17 research outputs found
The time-course linkage between hemolysis, redox, and metabolic parameters during red blood cell storage with or without uric acid and ascorbic acid supplementation
Oxidative phenomena are considered to lie at the root of the accelerated senescence observed in red blood cells (RBCs) stored under standard blood bank conditions. It was recently shown that the addition of uric (UA) and/or ascorbic acid (AA) to the preservative medium beneficially impacts the storability features of RBCs related to the handling of pro-oxidant triggers. This study constitutes the next step, aiming to examine the links between hemolysis, redox, and metabolic parameters in control and supplemented RBC units of different storage times. For this purpose, a paired correlation analysis of physiological and metabolism parameters was performed between early, middle, and late storage in each subgroup. Strong and repeated correlations were observed throughout storage in most hemolysis parameters, as well as in reactive oxygen species (ROS) and lipid peroxidation, suggesting that these features constitute donor-signatures, unaffected by the diverse storage solutions. Moreover, during storage, a general “dialogue” was observed between parameters of the same category (e.g., cell fragilities and hemolysis or lipid peroxidation and ROS), highlighting their interdependence. In all groups, extracellular antioxidant capacity, proteasomal activity, and glutathione precursors of preceding time points anticorrelated with oxidative stress lesions of upcoming ones. In the case of supplemented units, factors responsible for glutathione synthesis varied proportionally to the levels of glutathione itself. The current findings support that UA and AA addition reroutes the metabolism to induce glutathione production, and additionally provide mechanistic insight and footing to examine novel storage optimization strategies
Beta thalassemia minor is a beneficial determinant of red blood cell storage lesion
Blood donor genetics and lifestyle affect the quality of red blood cell (RBC) storage. Heterozygotes for beta thalassemia (bThal+) constitute a non-negligible proportion of blood donors in the Mediterranean and other geographical areas. The unique hematological profile of bThal+ could affect the capacity of enduring storage stress, however, the storability of bThal+ RBC is largely unknown. In this study, RBC from 18 bThal+ donors were stored in the cold and profiled for primary (hemolysis) and secondary (phosphatidylserine exposure, potassium leakage, oxidative stress) quality measures, and metabolomics, versus sex- and age-matched controls. The bThal+ units exhibited better levels of storage hemolysis and susceptibility to lysis following osmotic, oxidative and mechanical insults. Moreover, bThal+ RBC had a lower percentage of surface removal signaling, reactive oxygen species and oxidative defects to membrane components at late stages of storage. Lower potassium accumulation and higher uratedependent antioxidant capacity were noted in the bThal+ supernatant. Full metabolomics analyses revealed alterations in purine and arginine pathways at baseline, along with activation of the pentose phosphate pathway and glycolysis upstream to pyruvate kinase in bThal+ RBC. Upon storage, substantial changes were observed in arginine, purine and vitamin B6 metabolism, as well as in the hexosamine pathway. A high degree of glutamate generation in bThal+ RBC was accompanied by low levels of purine oxidation products (IMP, hypoxanthine, allantoin). The bThal mutations impact the metabolism and the susceptibility to hemolysis of stored RBC, suggesting good post-transfusion recovery. However, hemoglobin increment and other clinical outcomes of bThal+ RBC transfusion deserve elucidation by future studies
Redox Status, Procoagulant Activity, and Metabolome of Fresh Frozen Plasma in Glucose 6-Phosphate Dehydrogenase Deficiency
ObjectiveTransfusion of fresh frozen plasma (FFP) helps in maintaining the coagulation parameters in patients with acquired multiple coagulation factor deficiencies and severe bleeding. However, along with coagulation factors and procoagulant extracellular vesicles (EVs), numerous bioactive and probably donor-related factors (metabolites, oxidized components, etc.) are also carried to the recipient. The X-linked glucose 6-phosphate dehydrogenase deficiency (G6PD−), the most common human enzyme genetic defect, mainly affects males. By undermining the redox metabolism, the G6PD− cells are susceptible to the deleterious effects of oxidants. Considering the preferential transfusion of FFP from male donors, this study aimed at the assessment of FFP units derived from G6PD− males compared with control, to show whether they are comparable at physiological, metabolic and redox homeostasis levels.MethodsThe quality of n = 12 G6PD− and control FFP units was tested after 12 months of storage, by using hemolysis, redox, and procoagulant activity-targeted biochemical assays, flow cytometry for EV enumeration and phenotyping, untargeted metabolomics, in addition to statistical and bioinformatics tools.ResultsHigher procoagulant activity, phosphatidylserine positive EVs, RBC-vesiculation, and antioxidant capacity but lower oxidative modifications in lipids and proteins were detected in G6PD− FFP compared with controls. The FFP EVs varied in number, cell origin, and lipid/protein composition. Pathway analysis highlighted the riboflavin, purine, and glycerolipid/glycerophospholipid metabolisms as the most altered pathways with high impact in G6PD−. Multivariate and univariate analysis of FFP metabolomes showed excess of diacylglycerols, glycerophosphoinositol, aconitate, and ornithine but a deficiency in riboflavin, flavin mononucleotide, adenine, and arginine, among others, levels in G6PD− FFPs compared with control.ConclusionOur results point toward a different redox, lipid metabolism, and EV profile in the G6PD− FFP units. Certain FFP-needed patients may be at greatest benefit of receiving FFP intrinsically endowed by both procoagulant and antioxidant activities. However, the clinical outcome of G6PD− FFP transfusion would likely be affected by various other factors, including the signaling potential of the differentially expressed metabolites and EVs, the degree of G6PD−, the redox status in the recipient, the amount of FFP units transfused, and probably, the storage interval of the FFP, which deserve further investigation by future studies
