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.

Low-Dose Dietary Fish Oil Improves RBC Deformability without Improving Post-Transfusion Recovery in Mice.

Long-chain polyunsaturated fatty acids (LC-PUFAs) are important modulators of red blood cell (RBC) rheology. Dietary LC-PUFAs are readily incorporated into the RBC membrane, improving RBC deformability, fluidity, and hydration. Female C57BL/6J mice consumed diets containing increasing amounts of fish oil (FO) ad libitum for 8 weeks. RBC deformability, filterability, and post-transfusion recovery (PTR) were evaluated before and after cold storage. Lipidomics and lipid peroxidation markers were evaluated in fresh and stored RBCs. High-dose dietary FO (50%, 100%) was associated with a reduction in RBC quality (i.e., in vivo lifespan, deformability, lipid peroxidation) along with a reduced 24 h PTR after cold storage. Low-dose dietary FO (6.25-12.5%) improved the filterability of fresh RBCs and reduced the lipid peroxidation of cold-stored RBCs. Although low doses of FO improved RBC deformability and reduced oxidative stress, no improvement was observed for the PTR of stored RBCs. The improvement in RBC deformability observed with low-dose FO supplementation could potentially benefit endurance athletes and patients with conditions resulting from reduced perfusion, such as peripheral vascular disease.

Red Blood Cell Metabolism In Vivo and In Vitro.

Red blood cells (RBC) are the most abundant cell in the human body, with a central role in oxygen transport and its delivery to tissues. However, omics technologies recently revealed the unanticipated complexity of the RBC proteome and metabolome, paving the way for a reinterpretation of the mechanisms by which RBC metabolism regulates systems biology beyond oxygen transport. The new data and analytical tools also informed the dissection of the changes that RBCs undergo during refrigerated storage under blood bank conditions, a logistic necessity that makes >100 million units available for life-saving transfusions every year worldwide. In this narrative review, we summarize the last decade of advances in the field of RBC metabolism in vivo and in the blood bank in vitro, a narrative largely influenced by the authors' own journeys in this field. We hope that this review will stimulate further research in this interesting and medically important area or, at least, serve as a testament to our fascination with this simple, yet complex, cell.

Reticulocytes in donor blood units enhance red blood cell alloimmunization.

Although red blood cell (RBC) transfusions save lives, some patients develop clinically-significant alloantibodies against donor blood group antigens, which then have adverse effects in multiple clinical settings. Few effective measures exist to prevent RBC alloimmunization and/or eliminate alloantibodies in sensitized patients. Donor-related factors may influence alloimmunization; thus, there is an unmet clinical need to identify which RBC units are immunogenic. Repeat volunteer blood donors and donors on iron supplements have elevated reticulocyte counts compared to healthy non-donors. Early reticulocytes retain mitochondria and other components, which may act as danger signals in immune responses. Herein, we tested whether reticulocytes in donor RBC units could enhance RBC alloimmunization. Using a murine model, we demonstrate that transfusing donor RBC units with increased reticulocyte frequencies dose-dependently increased RBC alloimmunization rates and alloantibody levels. Transfusing reticulocyte-rich RBC units was associated with increased RBC clearance from the circulation and a robust proinflammatory cytokine response. As compared to previously reported post-transfusion RBC consumption patterns, erythrophagocytosis from reticulocyte-rich units was increasingly performed by splenic B cells. These data suggest that reticulocytes in a donated RBC unit impact the quality of blood transfused, are targeted to a distinct compartment, and may be an underappreciated risk factor for RBC alloimmunization.

Reticulocytes in donor RBC units enhance RBC alloimmunization.

Although red blood cell (RBC) transfusions save lives, some patients develop clinically-significant alloantibodies against donor blood group antigens, which then have adverse effects in multiple clinical settings. Few effective measures exist to prevent RBC alloimmunization and/or eliminate alloantibodies in sensitized patients. Donor-related factors may influence alloimmunization; thus, there is an unmet clinical need to identify which RBC units are immunogenic. Repeat volunteer blood donors and donors on iron supplements have elevated reticulocyte counts compared to healthy non-donors. Early reticulocytes retain mitochondria and other components, which may act as danger signals in immune responses. Herein, we tested whether reticulocytes in donor RBC units could enhance RBC alloimmunization. Using a murine model, we demonstrate that transfusing donor RBC units with increased reticulocyte frequencies dose-dependently increase RBC alloimmunization rates and alloantibody levels. Transfusing reticulocyte-rich RBC units was associated with increased RBC clearance from the circulation and a robust proinflammatory cytokine response. As compared to previously reported post-transfusion RBC consumption patterns, erythrophagocytosis from reticulocyte-rich units was increasingly performed by splenic B cells. These data suggest that reticulocytes in a donated RBC unit impact the quality of blood transfused, are targeted to a distinct compartment, and may be an underappreciated risk factor for RBC alloimmunization.

