red cell rigidity. etab decreased in 16 subjects, but increased in 8, due to a rise in hematocrit. Changes in RBC rigidity appeared to reflect weight loss and decrease in LDL cholesterol. Plasma viscosity was related to cholesterol and its training-induced changes are related to those of VO2 max ) but not to lipid oxidation. Red cell aggregability (Myrenne) reflected both the circulating lipids (Chol, HDL and LDL) and the ability to oxidize lipids at exercise. Factors associated to a post-training decrease in aggregability (M1) were weight loss and more precisely decrease in fat mass, improvement in lipid oxidation, rise in HDL-Chol, and decrease in fibrinogen. On the whole the major determinant of hemorheologic improvement was an increase in cardiorespiratory fitness (VO2 max ), correlated with a decrease in plasma viscosity, rather than an improvement in lipid metabolism, although RBC aggregability and deformability exhibited clear relationships with lipid metabolism. For which reason Hct increased in 30% of the patients during this kind of training remains unclear.
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.
Blood rheology is influenced by metabolism and nutrition. We investigated this issue in 41 elite athletes exercising 13+/-0.9 hr/wk (mean age: 23.9+/-0.67 yr; mean VO2max: 52.6+/-2.3 ml/min/kg body weight) with a standardised nutritional questionnaire suitable for sports medicine. Calorie intake (% of recommended intake) was negatively correlated with the RBC disaggregability threshold (r=-0.505, p=0.01). There were negative correlations between fibrinogen and protein intake (% of the total caloric intake r=-0.787, p=0.0008; amount in g/kg/day r=-0.597, p=0.03). Accordingly, the RBC disaggregability threshold was also correlated negatively with protein intake (r=-0.508, p=0.05). Lipid intake (g/kg/day) was negatively correlated with the RBC disaggregability threshold (r=-0.564, p=0.03) and positively to the hematocrit/viscosity ratio (r=0.531, p=0.03). Carbohydrate intake (g/kg/day) was positively correlated with whole blood viscosity (r=0.517, p=0.04) and negatively to the hematocrit/viscosity ratio (r=-0.4863, p=0.05). In addition fibrinogen was negatively correlated with hematocrit (r=-0.4129, p=0.036) and positively with a host of aggregation parameters (p<0.001). Therefore fibrinogen levels and red cell rheology exhibit correlations with the nutritional status in athletes. Low protein intake appears to be associated with (mildly) raised fibrinogen and aggregability, and low calorie intake is associated with lower RBC disaggregability.
The metabolic syndrome is a major health problem in western countries, due to the deleterious metabolic consequences of sedentarity and rich diet in the large part of the population who exhibits the so-called "thrifty phenotype". This syndrome, which is at high risk for diabetes and atherothrombosis is associated with hemorheologic abnormalities. Initially, insulin resistance was considered as the core of the syndrome. However, it becomes clear that the syndrome is a cluster in which the combined effects of obesity, insulin resistance, and hyperinsulinemia can be inconstantly associated. Thus, we investigated in 157 nondiabetic subjects (53 males and 104 females, age 35.6+/-1.1 yr, mean BMI 29.2+/-0.6 kg/m2) the respective importance of each of these factors. Subjects were divided in 6 groups according to BMI (cut-off point 25 kg/m2) and insulin sensitivity (SI) measured with the minimal model (lowest quartile SI<1.1 min(-1)/(microU/ml) x 10(-4), highest quartile SI>9.5, middle zone between 1.1 and 9.5). Results show that whole blood viscosity at high shear rate is higher in obese subjects (p<0.01). Plasma viscosity is also higher in obese subjects 1.41+/-0.02 vs 1.34+/-0.012 (p<0.01), and, in addition, in lean subjects, is lower when SI is in the upper quartile. RBC rigidity index "Tk" is higher in obese subjects. A worsening effect of insulin resistance (SI<1.1) on Tk is found only in obese subjects. The aggregability index "M1" is increased when SI<1.1 in both obese and nonobese subjects. No clear effect of either SI or obesity on hematocrit is observed. On the whole, obesity and insulin resistance both impair blood rheology by acting on red cell rigidity and plasma viscosity. Whole blood viscosity at high shear rate reflects rather obesity than insulin resistance. Myrenne "M1" aggregation is rather a marker of hyperinsulinemia. Thus, the hemorheologic picture of the metabolic syndrome is far to be only a reflect of insulin resistance alone.
