Antioxidant response and phagocytosis in senescent erythrocytes

LUJÁN M; RUCCI A; ACOSTA L; COTORRUELO C; GARCÍA BORRÁS S; RACCA L; BIONDI C; RACCA A

Macao - China

Congreso; XXX Internationa Congress of the ISBT; 2008

International Society of Blood Transfusion

Background: Red blood cells (RBC) ageing is a complex process affected by various parameters. It is closely associated with increased free radical production, which situation ultimately leads to membrane biomolecules modification. After a life span of 120 days in the bloodstream, the RBC expose removal markers that account for their selective recognition by macrophages and clearance from circulation. Aims: To investigate the antioxidant response during RBC ageing by studying the glucose-6-phosphate deshidrogenase (G6PD) and the NADH-cytochrome b5 reductase (b5R) activities. We also investigated the interaction between peripheral blood monocytes and erythrocytes of different ages. Methods: Anticoagulated blood samples from 17 O RhD+ volunteers donors were processed. All subjects gave informed consent to participate in the study and the protocol was approved by the Ethic Committee of the School of Medical Sciences of Rosario, Argentina. Young (Y) RBC and Senescent (Se) RBC were obtained by self-formed gradients of Percoll. Activities of G6PD were measured at 340 nm by the reduction of NADP+ to NADPH. Activities of soluble b5R (b5Rs) and membrane-bound b5R (b5Rm) were determined by following the oxidation of NADH at 340 nm. The interaction between monocytes and different RBC suspension was evaluated by the erythrophagocytosis assay. Blood monocytes were obtained through their glass-adhering property and incubated with 0.5% of SeRBC, YRBC and negative and positive controls using normal (N) RBC and anti-D sensitized (S) RBC respectively. Two hundred cells taken from different glass spots were analyzed to determine the percentage of monocytes with phagocytozed and adherent RBC (AM). Results: The activity of G6PD in SeRBC (5.01 ± 1.99 IU/g Hb) was significantly lower than that observed in YRBC (7.78 ± 1.45 IU/g Hb), (P<0.005). The level of b5Rm in SeRBC (450.25 ± 32.39 IU/mg protein) was significantly lower than that found in YRBC (488.02 ± 42.39 IU/mg protein), (P<0.001). No differences were found in b5Rs of both erythrocytes subpopulations: SeRBC (20.96 ± 1.85 IU/g Hb) and YRBC (21.09 ± 1.86 IU/g Hb), (P>0.05). The percentages of AM for the different RBC suspensions were: SeRBC: 16.81 ± 1.55; YRBC: 3.03 ± 1.15; NRBC: 2.71 ± 1.06; SRBC: 30.90 ± 2.77. The interaction of SeRBC with peripheral blood monocytes reflected an increase in the percentage of AM when compared to YRBC. The percentage of AM with SeRBC was lower than that obtained with SRBC, but higher than that with NRBC (P<0.001). Conclusions: The results indicate that the antioxidant capacity during RBC ageing is decreased due to a lower production of NADPH by G6PD. The decrease in the activity of b5Rm would indicate a loss in the antioxidant process associated RBC ageing. These findings would indicate that the oxidative changes of membrane occurring during the life span of the RBC might be relevant in the process of removal of SeRBC from the circulation.