As part of the ZOOMICS project, we set out to investigate common and diverging metabolic traits in the blood metabolome across various species by taking advantage of recent developments in high-throughput metabolomics. Here we provide the first comparative metabolomics analysis of fresh and stored human (n = 21, 10 males, 11 females), olive baboon (n = 20), and rhesus macaque (n = 20) red blood cells at baseline and upon 42 days of storage under blood bank conditions. The results indicated similarities and differences across species, which ultimately resulted in a differential propensity to undergo morphological alterations and lyse as a function of the duration of refrigerated storage. Focusing on purine oxidation, carboxylic acid, fatty acid, and arginine metabolism further highlighted species-specific metabolic wiring. For example, through a combination of steady state measurements and 13 C 6 15 N 4-arginine tracing experiments, we report an increase in arginine catabolism into ornithine in humans, suggestive of species-specific arginase 1 activity and nitric oxide synthesis-an observation that may impact the translatability of cardiovascular disease studies carried out in non-human primates (NHPs). Finally, we correlated metabolic measurements to storage-induced morphological alterations via scanning electron microscopy and hemolysis, which were significantly lower in human red cells compared to both NHPs.

BackgroundIncreases in the red blood cell (RBC) degree of fatty acid desaturation are reported in response to exercise, aging, or diseases associated with systemic oxidant stress. However, no studies have focused on the presence and activity of fatty acid desaturases (FADS) in the mature RBC.Study design and methodsSteady state metabolomics and isotope‐labeled tracing experiments, immunofluorescence approaches, and pharmacological interventions were used to determine the degree of fatty acid unsaturation, FADS activity as a function of storage, oxidant stress, and G6PD deficiency in human and mouse RBCs.ResultsIn 250 blood units from the REDS III RBC Omics recalled donor population, we report a storage‐dependent accumulation of free mono‐, poly‐(PUFAs), and highly unsaturated fatty acids (HUFAs), which occur at a faster rate than saturated fatty acid accumulation. Through a combination of immunofluorescence, pharmacological inhibition, tracing experiments with stable isotope‐labeled fatty acids, and oxidant challenge with hydrogen peroxide, we demonstrate the presence and redox‐sensitive activity of FADS2, FADS1, and FADS5 in the mature RBC. Increases in PUFAs and HUFAs in human and mouse RBCs correlate negatively with storage hemolysis and positively with posttransfusion recovery. Inhibition of these enzymes decreases accumulation of free PUFAs and HUFAs in stored RBCs, concomitant to increases in pyruvate/lactate ratios. Alterations of this ratio in G6PD deficient patients or units supplemented with pyruvate‐rich rejuvenation solutions corresponded to decreased PUFA and HUFA accumulation.ConclusionFatty acid desaturases are present and active in mature RBCs. Their activity is sensitive to oxidant stress, storage duration, and alterations of the pyruvate/lactate ratio.

Red blood cells have the special challenge of a large amount of reactive oxygen species (from their substantial iron load and Fenton reactions) combined with the inability to synthesize new gene products. Considerable progress has been made in elucidating the multiple pathways by which red blood cells neutralize reactive oxygen species via NADPH driven redox reactions. However, far less is known about how red blood cells repair the inevitable damage that does occur when reactive oxygen species break through anti-oxidant defenses. When structural and functional proteins become oxidized, the only remedy available to red blood cells is direct repair of the damaged molecules, as red blood cells cannot synthesize new proteins. Amongst the most common amino acid targets of oxidative damage is the conversion of asparagine and aspartate side chains into a succinimidyl group through deamidation or dehydration, respectively. Red blood cells express an L-Isoaspartyl methyltransferase (PIMT, gene name PCMT1) that can convert succinimidyl groups back to an aspartate. Herein, we report that deletion of PCMT1 significantly alters red blood cell metabolism in a healthy state, but does not impair the circulatory lifespan of red blood cells. Through a combination of genetic ablation, bone marrow transplantation and oxidant stimulation with phenylhydrazine in vivo or blood storage ex vivo, we use omics approaches to show that, when animals are exposed to oxidative stress, red blood cells from PCMT1 knockout undergo significant metabolic reprogramming and increased hemolysis. This is the first report of an essential role of PCMT1 for normal RBC circulation during oxidative stress.