ZOOMICS : comparative metabolomics of red blood cells from dogs, cows, horses and donkeys during refrigerated storage for up to 42 days.

BACKGROUND: The use of omics technologies in human transfusion medicine has improved our understanding of the red blood cell (RBC) storage lesion(s). Despite significant progress towards understanding the storage lesion(s) of human RBCs, a comparison of basal and post-storage RBC metabolism across multiple species using omics technologies has not yet been reported, and is the focus of this study. MATERIALS AND METHODS: Blood was collected in a standard bag system (CPD-SAG-Mannitol) from dogs (n=8), horses, bovines, and donkeys (n=6). All bags were stored at 4°C for up to 42 days (i.e., the end of the shelf life in Italian veterinary clinics) and sampled weekly for metabolomics analyses. In addition, data comparisons to our ongoing Zoomics project are included to compare this study's results with those of non-human primates and humans. RESULTS: Significant interspecies differences in RBC metabolism were observed at baseline, at the time of donation, with bovine showing significantly higher levels of metabolites in the tryptophan/kynurenine pathway; dogs showing elevated levels of high-energy compounds (especially adenosine triphosphate and S-adenosyl-methionine) and equine (donkey and horse) RBCs showing almost overlapping phenotypes, with the highest levels of free branched chain amino acids, glycolytic metabolites (including 2,3-diphosphoglycerate), higher total glutathione pools, and elevated metabolites of the folate pathway compared to the other species. Strikingly, previously described metabolic markers of the storage lesion(s) in humans followed similar trends across all species, though the rate of accumulation/depletion of metabolites in energy and redox metabolism varied by species, with equine blood showing the lowest degree of storage lesion(s). DISCUSSION: These results interrogate RBC metabolism across a range of mammalian species and improve our understanding of both human and veterinary blood storage and transfusion.

A randomized trial of blood donor iron repletion on red cell quality for transfusion and donor cognition and well-being.

Although altruistic regular blood donors are vital for the blood supply, many become iron deficient from donation-induced iron loss. The effects of blood donation-induced iron deficiency on red cell transfusion quality or donor cognition are unknown. In this double-blind, randomized trial, adult iron-deficient blood donors (n = 79; ferritin < 15 µg/L and zinc protoporphyrin >60 µMol/mol heme) who met donation qualifications were enrolled. A first standard blood donation was followed by the gold-standard measure for red cell storage quality: a 51-chromium posttransfusion red cell recovery study. Donors were then randomized to intravenous iron repletion (1 g low-molecular-weight iron dextran) or placebo. A second donation ∼5 months later was followed by another recovery study. Primary outcome was the within-subject change in posttransfusion recovery. The primary outcome measure of an ancillary study reported here was the National Institutes of Health Toolbox-derived uncorrected standard Cognition Fluid Composite Score. Overall, 983 donors were screened; 110 were iron-deficient, and of these, 39 were randomized to iron repletion and 40 to placebo. Red cell storage quality was unchanged by iron repletion: mean change in posttransfusion recovery was 1.6% (95% confidence interval -0.5 to 3.8) and -0.4% (-2.0 to 1.2) with and without iron, respectively. Iron repletion did not affect any cognition or well-being measures. These data provide evidence that current criteria for blood donation preserve red cell transfusion quality for the recipient and protect adult donors from measurable effects of blood donation-induced iron deficiency on cognition. This trial was registered at www.clinicaltrials.gov as NCT02889133 and NCT02990559.

Retention of functional mitochondria in mature red blood cells from patients with sickle cell disease.