Leukoreduction makes a difference: A pair proteomics study of extracellular vesicles in red blood cell units
Prestorage filtration of blood to remove contaminating donor leukocytes
and platelets has substantially increased the safety level of
transfusion therapy. We have previously shown that leukoreduction has a
mitigating effect on the storage lesion profile by lowering the extent
of hemolysis and of RBC aging and removal phenotypes, including surface
signaling and microvesiculation. Even though protein composition may
determine the fate of EVs in the recipient, the probable effect of
leukoreduction on the EV proteome has been scarcely investigated. In the
present paired study, we characterized the proteome of EVs released in
prestorage leukoreduced (L) and nonleukoreduced (N) RBC units prepared
from the same donors, by immunoblotting and qualitative proteomics
analyses at two storage intervals. Apart from common proteofrms
typically associated with the established EV biogenesis mechanisms, the
comparative proteomics analyses revealed that both leukoreduction and
storage duration affect the complexity of the EV proteome. Membrane and
cytoskeleton-related proteins and regulators, metabolic enzymes and
plasma proteins exhibited storage duration dependent variation in L- and
N-EVs. Specific proteoforms prevailed in each EV group, such as
transferrin in L-units or platelet glycoproteins, leukocyte surface
molecules, MHC HLA, histones and tetraspanin CD9 in N-units. Of note,
several unique proteins have been associated with immunomodulatory,
vasoregulatory, coagulatory and anti-bacterial activities or cell
adhesion events. The substantial differences between EV composition
under the two RBC preparation methods shed light in the underlying EV
biogenesis mechanisms and stimuli and may lead to different EV
interactions and effects to target cells post transfusion
Metabolic Linkage and Correlations to Storage Capacity in Erythrocytes from Glucose 6-Phosphate Dehydrogenase-Deficient Donors
ObjectiveIn glucose 6-phosphate dehydrogenase (G6PD) deficiency, decreased NADPH regeneration in the pentose phosphate pathway and subnormal levels of reduced glutathione result in insufficient antioxidant defense, increased susceptibility of red blood cells (RBCs) to oxidative stress, and acute hemolysis following exposure to pro-oxidant drugs and infections. Despite the fact that redox disequilibrium is a prominent feature of RBC storage lesion, it has been reported that the G6PD-deficient RBCs store well, at least in respect to energy metabolism, but their overall metabolic phenotypes and molecular linkages to the storability profile are scarcely investigated.MethodsWe performed UHPLC-MS metabolomics analyses of weekly sampled RBC concentrates from G6PD sufficient and deficient donors, stored in citrate phosphate dextrose/saline adenine glucose mannitol from day 0 to storage day 42, followed by statistical and bioinformatics integration of the data.ResultsOther than previously reported alterations in glycolysis, metabolomics analyses revealed bioactive lipids, free fatty acids, bile acids, amino acids, and purines as top variables discriminating RBC concentrates for G6PD-deficient donors. Two-way ANOVA showed significant changes in the storage-dependent variation in fumarate, one-carbon, and sulfur metabolism, glutathione homeostasis, and antioxidant defense (including urate) components in G6PD-deficient vs. sufficient donors. The levels of free fatty acids and their oxidized derivatives, as well as those of membrane-associated plasticizers were significantly lower in G6PD-deficient units in comparison to controls. By using the strongest correlations between in vivo and ex vivo metabolic and physiological parameters, consecutively present throughout the storage period, several interactomes were produced that revealed an interesting interplay between redox, energy, and hemolysis variables, which may be further associated with donor-specific differences in the post-transfusion performance of G6PD-deficient RBCs.ConclusionThe metabolic phenotypes of G6PD-deficient donors recapitulate the basic storage lesion profile that leads to loss of metabolic linkage and rewiring. Donor-related issues affect the storability of RBCs even in the narrow context of this donor subgroup in a way likely relevant to transfusion medicine
Short-term effects of hemodiafiltration versus conventional hemodialysis on erythrocyte performance
