Genetic regulation of carnitine metabolism controls lipid damage repair and aging RBC hemolysis in vivo and in vitro.

Recent large-scale multi-omics studies suggest that genetic factors influence the chemical individuality of donated blood. To examine this concept, we performed metabolomics analyses of 643 blood units from volunteers who donated units of packed red blood cells (RBCs) on two separate occasions. These analyses identified carnitine metabolism as the most reproducible pathway across multiple donations from the same donor. We also measured L-carnitine and acyl-carnitines in 13,091 packed RBC units from donors in the Recipient Epidemiology and Donor Evaluation (REDS) study. Genome wide association studies against 879,000 polymorphisms identified critical genetic factors contributing to inter-donor heterogeneity in end-of-storage carnitine levels, including common non-synonymous polymorphisms in genes encoding carnitine transporters (SLC22A16, SLC22A5, SLC16A9); carnitine synthesis (FLVCR1, MTDH) and metabolism (CPT1A, CPT2, CRAT, ACSS2), and carnitine-dependent repair of lipids oxidized by ALOX5. Significant associations between genetic polymorphisms on SLC22 transporters and carnitine pools in stored RBCs were validated in 525 Diversity Outbred mice. Donors carrying two alleles of the rs12210538 SLC22A16 Single Nucleotide Polymorphism exhibited the lowest L-carnitine levels, significant elevations of in vitro hemolysis, and the highest degree of vesiculation, accompanied by increases in lipid peroxidation markers. Separation of RBCs by age, via in vivo biotinylation in mice and Percoll density gradients of human RBCs, showed age-dependent depletions of L-carnitine and acyl-carnitine pools, accompanied by progressive failure of the reacylation process following chemically induced membrane lipid damage. Supplementation of stored murine RBCs with L-carnitine boosted post-transfusion recovery, suggesting this could represent a viable strategy to improve RBC storage quality.

Biological and Genetic Determinants of Glycolysis: Phosphofructokinase Isoforms Boost Energy Status of Stored Red Blood Cells and Transfusion Outcomes.

Mature red blood cells (RBCs) lack mitochondria, and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo and during storage in vitro in the blood bank. Here we identify an association between blood donor age, sex, ethnicity and end-of-storage levels of glycolytic metabolites in 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study. Associations were also observed to ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (which we detected in mature RBCs), hexokinase 1, and ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice, and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP levels, breakdown, and deamination into hypoxanthine were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions. Highlights: Blood donor age and sex affect glycolysis in stored RBCs from 13,029 volunteers;Ancestry, genetic polymorphisms in PFKP, HK1, CD38/BST1 influence RBC glycolysis;RBC PFKP boosts glycolytic fluxes when ATP is low, such as in stored RBCs;ATP and hypoxanthine are biomarkers of hemolysis in vitro and in vivo.

Regulation of kynurenine metabolism by blood donor genetics and biology impacts red cell hemolysis in vitro and in vivo.

ABSTRACT: In the field of transfusion medicine, the clinical relevance of the metabolic markers of the red blood cell (RBC) storage lesion is incompletely understood. Here, we performed metabolomics of RBC units from 643 donors enrolled in the Recipient Epidemiology and Donor Evaluation Study, REDS RBC Omics. These units were tested on storage days 10, 23, and 42 for a total of 1929 samples and also characterized for end-of-storage hemolytic propensity after oxidative and osmotic insults. Our results indicate that the metabolic markers of the storage lesion poorly correlated with hemolytic propensity. In contrast, kynurenine was not affected by storage duration and was identified as the top predictor of osmotic fragility. RBC kynurenine levels were affected by donor age and body mass index and were reproducible within the same donor across multiple donations from 2 to 12 months apart. To delve into the genetic underpinnings of kynurenine levels in stored RBCs, we thus tested kynurenine levels in stored RBCs on day 42 from 13 091 donors from the REDS RBC Omics study, a population that was also genotyped for 879 000 single nucleotide polymorphisms. Through a metabolite quantitative trait loci analysis, we identified polymorphisms in SLC7A5, ATXN2, and a series of rate-limiting enzymes (eg, kynurenine monooxygenase, indoleamine 2,3-dioxygenase, and tryptophan dioxygenase) in the kynurenine pathway as critical factors affecting RBC kynurenine levels. By interrogating a donor-recipient linkage vein-to-vein database, we then report that SLC7A5 polymorphisms are also associated with changes in hemoglobin and bilirubin levels, suggestive of in vivo hemolysis in 4470 individuals who were critically ill and receiving single-unit transfusions.

Modeling Red Blood Cell Metabolism in the Omics Era.

Red blood cells (RBCs) are abundant (more than 80% of the total cells in the human body), yet relatively simple, as they lack nuclei and organelles, including mitochondria. Since the earliest days of biochemistry, the accessibility of blood and RBCs made them an ideal matrix for the characterization of metabolism. Because of this, investigations into RBC metabolism are of extreme relevance for research and diagnostic purposes in scientific and clinical endeavors. The relative simplicity of RBCs has made them an eligible model for the development of reconstruction maps of eukaryotic cell metabolism since the early days of systems biology. Computational models hold the potential to deepen knowledge of RBC metabolism, but also and foremost to predict in silico RBC metabolic behaviors in response to environmental stimuli. Here, we review now classic concepts on RBC metabolism, prior work in systems biology of unicellular organisms, and how this work paved the way for the development of reconstruction models of RBC metabolism. Translationally, we discuss how the fields of metabolomics and systems biology have generated evidence to advance our understanding of the RBC storage lesion, a process of decline in storage quality that impacts over a hundred million blood units transfused every year.

Sphingosine 1-phosphate has a negative effect on RBC storage quality.

Blood storage promotes the rapid depletion of red blood cell (RBC) high-energy adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG), which are critical regulators of erythrocyte physiology and function, as well as oxygen kinetics and posttransfusion survival. Sphingosine-1-phosphate (S1P) promotes fluxes through glycolysis. We hypothesized that S1P supplementation to stored RBC units would improve energy metabolism and posttransfusion recovery. We quantified S1P in 1929 samples (n = 643, storage days 10, 23, and 42) from the REDS RBC Omics study. We then supplemented human and murine RBCs from good storer (C57BL6/J) and poor storer strains (FVB) with S1P (1, 5, and 10 µM) before measurements of metabolism and posttransfusion recovery. Similar experiments were repeated for mice with genetic ablation of the S1P biosynthetic pathway (sphingosine kinase 1 [Sphk1] knockout [KO]). Sample analyses included metabolomics at steady state, tracing experiments with 1,2,3-13C3-glucose, proteomics, and analysis of end-of-storage posttransfusion recovery, under normoxic and hypoxic storage conditions. Storage promoted decreases in S1P levels, which were the highest in units donated by female or older donors. Supplementation of S1P to human and murine RBCs boosted the steady-state levels of glycolytic metabolites and glycolytic fluxes, ie the generation of ATP and DPG, at the expense of the pentose phosphate pathway. Lower posttransfusion recovery was observed upon S1P supplementation. All these phenomena were reversed in Sphk1 KO mice or with hypoxic storage. S1P is a positive regulator of energy metabolism and a negative regulator of antioxidant metabolism in stored RBCs, resulting in lower posttransfusion recoveries in murine models.