Sickle cell disease (SCD) is an inherited blood disorder characterized by sickled red blood cells (RBCs), which are more sensitive to haemolysis and can contribute to disease pathophysiology. Although treatment of SCD can include RBC transfusion, patients with SCD have high rates of alloimmunization. We hypothesized that RBCs from patients with SCD have functionally active mitochondria and can elicit a type 1 interferon response. We evaluated blood samples from more than 100 patients with SCD and found elevated frequencies of mitochondria in reticulocytes and mature RBCs, as compared to healthy blood donors. The presence of mitochondria in mature RBCs was confirmed by flow cytometry, electron microscopy, and proteomic analysis. The mitochondria in mature RBCs were metabolically competent, as determined by enzymatic activities and elevated levels of mitochondria-derived metabolites. Metabolically-active mitochondria in RBCs may increase oxidative stress, which could facilitate and/or exacerbate SCD complications. Coculture of mitochondria-positive RBCs with neutrophils induced production of type 1 interferons, which are known to increase RBC alloimmunization rates. These data demonstrate that mitochondria retained in mature RBCs are functional and can elicit immune responses, suggesting that inappropriate retention of mitochondria in RBCs may play an underappreciated role in SCD complications and be an RBC alloimmunization risk factor.

Deuterated Linoleic Acid Attenuates the RBC Storage Lesion in a Mouse Model of Poor RBC Storage.

Background: Long-chain polyunsaturated fatty acids (PUFAs) are important modulators of red blood cell (RBC) rheology. Dietary PUFAs are readily incorporated into the RBC membrane, improving RBC deformability, fluidity, and hydration. However, enriching the lipid membrane with PUFAs increases the potential for peroxidation in oxidative environments (e.g., refrigerated storage), resulting in membrane damage. Substitution of bis-allylic hydrogens with deuterium ions in PUFAs decreases hydrogen abstraction, thereby inhibiting peroxidation. If lipid peroxidation is a causal factor in the RBC storage lesion, incorporation of deuterated linoleic acid (DLA) into the RBC membrane should decrease lipid peroxidation, thereby improving RBC lifespan, deformability, filterability, and post-transfusion recovery (PTR) after cold storage. Study Design and Methods: Mice associated with good (C57BL/6J) and poor (FVB) RBC storage quality received diets containing 11,11-D2-LA Ethyl Ester (1.0 g/100 g diet; deuterated linoleic acid) or non-deuterated LA Ethyl Ester (control) for 8 weeks. Deformability, filterability, lipidomics, and lipid peroxidation markers were evaluated in fresh and stored RBCs. Results: DLA was incorporated into RBC membranes in both mouse strains. DLA diet decreased lipid peroxidation (malondialdehyde) by 25.4 and 31% percent in C57 mice and 12.9 and 79.9% in FVB mice before and after cold storage, respectively. In FVB, but not C57 mice, deformability filterability, and post-transfusion recovery were significantly improved. Discussion: In a mouse model of poor RBC storage, with elevated reactive oxygen species production, DLA attenuated lipid peroxidation and significantly improved RBC storage quality.

Physiologically based serum ferritin thresholds for iron deficiency in women of reproductive age who are blood donors.

Our objective is to develop a physiologically based method to determine serum ferritin thresholds for iron deficiency in healthy individuals. The current World Health Organization threshold of <15 µg/L for iron deficiency in women is based on expert opinion. We examined the relationship between serum ferritin and 2 independently measured indicators of iron-deficient erythropoiesis, soluble transferrin receptor (sTfR) and hemoglobin, in baseline data from 286 women, 20 to 49 years, who were first-time or reactivated donors in the Retrovirus Epidemiology Donor Study-II Donor Iron Status Evaluation (REDS-RISE) study. At lower serum ferritin concentrations, median sTfR increased as hemoglobin decreased. Using restricted cubic spline regression analysis to determine thresholds for iron-deficient erythropoiesis, the thresholds identified by sTfR (serum ferritin < 25.4 µg/L) and hemoglobin (serum ferritin < 25.3 µg/L) did not differ significantly. The thresholds found in the REDS-RISE study do not differ from those identified by sTfR (serum ferritin < 25.5 µg/L) and hemoglobin (serum ferritin < 26.6 µg/L) in a previous study of 5442 women, 20 to 49 years, in the US National Health and Nutrition Examination Survey 2003 to 2018 (P = .98 and 0.83, respectively). Although international comparisons are needed, these results with US data provide additional evidence for the potential usefulness of a physiologically based method to identify serum ferritin thresholds for iron deficiency.

ZOOMICS: Comparative Metabolomics of Red Blood Cells From Guinea Pigs, Humans, and Non-human Primates During Refrigerated Storage for Up to 42 Days.

