Liquid chromatography - high resolution mass spectrometry-based metabolomic approach for the detection of Continuous Erythropoiesis Receptor Activator effects in horse doping control.

Erythropoiesis Stimulating Agents (ESAs) were developed for therapeutic purposes to stimulate red blood cell (RBC) production. Consequently, tissue oxygenation is enhanced as athlete's endurance and ESAs misuse now benefits doping. Our hypothesis is that most of ESAs should have similar mechanisms and thus have the same effects on metabolism. Studying the metabolome variations could allow suspecting the use of any ESAs with a single method by targeting their effects. In this objective, a metabolomic study was carried out on 3 thoroughbred horses with a single administration of 4.2µg/kg of Mircera®, also called Continuous Erythropoiesis Receptor Activator (CERA). Blood and urine samples were collected from D-17 to D+74 and haematological parameters were followed throughout the study as plasmatic CERA concentration (ELISA). Urine and plasma metabolic fingerprints were recorded by Liquid Chromatography coupled to High Resolution Mass Spectrometry (LC-HRMS) in positive and negative mode. After preprocessing steps, normalized data were analyzed by multivariate statistics to build OPLS models. Hemoglobin concentration and hematocrit showed a significant increase after CERA administration unlike reticulocytes. CERA concentration showed a high intensity peak and then a slow decrease until becoming undetectable after D+31. Models built with multivariate statistics allow a discrimination between pre and post-administration plasma and urine samples until 74days after administration, i.e. 43days longer than ELISA method. By reducing and studying variables (ions), some potential candidate biomarkers were found.

Segmental bioelectrical impedance analysis (SBIA) and blood rheology: Reducing the gap between in vivo and in vitro?

Bioelectrical impedancemetry (BIA) has been used to evaluate hematocrit and red cell aggregability in vitro but whole body impedance measurements are also correlated to some hemorheologic factors, suggesting a relationship between viscosity factors and electric properties of blood. We repeatedly reported correlations with whole body BIA and hematocrit, whole blood viscosity and plasma viscosity, red cell rigidity and RBC aggregation. The SBIA Inbody 770 modelizes body as 5 cylinders and measures impedance at 1, 5, 50, 250, 500, and 1000 kHz. With the SBIA we found that hematocrit is best correlated to leg reactance at 50 kHz but also to leg impedance at 1 and 5 kHz and trunk reactance. RBC aggregation "M" is best correlated to arm reactance at 5 kHz but also to most measurements of segmental impedance (28 correlations found). RBC aggregation "M1" is best correlated to arm reactance at 5 kHz and to 19 other impedance measurements. A predictive equation for "M" from the mean between the two arm reactances at 5 kHz (maXc5) is found: M = 2.1845maXc5-23.958 (r = 0.665, p < 0.001) that provides a satisfactory Bland-Altman plot (mean difference: 0.000524 range [-1.6;+1.6]. This study suggests that previously reported correlations between BIA and viscosity factors were not spurious, and that in a narrow cylinder such as the arm the structure of circulating blood (hematocrit, red cell aggregation) may influence the passage of an electric current by increasing reactance.

Rise in RBC aggregability and concomitant decrease in blood pressure 10 days after injection of the long acting erythropoietin analogue methoxy polyethylene glycol-epoetin-ß (MIRCERA®).

Erythropoietin (EPO) is a major regulator of blood viscosity. Its long lasting action analogue methoxy polyethylene glycol-epoetin-ß (MIRCERA®) seems to be also employed in modern doping. We took the opportunity of a study aiming at developing a detection of recent MIRCERATM injection in the context of doping detection to assess the effects of this EPO analogue on red blood cells (RBC) aggregation. A single dose 200 µg of MIRCERA® was injected to 10 male volunteers and blood samplings were drawn over 24 days. After injection a decrease in mean corpuscular volume at day 2 (p < 0.01) and day 10 (p < 0.02), a rise in reticulocyte count (p < 0.001) between day 4 and day 17 and a decrease in ferritin a day 5 (p < 0.05) was observed. Hemoglobin decreased at day 4 (p < 0.005). Hematocrit was unchanged. There was a dramatic (+67%) increase in RBC aggregation index "M" (from 9.49±1.01 to 17.66±1.8, p < 0.01). A decrease in systolic blood pressure was observed during the period from day 4 to day 17 (at day 10: -11.90±2.28 mmHg, p < 0.001; at day 17: -15.80±2.83, p < 0.001). There was also a decrease in diastolic blood pressure, mean and pulse pressure. Correlations between this decrease in blood pressure and "M" did not reach significance but pulse pressure was positively correlated to "M" (r = 0.743, p < 0.05).These data show that the long acting erythropoietin analogue MIRCERA® strongly increases RBC aggregation parallel to a decrease in blood pressure, but a possible causative link between the two events is not clearly evidenced.