Hemodiafiltration (HDF) is a renal replacement therapy which is based on the principles of diffusion and convection for the elimination of uremic toxins. A significant and increasing number of end stage renal disease (ESRD) patients are treated with HDF, even in the absence of definite and conclusive survival and anemia treatment data. However, its effects on red blood cell (RBC) physiological features have not been examined in depth. In this study, ESRD patients under regular HDF or conventional hemodialysis (cHD) treatment were examined for RBC-related parameters, including anemia, hemolysis, cell shape, redox status, removal signaling, membrane protein composition and microvesiculation, in repeated paired measurements accomplished before and right after each dialysis session. HDF-group was characterized by better redox potential and suppressed exovesiculation of blood cells compared to the cHD-group pre-dialysis. However, HDF was associated with a temporary but acute, oxidative stress-driven increase in hemolysis, RBC removal signaling and stomatocytosis, probably associated with the effective clearance of dialyzable natural antioxidant components, including uric acid, from the uremic plasma. The nature of these adverse short-term effects of HDF on post-dialysis plasma and RBCs strongly suggests the use of a parallel antioxidant therapy during the HDF session.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author
Red Blood Cell Proteasome in Beta-Thalassemia Trait: Topology of Activity and Networking in Blood Bank Conditions
Proteasomes are multi-catalytic complexes with important roles in
protein control. Their activity in stored red blood cells (RBCs) is
affected by both storage time and the donor’s characteristics. However,
apart from their abundancy in the membrane proteome, not much is known
about their topology, activity, and networking during the storage of
RBCs from beta-thalassemia trait donors (beta Thal(+)). For this
purpose, RBC units from fourteen beta Thal(+) donors were fractionated
and studied for proteasome activity distribution and interactome through
fluorometric and correlation analyses against units of sex- and
aged-matched controls. In all the samples examined, we observed a
time-dependent translocation and/or activation of the proteasome in the
membrane and a tight connection of activity with the oxidative burden of
cells. Proteasomes were more active in the beta Thal(+) membranes and
supernatants, while the early storage networking of 20S core particles
and activities showed a higher degree of connectivity with chaperones,
calpains, and peroxiredoxins, which were nonetheless present in all
interactomes. Moreover, the beta Thal(+) interactomes were specially
enriched in kinases, metabolic enzymes, and proteins differentially
expressed in beta Thal(+) membrane, including arginase-1, piezo-1, and
phospholipid scramblase. Overall, it seems that beta Thal(+)
erythrocytes maintain a considerable “proteo-vigilance” during
storage, which is closely connected to their distinct antioxidant
dynamics and membrane protein profile
Deciphering the Relationship Between Free and Vesicular Hemoglobin in Stored Red Blood Cell Units
Red blood cells (RBCs) release hemoglobin (Hb)-containing extracellular
vesicles (EVs) throughout their lifespan in the circulation, and
especially during senescence, by spleen-facilitated vesiculation of
their membrane. During ex vivo aging under blood bank conditions, the
RBCs lose Hb, both in soluble form and inside EVs that accumulate as a
part of storage lesion in the supernatant of the unit. Spontaneous
hemolysis and vesiculation are increasingly promoted by the storage
duration, but little is known about any physiological linkage between
them. In the present study, we measured the levels of total
extracellular and EV-enclosed Hb (EV-Hb) in units of whole blood (n =
36) or packed RBCs stored in either CPDA-1 (n = 99) or in CPD-SAGM
additive solution (n = 46), in early, middle, and late storage. The
spectrophotometry data were subjected to statistical analysis to detect
possible correlation(s) between storage hemolysis and EV-Hb, as well as
the threshold (if any) that determines the area of this dynamic
association. It seems that the percentage of EV-Hb is negatively
associated with hemolysis levels from middle storage onward by showing
low to moderate correlation profiles in all strategies under
investigation. Moreover, 0.17% storage hemolysis was determined as the
potential cut-off, above which this inverse correlation is evident in
non-leukoreduced CPDA units. Notably, RBC units with hemolysis levels >
0.17% are characterized by higher percentage of nanovesicles (<100 nm)
over typical microvesicles (100-400 nm) compared with the lower
hemolysis counterparts. Our results suggest an ordered loss of Hb during
RBC accelerated aging that might fuel targeted research to elucidate its
mechanistic basis
Corpuscular Fragility and Metabolic Aspects of Freshly Drawn Beta-Thalassemia Minor RBCs Impact Their Physiology and Performance Post Transfusion: A Triangular Correlation Analysis In Vitro and In Vivo
The clarification of donor variation effects upon red blood cell (RBC) storage lesion and transfusion efficacy may open new ways for donor–recipient matching optimization. We hereby propose a “triangular” strategy for studying the links comprising the transfusion chain—donor, blood product, recipient—as exemplified in two cohorts of control and beta-thalassemia minor (βThal+) donors (n = 18 each). It was unraveled that RBC osmotic fragility and caspase-like proteasomal activity can link both donor cohorts to post-storage states. In the case of heterozygotes, the geometry, size and intrinsic low RBC fragility might be lying behind their higher post-storage resistance to lysis and recovery in mice. Moreover, energy-related molecules (e.g., phosphocreatine) and purine metabolism factors (IMP, hypoxanthine) were specifically linked to lower post-storage hemolysis and phosphatidylserine exposure. The latter was also ameliorated by antioxidants, such as urate. Finally, higher proteasomal conservation across the transfusion chain was observed in heterozygotes compared to control donors. The proposed “triangularity model” can be (a) expanded to additional donor/recipient backgrounds, (b) enriched by big data, especially in the post-transfusion state and (c) fuel targeted experiments in order to discover new quality biomarkers and design more personalized transfusion medicine schemes