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A. D’Alessandro et al.
of oxidant stress. Of note, this observation in parts recon- ciles the impact of donor sex, age and ethnicity on the propensity to hemolyse following oxidant insult. Indeed, G6PD is coded by a gene on chromosome X, which makes female donors – who carry two copies of this chromosome - less likely to be fully deficient.
On the other hand, G6PD activity declines with the RBC age and human aging,43,44 which makes our present findings counterintuitive, since higher levels of reduced GSH and improved redox homeostasis were observed in RBC from old donors. A simple explanation may be due to a sex (male) and ethnicity bias of our younger recall donor population in this study. Alternatively, it has been previously shown that RBC lifespans are shorter in older subjects.45 Though merely speculative at this stage, a potential alternative explanation to the observed lower oxidative hemolysis in female and older donors may be associated with alterations to iron metabolism, including for example iron deficiency or decreased ferritin levels in blood from older, female donors. This would in turn limit the availability of iron, a key substrate for radical-gener- ating reactions. Further studies are currently in process to test blood ferritin levels in this cohort.46 In the meantime, it has been noted that frequent blood donors in this cohort tended to be white, older age (56 + 5 years) male who were characterized by lower oxidative hemolysis.47 Alternatively, it could be hypothesized that RBC from older or pre-menopausal female donors are either exposed to higher basal levels of stress or more rapidly renewed in the bloodstream (owing to either a shorter physiological lifespan in the former group or because of other sex-related confounders in the latter). Further stud- ies will be necessary to identify the impact of stress ery- thropoiesis on the maturation of erythrocytes in a back- ground of inflammation in aging48 or as a function of donor sex and sex-defining hormones.32
The use of metabolomics to complement existing genomics information from the RBC-Omics study allowed us to identify as of yet unexplored pathways with respect to blood storage and the storage lesion. Of note, several oxylipins – markers of post-transfusion recovery in murine models14 – were amongst the top pos- itive correlates of oxidative hemolysis. Donors with high oxidative hemolysis, including G6PD deficient donors – an extreme subgroup of these high hemolysers – were characterized by a decreased capacity to detoxify lipids through aldehyde dehydrogenase 1-dependent reactions. Of note, ALDH1 activity depends upon NADPH avail- ability, in like fashion to the ferroreductase STEAP 3 – a genetic determinant of the heterogeneity of post-transfu- sion recovery across mouse strains.14
In a recent assessment of intradonor hemolytic propen- sity within the framework of the RBC-Omics study, Lanteri and colleagues have reported that oxidative hemolysis has a strong donor-dependency across multiple donations.35 However, the authors also noted that oxida- tive hemolysis was less reproducible across multiple donations than osmotic hemolysis. This observation is in part explained by the impact of environmental factors rather than donor genetics alone on RBC propensity to hemolysis following oxidant insults, as our metabolomics results suggest here, in that we noted significant negative correlations between the levels of dietary antioxidant metabolites (e.g., epigallocatechin and ergothioneine, abundant in tea – Figure 1D) and oxidative hemolysis. If
confirmed in mechanistic studies, this observation could pave the way for considering the introduction of dietary supplements prior to donation in recurring donors. While this study focused on donor biology, other factors such as donor habits (e.g., smoking49) or environmental exposures beyond diet could play a role in the observed hetereo- geneity in oxidative hemolysis across donors and the par- tial longitudinal reproducibility of this parameter in the same donor across multiple donations.35 Finally, we antic- ipate that our findings will be relevant not just to a read- ership of transfusion medicine experts, but they will also impact those fields where appreciation of RBC propensi- ty to hemolyse as a function of oxidative injury is critical to the pathophysiology of diseases like sickle cell disease, pulmonary hypertension, Parkinson’s or Alzheimer’s, sepsis or aging.50
RBC-Omics study group members
The NHLBI Recipient Epidemiology Donor Evaluation Study- III (REDS-III), Red Blood Cell (RBC)-Omics study, is the responsibility of the following persons: Hubs: AE Mast, JL Gottschall, WB, LA, JM, AH, ZU, and VJ, BloodCenter of Wisconsin, Milwaukee, WI, USA; DJT, JEK and PAD’A, The Institute for Transfusion Medicine (ITXM), Pittsburgh, PA, USA; E. L. Murphy and AMG, University of California, San Francisco, San Francisco, CA, USA; RGC, BRS, and STJ; American Red Cross Blood Services, Farmington, CT, USA; Data coordinating center: DJB, MTS, SME, GPP, YG, NH, DR, and BCS; RTI International, Rockville, MD, USA; Central and testing laboratories: MPB, MCL, MS, and SK, Blood Systems Research Institute, San Francisco, CA, USA; TK and MG, Pittsburgh Heart, Lung, Blood, and Vascular Medicine Institute, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA; Steering committee chairman: SHK, University of British Columbia, Victoria, BC, Canada; National Heart, Lung, and Blood Institute, National Institutes of Health: SAG, KBM and AMC.
Disclosures
Though unrelated to the contents of this manuscripts, the authors declare that AD is a founder of Omix Technologies Inc and Altis Biosciencens LLC. James C Zimring serves as a con- sultant for Rubius Therapeutics. All the other authors disclose no conflicts of interest relevant to this study. Angelo D’Alessandro is a consultant for Hemanext Inc. James C Zimring serves on the scientific advisory board for Rubius Therapeutics. All the other authors disclose no conflicts of interest relevant to this study.
Contributions
TK, SK, MTG, ML, MS, MPB, GP, JCZ designed the study. AD, XF, JAR, RCH, JCZ performed metabolomics analy- ses; YG, GP performed genomics analyses; TK performed oxidative hemolysis measurements; AD prepared figures and wrote the first version of the manuscript. All the authors con- tributed to the finalization of the manuscript.
Acknowledgments
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors would like to express their grat- itude Dr. Simone Glynn of NHLBI for her support throughout this study, the RBC-Omics research staff at all participating blood centers and testing labs for their contribution to this project, and to all blood donors who agreed to participate in this study.
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haematologica | 2021; 106(5)