Unlike other rodents, guinea pigs (Cavia porcellus) have evolutionarily lost their capacity to synthesize vitamin C (ascorbate) de novo and, like several non-human primates and humans, rely on dietary intake and glutathione-dependent recycling to cope with oxidant stress. This is particularly relevant in red blood cell physiology, and especially when modeling blood storage, which exacerbates erythrocyte oxidant stress. Herein we provide a comprehensive metabolomics analysis of fresh and stored guinea pig red blood cell concentrates (n = 20), with weekly sampling from storage day 0 through 42. Results were compared to previously published ZOOMICS studies on red blood cells from three additional species with genetic loss of L-gulonolactone oxidase function, including humans (n = 21), olive baboons (n = 20), and rhesus macaques (n = 20). While metabolic trends were comparable across all species, guinea pig red blood cells demonstrated accelerated alterations of the metabolic markers of the storage lesion that are consistent with oxidative stress. Compared to the other species, guinea pig red blood cells showed aberrant glycolysis, pentose phosphate pathway end product metabolites, purine breakdown products, methylation, glutaminolysis, and markers of membrane lipid remodeling. Consistently, guinea pig red blood cells demonstrated higher end storage hemolysis, and scanning electron microscopy confirmed a higher degree of morphological alterations of their red blood cells, as compared to the other species. Despite a genetic inability to produce ascorbate that is common to the species evaluated, guinea pig red blood cells demonstrate accelerated oxidant stress under standard storage conditions. These data may offer relevant insights into the basal and cold storage metabolism of red blood cells from species that cannot synthesize endogenous ascorbate.

Donor genetic and nongenetic factors affecting red blood cell transfusion effectiveness.

BACKGROUNDRBC transfusion effectiveness varies due to donor, component, and recipient factors. Prior studies identified characteristics associated with variation in hemoglobin increments following transfusion. We extended these observations, examining donor genetic and nongenetic factors affecting transfusion effectiveness.METHODSThis is a multicenter retrospective study of 46,705 patients and 102,043 evaluable RBC transfusions from 2013 to 2016 across 12 hospitals. Transfusion effectiveness was defined as hemoglobin, bilirubin, or creatinine increments following single RBC unit transfusion. Models incorporated a subset of donors with data on single nucleotide polymorphisms associated with osmotic and oxidative hemolysis in vitro. Mixed modeling accounting for repeated transfusion episodes identified predictors of transfusion effectiveness.RESULTSBlood donor (sex, Rh status, fingerstick hemoglobin, smoking), component (storage duration, γ irradiation, leukoreduction, apheresis collection, storage solution), and recipient (sex, BMI, race and ethnicity, age) characteristics were associated with hemoglobin and bilirubin, but not creatinine, increments following RBC transfusions. Increased storage duration was associated with increased bilirubin and decreased hemoglobin increments, suggestive of in vivo hemolysis following transfusion. Donor G6PD deficiency and polymorphisms in SEC14L4, HBA2, and MYO9B genes were associated with decreased hemoglobin increments. Donor G6PD deficiency and polymorphisms in SEC14L4 were associated with increased transfusion requirements in the subsequent 48 hours.CONCLUSIONDonor genetic and other factors, such as RBC storage duration, affect transfusion effectiveness as defined by decreased hemoglobin or increased bilirubin increments. Addressing these factors will provide a precision medicine approach to improve patient outcomes, particularly for chronically transfused RBC recipients, who would most benefit from more effective transfusion products.FUNDINGFunding was provided by HHSN 75N92019D00032, HHSN 75N92019D00034, 75N92019D00035, HHSN 75N92019D00036, and HHSN 75N92019D00037; R01HL126130; and the National Institute of Child Health and Human Development (NICHD).

Biological and Clinical Factors Contributing to the Metabolic Heterogeneity of Hospitalized Patients with and without COVID-19.