Blood lactate increases during exercise. Although this increase was classically interpreted as a "Pasteur-like effect" resulting from anaerobiosis, it is now clear that it mostly results from a shift in the balance of oxidation of substrates in the muscle, with carbohydrate becoming the predominant fuel. However, we have repeatedly observed that the rise in blood lactate during exercise is correlated to blood viscosity and red cell aggregation. More recently we investigated this issue with the modelling of postexercise lactate kinetics, that allows a fair evaluation of lactate production by muscles (gamma1) and lactate disappearance (gamma2). Postexercise red cell aggregation (Myrenne M1) appears to be correlated to gamma2. Thus microcirculatory adaptations influenced by red cell aggregation may influence lactate disposal, adding its effect to that of the balance between carbohydrates and fat. On the other hand, the rise in blood lactate seems to induce some alterations in erythrocyte rheology at exercise. Correlations between its concentrations during exercise and erythrocyte rigidity support the concept that lactate, at least when it rises above the 4 mmol.l(-1) threshold impairs red cell deformability. Moreover, it seems that endurance training influences erythrocyte response to lactate. While lactate did not in vitro affect erythrocyte aggregation, it impaired (as expected) erythrocyte deformability in sedentary subjects but it (unexpectedly) improved it in trained subjects. This difference may be due to training-induced adaptations in erythrocyte metabolism, including transmembrane transfer via monocarboxylate transporters which show marked alterations in this context. This specific training-induced pattern of response to lactate may provide an alternative explanation to the exercise-induced arterial hypoxemia that occurs in such athletes.
We previously reported in rugbywomen correlations between RBC deformability and the ability to oxidize at exercise more lipids. This surprising finding might of course be spurious, or reflect the importance of the balance of substrates at exercise on baseline parameters that regulate blood rheology. Actually, the capacity of skeletal muscle to utilize either lipid or carbohydrate as fuels strongly influences whole body metabolism both at rest and during exercise. While the healthy skeletal muscle has substantial metabolic flexibility and is able to switch from predominantly lipid o oxidation during fasting or endurance exercise to increased glucose oxidation in conditions of insulin stimulation, obese individuals and those with type 2 diabetes manifest higher lipid oxidation during insulin-stimulated conditions despite lower rates of lipid oxidation during fasting or prolonged exercise. A low ability to oxidize and to periodically deplete triglyceride in muscle is associated with raised blood lipids. In addition, high carbohydrate oxidation rates in the mitochondrion are likely to promote more free radical generation. An increase in either blood lipids or free radicals is likely to induce profound hemorheological effects. We present here hemorheological studies in various populations with the use of exercise calorimetry in order to assess this switch of substrates. These studies further evidence negative correlations between the ability to oxidize lipids at exercise and parameters of blood viscosity. Correlations found between RBC deformability and the ability to oxidize at exercise more lipids may be due to effects of endurance training on lipid oxidation which may in turn modify both lipid metabolism and free radical generation, thus influencing RBC rheology.
Among hemorheologic parameters, plasma viscosity is one of the most studied in epidemiology, so that it has emerged as an independent risk factor. In diabetes, plasma viscosity is frequently elevated. For this reason we tried to define characteristics of non-insulin dependent diabetics with high plasma viscosity (>1.45 mPa.s) and whether they were more insulin resistant and/or exhibited other hemorheologic disturbances. 12 subjects (age 56.1+/-11.7; BMI 28.6+/-4.8) were thus found to have a value of plasma viscosity >1.45 mPa.s. They were compared to 20 age and BMI-matched NIDDMs. Patients have similar insulin sensitivity, HbA1c, and fibrinogen. RBC aggregation, rigidity and hematocrit were not significantly different. Whole blood viscosity at high shear rate was slightly higher (p=0.05). When corrected for hematocrit whole blood viscosity is no longer different. However, hematocrit was not lower in subjects with hpl >1.45. By contrast blood pressure was markedly higher (systolic: 177.5+/-2.5 mmHg vs 140+/-8 mmHg, p<10(-8); diastolic 110+/-14 vs 83+/-9 mmHg, p<10(-9); mean 132+/-18 mmHg vs 102+/-7 mmHg p<10(-9)). Therefore, in NIDDM, higher plasma viscosity, regardless insulin resistance and adiposity, is strongly related to blood pressure.
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.
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.
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).