Increased 5-oxoproline and 5-oxoprolinase enzymes in stored RBCs: Biomarker for homologous blood doping

Abstract

Blood transfusion increases red blood cells (RBCs), it significantly improves physical performance. However, the International Olympic Committee has banned blood transfusion methods ever since 1988. Heterogenous blood transfusion can be tested and detected in the doping labs. However, homologous blood doping is still critical for blood doping laboratories. For years now, doping labs have been investigating for markers related to homologous blood doping. In 1st study, we have stored human blood at 4° C in 2 different groups. Group one was kept for 3 days, and group two was kept for 20 days. Later we have collected the storage buffer and measured the 5-oxoprolinase enzyme. In 2nd experiment, we have stored RBCs at 4° C in a cell stabilizing buffer for 14 days. We collected RBC and buffer samples daily and stored collected cells and buffer at -20° C for further analysis. Later, we measured 5-oxoproline, glutamate, and GSH inside RBCs and 5-oxoprolinase enzyme in the buffer stored the RBCs. We found that the 5-oxoprolinase enzyme released in cell stabilizing buffer was significantly higher on day 20 than on day 3. Moreover, In RBC cells, we found a time kinetic increase of 5-oxoproline and glutamate from day 1 to day 14. On the other hand, the 5-oxoprolinase enzyme level increased in the storage buffer until day 13, when the level was significantly higher. The level of increasing 5-oxoproline inside cells and 5-oxoprolinase enzymes in the buffer showed a significant correlation. We conclude that storage aging has a significant relationship with 5-oxoproline and 5-oxoprolinase enzyme levels in RBC. The average human body does not have 5-oxoproline and 5-oxoprolinase enzymes at the detectable level. In homologous blood doping, athletes take their preserved RBCs for doping. The intake of storage RBCs in the blood may increase 5-oxoproline and 5-oxoprolinase enzymes to the detectable level, thus establishing a biomarker for homologous blood doping. Further experiments on humans are needed to confirm these doping markers to include them in daily laboratory detection routines.