Untargeted Metabolomics Identifies a Novel Panel of Markers for Autologous Blood Transfusion

Abstract

Untargeted metabolomics was used to analyze serum and urine samples for biomarkers of autologous blood transfusion (ABT). Red blood cell concentrates from donated blood were stored for 35–36 days prior to reinfusion into the donors. Participants were sampled at different time points post-donation and up to 7 days post-transfusion. Metabolomic profiling was performed using ACQUITY ultra performance liquid chromatography (UPLC), Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The markers of ABT were determined by principal component analysis and metabolites that had p < 0.05 and met ≥ 2-fold change from baseline were selected. A total of 11 serum and eight urinary metabolites, including two urinary plasticizer metabolites, were altered during the study. By the seventh day post-transfusion, the plasticizers had returned to baseline, while changes in nine other metabolites (seven serum and two urinary) remained. Five of these metabolites (serum inosine, guanosine and sphinganine and urinary isocitrate and erythronate) were upregulated, while serum glycourdeoxycholate, S-allylcysteine, 17-alphahydroxypregnenalone 3 and Glutamine conjugate of C6H10O2 (2)* were downregulated. This is the first study to identify a panel of metabolites, from serum and urine, as markers of ABT. Once independently validated, it could be universally adopted to detect ABT.

Indirect biomarkers of blood doping: A systematic review

“…61 On the other hand, a second investigation reported 11 serum and 8 urinary metabolites altered at different time points after blood donation and up to 7 days post-reinfusion. 63 Although plasticizer markers tend to be rapidly eliminated, nine other serum and urine metabolites showed a detection window beyond 7 days, making their monitoring particularly promising for ABT screening. Therefore, based on these promising results, metabolomics will definitely be the field of forthcoming research in the near future, 137 although external influences can be sources of disturbances during a longitudinal follow-up (e.g., intense exercise or nutritional supplements).…”

“…Focusing on urine samples, a first study suggested several metabolites from plasticizers as the most relevant markers after blood reinfusion 61 . On the other hand, a second investigation reported 11 serum and 8 urinary metabolites altered at different time points after blood donation and up to 7 days post‐reinfusion 63 . Although plasticizer markers tend to be rapidly eliminated, nine other serum and urine metabolites showed a detection window beyond 7 days, making their monitoring particularly promising for ABT screening.…”

Future opportunities for the Athlete Biological Passport

“…If transcriptomic and proteomic investigations have shown their interest ( 33 ), a metabolomic approach has recently demonstrated an interesting perspective ( 34 ). In this context, a recent study identified a panel of metabolites following autologous blood transfusion ( 35 ). Also applied for the screening of growth hormone ( 36 ) and testosterone misuse ( 37 ), individualized reference ranges seem to be the most promising approach ( 34 ).…”

Contemporary blood doping—Performance, mechanism, and detection

“…115,116 Accordingly, it appears unlikely that other metabolites in urine are of interest for detection of ABT, although a small and uncontrolled study indicated that other urinary biomarkers could be of value. 117 However, metabolome changes within the plasma or RBC may prove valuable, especially as the total hemoglobin mass can remain elevated for more than 4 weeks following blood reinfusion, 118 indicating that the transfused cells are present for a long period of time.…”