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D. Stefanoni et al.
that fresh and stored human and RM RBC have compara- ble glycolytic and PPP fluxes. However, RM RBC have higher baseline levels of glutathione oxidation and turnover (i.e., the gamma-glutamyl cycle), along with increased transamination reactions (e.g., higher levels of glutamine, aspartate, alanine, and glutamate) and sulfur metabolism (e.g., cysteine, taurine, and SAM). Glutathionylated lipid and sugar oxidation products (e.g., GS-HNE and lactoyl-glutathione) suggest that increased sulfur metabolism in this species could result from enhanced basal oxidant stress and/or decreased activity of the deglutathionylation protein machinery. These results were confirmed by tracing experiments, highlighting sig- nificant activation of the glyoxylate pathway in RM, as compared to human, RBC; this pathway is usually associ- ated with oxidant stressors (e.g., diabetes40). Alternatively, this observation suggests that evolution has driven species- specific changes in sulfur metabolism resulting from sulfur- rich diets in the wild, as suggested by increased plant- derived41 phytochelatins in RM RBC. Combining targeted and untargeted metabolomics identified additional dietary metabolites of plant origin in RM, supporting a significant impact of diet on the molecular composition of their RBC. Based on this observation, one could test whether differ- ences in free fatty acids, fatty acyl composition of car- nitines and phosphatidylethanolamines (but not phos- phatidylserines) in RM RBC result from differing fatty acyl contents of their dietary lipids, as compared to humans. Since fatty acyl-conjugation to carnitines depends on high- energy ATP and co-enzyme A availability, and is involved in lipid damage repair, one could speculate that differential levels of acyl-carnitines in humans and RM could be explained by a differential species-specific capacity to pre- serve ATP stores or prevent/repair lipid damage during storage. In this view, species-specific differences in carni- tine metabolism suggest a differential impact of storage on membrane phospholipid homeostasis.42 Such phenomena may be explained, in part, by simply considering that the RBC storage additive used here was designed to optimize human RBC storage. Higher phosphatidylserine levels in RM RBC suggest the potential for increased erythrophago- cytosis by mononuclear phagocytes; this could be tested by post-transfusion RBC recovery studies. Nonetheless, the present study assessed the total RBC phosphatidylser- ine content, rather than its cellular compartmentalization (i.e., exposure on the RBC membrane outer leaflet43). On the other hand, phthalate plasticizers, which accumulate up to millimolar levels in human RBC by the end of stor- age, appeared to be present at even higher levels in RM RBC, despite virtually identical storage conditions; one possible explanation, consistent with the current data, is an increase in lipid oxidation and remodeling in RM RBC.
Xenometabolites, including environmental and dietary metabolites, can affect RBC integrity. Interestingly, all human blood donors in this study consumed caffeine (a purine metabolite that could modulate stored RBC metabolism via signaling through adenosine receptors44), based on detecting the parent compound and its metabo- lites. Additionally, cotinine (a nicotine metabolite and marker of smoking45) was detected in two of 21 donors, similar to the overall smoking incidence in USA donors (14% in 2017). Similarly, metabolites of chemical expo- sure, such as aniline, were only detected in human donor RBC, with broad inter-donor variability.
Sex-specific signatures were detected, especially in RM,
consistent with recent observations about the potential impact of sex on RBC storability and capacity to cope with oxidant and osmotic stressors; these parameters appear to be improved in female donor RBC.16,46 Metabolic pathways affected by sex include arginine, car- boxylic acid metabolism, and purine oxidation, which were validated using targeted quantitative measure- ments.
Perhaps the study’s most interesting finding is that purine oxidation products were all higher in RM, as com- pared to human, RBC, except for the antioxidant urate.47 This is particularly relevant in light of the recently described role of ATP breakdown and oxidation products upstream of urate (e.g., hypoxanthine as a critical marker of post-transfusion recovery in humans35). Similarly, argi- nine and asymmetric dimethylarginine (isobaric isomers could not be resolved in this study) were >100-fold high- er in RM RBC throughout storage. This suggests that RM RBC have an altered capacity for metabolizing arginine to ornithine or citrulline via arginase and nitric oxide synthase, a phenomenon previously connected to a potential impact of blood storage and transfusion on nitric oxide metabolism and transfusion-related vasodila- tory capacity.48
There are limitations to the study despite our attempt to keep blood experimental conditions constant between RM and humans. For example, blood collection from RM required mild anesthesia/sedation with ketamine- dexmedetomidine. In one study, anesthetics and seda- tives alone or in combination transiently increased circu- lating glucose levels and promoted insulin resistance (within the hour after dosing).49 Although we are unaware of any association between donor age and RBC storage quality, the RM RBC evaluated in this study were donated by adolescent or young adult animals, whereas the human donors represented the general age range of individuals volunteering for blood donation. The RM RBC were collected from animals originating on Morgan Island, South Carolina, whose ancestors were transferred from the Caribbean Primate Research Center in Puerto Rico in 1979-1980. The Morgan Island RM form a large, free-ranging colony, with more than 4,000 animals and ~75% female predominance.50 The colony comprises ani- mals that descended from India with little or no known Chinese RM introgression. Although most large national primate research centers in the USA conduct extensive genetic testing of animals in their care to promote gene flow, diverse genetic composition remains a challenge. Conversely, little genetic testing was performed on the Morgan Island colony. Nonetheless, although it is not expected that mixed Chinese-India hybrid animals occur there, it is likely that this colony experiences some genet- ic drift and genetic homozygosity due to the potential for inbreeding.
Conclusion
By describing the metabolic landscape of human and RM RBC throughout 42 days of refrigerated storage, we identified storage-, diet-, and sex-specific metabolites that may affect human biology and the potential translatabili- ty of future pre-clinical studies using RM as a model for RBC storage and transfusion. Although species-specific differences were certainly anticipated, identifying molec-
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haematologica | 2020; 105(8)