The Corona Virus Disease 2019 (COVID-19) pandemic represents an ongoing worldwide challenge. The present large study sought to understand independent and overlapping metabolic features of samples from acutely ill patients (n = 831) that tested positive (n = 543) or negative (n = 288) for COVID-19. High-throughput metabolomics analyses were complemented with antigen and enzymatic activity assays on plasma from acutely ill patients collected while in the emergency department, at admission, or during hospitalization. Lipidomics analyses were also performed on COVID-19-positive or -negative subjects with the lowest and highest body mass index (n = 60/group). Significant changes in amino acid and fatty acid/acylcarnitine metabolism emerged as highly relevant markers of disease severity, progression, and prognosis as a function of biological and clinical variables in these patients. Further, machine learning models were trained by entering all metabolomics and clinical data from half of the COVID-19 patient cohort and then tested on the other half, yielding ~78% prediction accuracy. Finally, the extensive amount of information accumulated in this large, prospective, observational study provides a foundation for mechanistic follow-up studies and data sharing opportunities, which will advance our understanding of the characteristics of the plasma metabolism in COVID-19 and other acute critical illnesses.

Mouse background genetics in biomedical research: The devil's in the details.

BACKGROUND: Genetically modified mice are used widely to explore mechanisms in most biomedical fields-including transfusion. Concluding that a gene modification is responsible for a phenotypic change assumes no other differences between the gene-modified and wild-type mice besides the targetted gene. STUDY DESIGN AND METHODS: To test the hypothesis that the N-terminus of Band3, which regulates metabolism, affects RBC storage biology, RBCs from mice with a modified N-terminus of Band3 were stored under simulated blood bank conditions. All strains of mice were generated with the same initial embryonic stem cells from 129 mice and each strain was backcrossed with C57BL/6 (B6) mice. Both 24-h recoveries post-transfusion and metabolomics were determined for stored RBCs. Genetic profiles of mice were assessed by a high-resolution SNP array. RESULTS: RBCs from mice with a mutated Band3 N-terminus had increased lipid oxidation and worse 24-h recoveries, "demonstrating" that Band3 regulates oxidative injury during RBC storage. However, SNP analysis demonstrated variable inheritance of 129 genetic elements between strains. Controlled interbreeding experiments demonstrated that the changes in lipid oxidation and some of the decreased 24-hr recovery were caused by inheritance of a region of chromosome 1 of 129 origin, and not due to the modification of Band 3. SNP genotyping of a panel of commonly used commercially available KO mice showed considerable 129 contamination, despite wild-type B6 mice being listed as the correct control. DISCUSSION: Thousands of articles published each year use gene-modified mice, yet genetic background issues are rarely considered. Assessment of such issues are not, but should become, routine norms of murine experimentation.

Approaching the Interpretation of Discordances in SARS-CoV-2 Testing.

The coronavirus disease 2019 pandemic has upended life throughout the globe. Appropriate emphasis has been placed on developing effective therapies and vaccines to curb the pandemic. While awaiting such countermeasures, mitigation efforts coupled with robust testing remain essential to controlling spread of the disease. In particular, serological testing plays a critical role in providing important diagnostic, prognostic, and therapeutic information. However, this information is only useful if the results can be accurately interpreted. This pandemic placed clinical testing laboratories and requesting physicians in a precarious position because we are actively learning about the disease and how to interpret serological results. Having developed robust assays to detect antibodies generated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and serving the hardest-hit areas within the New York City epicenter, we found 3 types of discordances in SARS-CoV-2 test results that challenge interpretation. Using representative clinical vignettes, these interpretation dilemmas are highlighted, along with suggested approaches to resolve such cases.

Hematologic and systemic metabolic alterations due to Mediterranean class II G6PD deficiency in mice.

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (approximately 5%). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress. Moreover, G6PD deficiency is associated with kidney disease, diabetes, pulmonary hypertension, immunological defects, and neurodegenerative diseases. To date, the only available mouse models have decreased levels of WT stable G6PD caused by promoter mutations. However, human G6PD mutations are missense mutations that result in decreased enzymatic stability. As such, this results in very low activity in red blood cells (RBCs) that cannot synthesize new protein. To generate a more accurate model, the human sequence for a severe form of G6PD deficiency, Med(-), was knocked into the murine G6PD locus. As predicted, G6PD levels were extremely low in RBCs, and deficient mice had increased hemolytic sequelae to oxidant stress. Nonerythroid organs had metabolic changes consistent with mild G6PD deficiency, consistent with what has been observed in humans. Juxtaposition of G6PD-deficient and WT mice revealed altered lipid metabolism in multiple organ systems. Together, these findings both establish a mouse model of G6PD deficiency that more accurately reflects human G6PD deficiency and advance our basic understanding of altered metabolism in this setting.