Arginine Metabolism Is a Biomarker of Red Blood Cell and Human Aging

Introduction: Increases in life expectancy worldwide present a pressing burden as occurrences of age-associated diseases rise accordingly. Defining the molecular phenotypes of organismal aging is a required step towards providing updated interventions for addressing age-related disease and enhancing the quality of years lived. Red blood cells (RBCs), the most numerous host cell in humans, provide a tractable means of assessing both the molecular changes occurring across the aging spectrum and the relevance of age-related phenotypes in transfusion medicine. Understanding the impacts of aging on RBC metabolism is critical to transfusion medicine given the role of the metabolic storage lesion as a marker of storage quality and post-transfusion efficacy, and in light of global aging trends of blood donor and recipient populations. Methods: RBCs from four cohorts, including healthy individuals and patients with sickle cell disease (SCD), were characterized by metabolomics approaches to define age-associated changes in RBC metabolism. Over 15,700 specimens from 13,757 humans were examined, a major expansion over previous studies of RBCs in aging. Findings were assessed in the context of donor demographics, including sex, body mass index, and genetic ancestry. RBCs from 525 diversity outbred mice were utilized to investigate the effects of blood storage on age-associated metabolite changes. Integration of omics data with metabolite quantitative trait loci (mQTL) analysis identified genetic bases for the altered metabolic phenotypes. Finally, correlations with a vein-to-vein database linked metabolic profiles in blood donors with transfusion outcomes in recipients. Results: The arginine pathway was identified as the highest correlated hub of age-related alterations in human RBCs. Arginine catabolites ornithine and citrulline were two of the top positive aging correlates along with kynurenine, a biomarker of osmotic fragility in stored RBCs; in addition, arginine decreased as a function of donor age. Changes to arginine metabolites were consistent across healthy and SCD cohorts and were influenced by donor biology; for example, RBCs from females had lower arginine at all ages while a sex dimorphism for citrulline and ornithine was present until age 50. During refrigerated RBC storage (days 10, 23, and 42), arginine and ornithine increased while citrulline decreased. Total vesicle counts also increased linearly during storage, and arginine was the top positive correlate with vesiculation. Integrating omics data and mQTLs in humans and mice uncovered the association of vesicle counts with arginase 1 polymorphisms, functionally associating arginine metabolism during storage with increased vesiculation. Leveraging the REDS-III vein-to-vein database, which links donor biology parameters to recipient transfusion outcomes, identified significant associations between age-altered metabolites and hemoglobin (Hb) increments, in particular: a) negative correlation between arginine levels and Hb increments; b) positive correlation between citrulline:arginine ratios and Hb increments. These effects were most significant with donors <50 years old and with units stored for >35 days. Conclusion: These data amplify the relevance of modulations of RBC arginine metabolism in vivo to human aging, while also implicating this pathway both in ex vivo RBC aging in stored units and transfusion efficacy in vivo of long-term stored units. These results suggest that transfusing RBC units with higher arginine levels (i.e., associated with increased vesiculation) will yield smaller Hb increments when transfused near their outdate. Thus, RBC arginine metabolism provides an organismal aging biomarker, suggesting potential new targets for addressing the sequelae of aging.