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

ABSTRACT: Recent large-scale multiomics 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 2 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 study. Genome-wide association studies against 879 000 polymorphisms identified critical genetic factors contributing to interdonor heterogeneity in end-of-storage carnitine levels, including common nonsynonymous polymorphisms in genes encoding carnitine transporters (SLC22A16, SLC22A5, and SLC16A9); carnitine synthesis (FLVCR1 and MTDH) and metabolism (CPT1A, CPT2, CRAT, and 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 2 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 after chemically induced membrane lipid damage. Supplementation of stored murine RBCs with l-carnitine boosted posttransfusion recovery, suggesting this could represent a viable strategy to improve RBC storage quality.

The Role of Ergothioneine in Red Blood Cell Biology: A Review and Perspective.

Oxidative stress can damage tissues and cells, and their resilience or susceptibility depends on the robustness of their antioxidant mechanisms. The latter include small molecules, proteins, and enzymes, which are linked together in metabolic pathways. Red blood cells are particularly susceptible to oxidative stress due to their large number of hemoglobin molecules, which can undergo auto-oxidation. This yields reactive oxygen species that participate in Fenton chemistry, ultimately damaging their membranes and cytosolic constituents. Fortunately, red blood cells contain robust antioxidant systems to enable them to circulate and perform their physiological functions, particularly delivering oxygen and removing carbon dioxide. Nonetheless, if red blood cells have insufficient antioxidant reserves (e.g., due to genetics, diet, disease, or toxin exposure), this can induce hemolysis in vivo or enhance susceptibility to a "storage lesion" in vitro, when blood donations are refrigerator-stored for transfusion purposes. Ergothioneine, a small molecule not synthesized by mammals, is obtained only through the diet. It is absorbed from the gut and enters cells using a highly specific transporter (i.e., SLC22A4). Certain cells and tissues, particularly red blood cells, contain high ergothioneine levels. Although no deficiency-related disease has been identified, evidence suggests ergothioneine may be a beneficial "nutraceutical." Given the requirements of red blood cells to resist oxidative stress and their high ergothioneine content, this review discusses ergothioneine's potential importance in protecting these cells and identifies knowledge gaps regarding its relevance in enhancing red blood cell circulatory, storage, and transfusion quality.

Rh disease in Mexico: evaluating regional and institutional differences in treatment availability and disease management.

BACKGROUND: Rh disease occurs following maternal alloimmunization, which can develop due to RhD blood group antigen incompatibility between a mother and her fetus. Despite developing robust clinical protocols for effective immunoprophylaxis over the last 50+ years, a significant global burden of Rh disease still exists, particularly in low/middle-income countries such as Mexico. MATERIALS AND METHODS: This study examined disparities in the allocation of maternal and child health resources, as well as clinical knowledge regarding Rh disease, to gain insight into why Rh disease remains prevalent in Mexico. To this end, an 11-question survey was sent to members of the Federación Mexicana de Colegios de Obstetricia y Ginecología (FEMECOG) to evaluate their knowledge of the availability and implementation of anti-RhD immunoglobulin prophylaxis in their practices and institutions, and about managing Rh disease by monitoring fetal anemia risk and providing intrauterine treatment when necessary. Responses were separated by region, and chi-square two-by-two contingency tests were performed to evaluate regional and institutional differences. RESULTS: Significant variations in prevention and treatment were found within the Mexican healthcare system, particularly, with regard to providing anti-RhD immunoglobulin to prevent alloimmunization, which is critically important for preventing Rh disease. Specifically, Regions 5, 6, and 7 were most lacking in this regard. DISCUSSION: This study highlights differences in the Mexican healthcare system in preventing and treating Rh disease. Closing the gap in the availability of anti-RhD immunoglobulin should take priority in future efforts aimed at providing equitable care, because this will lead to the more preferable outcome of preventing Rh disease, rather than forcing patients to seek out more complex measures for treating Rh disease after it develops. These data can be used to create strategies to understand and eliminate these healthcare disparities.

Ferroptosis regulates hemolysis in stored murine and human red blood cells.

Red blood cell (RBC) metabolism regulates hemolysis during aging in vivo and in the blood bank. Here, we leveraged a diversity outbred mouse population to map the genetic drivers of fresh/stored RBC metabolism and extravascular hemolysis upon storage and transfusion in 350 mice. We identify the ferrireductase Steap3 as a critical regulator of a ferroptosis-like process of lipid peroxidation. Steap3 polymorphisms were associated with RBC iron content, in vitro hemolysis, and in vivo extravascular hemolysis both in mice and 13,091 blood donors from the Recipient Epidemiology and Donor evaluation Study. Using metabolite Quantitative Trait Loci analyses, we identified a network of gene products (FADS1/2, EPHX2 and LPCAT3) - enriched in donors of African descent - associated with oxylipin metabolism in stored human RBCs and related to Steap3 or its transcriptional regulator, the tumor protein TP53. Genetic variants were associated with lower in vivo hemolysis in thousands of single-unit transfusion recipients. Highlights: Steap3 regulates lipid peroxidation and extravascular hemolysis in 350 diversity outbred miceSteap3 SNPs are linked to RBC iron, hemolysis, vesiculation in 13,091 blood donorsmQTL analyses of oxylipins identified ferroptosis-related gene products FADS1/2, EPHX2, LPCAT3Ferroptosis markers are linked to hemoglobin increments in transfusion recipients.

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 or storage in the blood bank. Here we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify an association between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs), hexokinase 1, 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 and hypoxanthine levels - and the genetic traits linked to them - 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, suggesting their potential as markers to improve transfusion outcomes. 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;Modeled PFKP effects relate to preventing loss of the total AXP pool in stored RBCs;ATP and hypoxanthine are biomarkers of hemolysis in vitro and in vivo.

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 or storage in blood banks. Here, we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify associations between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs); hexokinase 1 (HK1); 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 and hypoxanthine (HYPX) levels-and the genetic traits linked to them-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, suggesting their potential as markers to improve transfusion outcomes.