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A. D’Alessandro et al.
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Figure 7. Glucose 6-phosphate dehydrogenase (G6PD) deficient donor from the REDS-III Omics study (A) had higher oxidative hemolysis (B), lower glutathione and lower pentose phosphate pathway activation than G6PD sufficient donors (C). By limiting the availability of reduced glutathione (GSH) and NADPH, G6PD deficiency negatively impact recycling of peroxidized lipids through the glyoxalase pathway and aldehyde dehydrogenase 1 (ALDH1)-dependent steps (D). 4HNE: 4-hydroxynone- nal; 6PG: 6-phosphogluconate; ALDH1: aldehyde dehydrogenase 1; ATP: adenosine triphosphate; DPG: 2,3-diphosphoglycerate; E4P: erythrose phosphate; FBP: fruc- tose bisphosphate; G3P: glyceraldehyde 3-phosphate; GLN: glutamine; GLUC: glucose; GSDHN: glutathionyl dihydroxynonenal; GSH: reduced glutathione; HNA: hydroxynonenoic acid; HPX: hypoxanthine; LAC: lactate; LACTALDH: lactaldehyde; METH: methionine; PEP: phosphoenolpyruvate; PGLY: phosphoglycerate; RibP: ribose phosphate (isomers); SAM/SAH: S-adenosylmethionine to S-Adenosylhomocysteine ratios;
mocysteine (SAH) levels, suggestive of decreased basal oxidant stress in female RBC.
Similarly, donor age explained ~5% of the total meta- bolic variance across donors (second principal component [PC2] in the principal component analysis in Figure 5A). Unexpectedly, RBC from young donors had higher oxida- tive hemolysis (Figure 5B-C) and lower levels of GSH (Figure 5D), which was accompanied by altered glycoly- sis, lower activation of the PPP, altered methionine and citrulline metabolism independently from storage dura- tion (Figure 5).
Correlations of oxidative hemolysis and energy and antioxidant metabolism relative to storage additives and donor race-ethnicities
Expanding on prior work,36 we confirmed a significant impact (~11%) of storage additives on the metabolic het- erogeneity of the 599 tested units, which was here corre- lated to decreased oxidative hemolysis in storage additive AS-3 RBC (Online Supplementary Figure S4A-C). Lower oxidative hemolysis was accompanied by higher levels of GSH in AS-3 RBC, faster glycolysis, higher ATP and DPG, higher PPP, decreased purine oxidation and protein dam- age-repair pathway activity (SAH) in AS-3 RBC independ- ently of storage duration (Online Supplementary Figure S4D- E). Donor ethnicity had a significant impact on oxidative hemolysis as well (~9.2% of the total variance), with RBC from Hispanic/Latino donors having significantly higher oxidative hemolysis than RBC from Asian and Southern Asian donors (Online Supplementary Figure S5). Of note, RBC from African American donors had high levels of phosphoglycerate and ribose phosphate, methionine and arginine in comparison to RBC from Asian donors.
Altered glutathione metabolism and recycling underlying increases in oxidative hemolysis may be explained by glucose 6-phosphate dehydrogenase status
G6PD is the rate-limiting enzyme of the PPP, a key antioxidant pathway in the mature erythrocyte for the recycling of oxidized GSH (Figure 6A). Within the frame- work of the RBC-Omics genome wide association study (GWAS), a specific single nucleotide polymorphisms (SNP) to G6PD was found to correlate significantly to oxidative hemolysis (GWAS analysis – Figure 6B). Such polymorphism, better described in Page et al. (under review), include point mutations resulting from SNP on chromosome X, rs1050828 (ASN126ASP) which is com- mon in individuals of African descent (hence named G6PD A- variant). This finding was expected, in that the oxidative hemolysis test used to screen the donors in the present study is similar to the clinical test for G6PD defi- ciency in the clinical setting. Among the subjects tested in the present study, we identified SNP in G6PD potentially associated with oxidative hemolysis in the seven recalled samples that underwent metabolomics in the present study (Figure 6C). Bioinformatics integration of the top 100 hits from the GWAS and metabolomics analysis against oxidative hemolysis was performed via OmicsNet; elaborations include a gene-centric (top) and metabolite- centric (bottom) network view, both analyses confirming a central role for G6PD in mediating energy and redox homeostasis (Online Supplementary Figure 6D). This unsu- pervised analysis identified the centrality of the G6PD net- work and branching pathways (including NADPH- and GSH-dependent pathways) in the REDS-III omics recalled donor cohort. We thus used the metabolomics data to per-
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