Over the last 50 years, almost all computational efforts to simulate the RBC metabolome ex vivo were limited to the pathways described above [61,62,63]. The recent implementation of omics technologies to study RBCs has revealed an unanticipated complexity of the erythrocyte proteome, now counting ~2500–3000 unique proteins; indeed, functional metabolic tracing experiments suggest that even this list may be incomplete [8,59,64,65,66,67,68,69]. Leveraging these recent datasets, systems biology experts are redrawing connectivity maps of the human RBC metabolome (Figure 1), of relevance for basic science and translational applications [70,71]. With >77 active transporters, circulating RBCs can take up and release many metabolites from peripheral tissues, making RBCs a unique window into system health [72]. From creatinine and carnitine (as markers of renal function [73]) to conjugated bile acids (from the gut microbiome [74]), from transamination products (e.g., alanine, glutamate, aspartate) to neurotransmitters (e.g., serotonin, dopamine, acetylcholine), RBCs can directly and indirectly participate in systems metabolism throughout the body.

During erythropoiesis, amino acid metabolism (e.g., glycine, glutamine, branched-chain amino acids) is essential to heme synthesis as both direct and indirect substrates (e.g., succinyl-CoA from glutaminolysis and branched-chain amino acid catabolism). This process concomitantly releases ammonium, making erythropoiesis and de novo glutamine synthesis two of the leading pathways contributing to ammonium homeostasis in humans [75]. Genetic defects of rate-limiting genes in the branched-chain amino acid catabolism (e.g., in propionic [76] or methylmalonic acidemias) are associated with defects in erythropoiesis [77]. Along with cysteine, both glycine and glutamine-derived glutamate contribute to the synthesis of the glutathione tripeptide. The exchange of cystine—a cysteine disulfide—to glutamate is a critical regulator of a process previously referred to as eryptosis [78] and, more recently, recognized as ferroptosis [79] owing to its expanded relevance in other cells besides RBCs.

When considering erythropoiesis, one-carbon metabolism is one of the first pathways that is referenced. Folates and methyl group donors such as methionine, choline and betaine—along with B6, B12 and B5 cofactors [80,81]—are essential for de novo purine nucleotide synthesis to support proliferation, as well as for generating glycine to contribute to heme synthesis described above. However, methyl-group donors also participate in RBC redox homeostasis and its dysregulation, such as in the context of folate dietary deficiency or excess [81]. This is also relevant in diseases associated with alterations of this pathway such as homocystinuria [82] or Down syndrome [83], owing to gene dosage of cystathionine beta synthase resulting in a “folate trap”-like phenotype [84]. These conditions present with macrocytic or megaloblastic anemia [82]. In addition, in keeping with the central role of oxidant stress in the economy of RBC metabolism, methionine uptake and consumption in RBCs fuel a pathway of isoaspartyl protein damage repair. As a hallmark of RBC aging in vivo [85], deamidation of asparagine residues in key structural membrane proteins and glycolytic enzymes [86,87,88,89,90,91,92] alters the protein backbone, with important structural/functional implications. Since RBCs cannot replace damaged components by de novo protein synthesis, repairing this damage is essential to their survival. The methylation of deamidated residues favors the formation of a succinimide intermediate, ultimately promoting the rescue of the protein backbone structure in 15–30% of the cases, a reaction catalyzed by the enzyme “protein isoaspartyl o-methyltransferase” (PIMT). Increased uptake of methyl donors such as methionine is observed in RBCs in response to oxidative challenges (e.g., incubation with hydrogen peroxide [93], refrigerated storage under blood bank conditions [92]). Thus, one can speculate that RBC uptake and consumption of methyl-group donors (e.g., methionine, choline) could compete with other tissues where these substrates are used to fuel epigenetic regulatory mechanisms, such as methylation of proteins (e.g., histones), RNA (N6-methyladenosine) and DNA (CpG islands), making RBCs an indirect player in systemic long-term responses to oxidant stress events.

In response to oxidant stress, ATP synthesis is reduced because of the redox sensitivity of glycolytic enzymes and the band 3-dependent mechanism, as described above. In this case, ATP breaks down into lower energy phosphate compounds, ADP and AMP, the latter being prone to deamination to IMP by RBC-specific AMP deaminase 3 [94]. Phosphoribolysis of IMP releases hypoxanthine, a substrate for xanthine oxidase to generate xanthine and urate, with concomitant production of hydrogen peroxide [94]. The presence of an active xanthine oxidase in mature RBCs was reported and challenged [95]. Exogenous urate is a potent antioxidant in mature RBCs [96]. Similarly, the breakdown of AMP into adenosine, with adenosine oxidation by adenosine deaminase was reported as contributing to hypoxanthine accumulation [97]. The recycling of hypoxanthine by X-linked hypoxanthine guanosine phosphoribosyltransferase (HPRT) preserves IMP/GMP homeostasis in RBCs. Lesch Nyhan syndrome patients exhibit genetic mutations of this enzyme and present with a range of clinical manifestations, including macrocytic anemia [98]. Purine homeostasis is relevant to RBC homeostasis for additional reasons beyond those described above. For example, ATP release by RBCs has regulatory effects on endothelial cells and on the RBCs themselves, as a type of autocrine signaling. The breakdown of extracellular ATP to ADP, AMP and adenosine by ectonucleotidases (e.g., CD38) generates agonists of P2Y receptors on endothelial cells, but adenosine can also be imported by equilibrative nucleotide transporters (ENT1) or stimulate adenosine receptors (e.g., ADORA2b). Both of these pathways contribute to RBC responses to hypoxia, the former, for example, by limiting circulating adenosine, a process that is counteracted by irreversible ENT1 degradation to facilitate acclimatization to high altitude hypoxia upon reascent [99]. In addition, ADORA2b transduces an intracellular signaling pathway that activates downstream protein kinase A and AMP-dependent kinase (AMPK), both contributing to metabolic reprogramming; for example, via phosphorylation-mediated activation of bisphosphoglycerate kinase [97,100].

Recent applications of unsupervised metabolomics and lipidomics approaches identified a sphingolipid sphingosine 1-phosphate (S1P) as a relevant metabolite regulating RBC responses to hypoxia [101]. For example, in response to high altitude-induced hypoxia [101] S1P can bind to deoxyhemoglobin following its stabilization by DPG, further promoting oxygen off-loading [102]. By further stabilizing deoxyhemoglobin, S1P also contributes to energy metabolism by promoting the release of glycolytic enzymes from band 3 into the cytosol, with subsequent activation of glycolysis at the expense of the pentose phosphate pathway. Although this adaptation is beneficial in hypoxia, it is deleterious when RBCs are challenged by oxidant stress, such as during refrigerated storage in the blood bank [103] or in sickle cell disease, where deoxy-sickle hemoglobin stabilization further promotes its crystallization [102].