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 steady-state metabolomics, tracing experiments with 1,2,3-13C3-glucose, proteomics, and end-of-storage posttransfusion recovery analysis under normoxic and hypoxic storage conditions. Storage promoted decreases in S1P levels, 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.

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Introduction

Red blood cell (RBC) transfusion is a life-saving intervention for millions of recipients worldwide every year. However, the quality of packed RBC products declines during storage in the blood bank, which promotes a series of morphological1,2 and biochemical changes3 that ultimately affect erythrocyte physiology and posttransfusion performances. The quality of stored RBCs, as per the US Food and Drug Administration (FDA) and the European Council guidelines, is defined by hemolytic propensity and the capacity of transfused RBCs to circulate at 24 hours after transfusion (henceforth, posttransfusion recovery [PTR]); these 2 parameters increase and decrease, respectively, as a function of storage duration.

It has been argued that oxidative stress is the main driver of the storage lesion.4 RBCs are well equipped to counteract oxidant stress through different systems, among which the pentose phosphate pathway (PPP) represents a critical lynchpin. The PPP is the main pathway generating the key reducing equivalent nicotinamide adenine dinucleotide phosphate, NADPH, which participates in the scavenging of reactive oxygen species and recycling of oxidized soluble small molecules (for example, the glutathione system) and enzyme-catalyzed antioxidant batteries.5 Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the PPP, and it is highly polymorphic in humans.6 Of note, antioxidant capacity,7 storage quality,8 hemolytic propensity,9 PTRs,10 and posttransfusion hemoglobin increments11 are poorer in packed RBCs donated by volunteers suffering from G6PD deficiency, a condition that affects ~500 million individuals to a variable extent, owing to a plethora of genetic mutations, all resulting in a hypomorphic or unstable G6PD enzyme.6 Because G6PD is a chromosome X–linked gene, whose activity declines with age12 and affects lifespan in mammals,13 it is interesting to note that donor sex and age8,14,15 affect RBC antioxidant capacity and posttransfusion RBC performances beyond the chronological age of the unit, that is, the days elapsed since the time of donation.16,17

Others have noted that the rapid depletion of high-energy phosphate compounds (adenosine triphosphate [ATP] and 2,3- diphosphoglycerate [DPG]) is a hallmark of the RBC metabolic storage lesion.18 ATP consumption is accompanied by dysregulated calcium ion pumps,19 resulting in intracellular calcium accumulation and cryptos is.20 Reductions in ATP deprive erythrocytes of a rate-limiting substrate for phosphorylation of structural membrane proteins, which is critical to morphological homeostasis of the stored RBC.21 By fueling ATP-dependent flippase, ATP depletion in the stored RBC results in phosphatidylserine exposure to the outer membrane leaflet and untimely removal upon transfusion.3,22,23 DPG exhaustion by storage weeks 2 to 3 is mechanistically linked to altered oxygen kinetics.22 Indeed, depletion of DPG levels promotes increases in oxygen saturation (SO2) by shifting the oxygen dissociation curve to the left,24 which in turn promotes the concomitant storage-dependent formation of reactive oxygen species2 that are triggered by Haber-Weiss chemistry in the presence of elevated O2 levels. Indeed, heterogeneity in baseline SO2 levels after processing is associated with heterogeneity in storage quality,25 to the extent that SO2 manipulation by hypoxic storage improves energy metabolism, prevents the oxidative storage lesion,26,27 and boosts PTRs in randomized clinical trials in humans,28 as well as transfusion efficacy in rodent models of trauma and shock.29 Among the mechanisms identified as key drivers of the benefits of hypoxic storage is the alkalinization of intracellular pH,30 and the mitigation of cysteine oxidation27 and asparagine deamidation26 of key glycolytic enzymes. These factors, combined with deoxyhemoglobin competitive binding to the N-terminus of band 3 that displaces otherwise bound/inhibited glycolytic enzymes,31-33 all contribute to boosting glycolysis and ATP generation capacity in hypoxically stored RBCs, which ultimately preserves oxygen kinetics of these products.34 Of note, even though DPG and ATP synthesis is slowly restored upon transfusion in vivo,35 the kinetics may be insufficient to meet the oxygen requirements in the hypoxic, massively transfused recipient (for example, patients with trauma).

In previous mechanistic studies on high-altitude hypoxia and sickle cell disease,36,37 we showed that sphingosine-1-phosphate (S1P) supplementation promotes fluxes through glycolysis by mediating hemoglobin binding to the N-terminus domain of band 3,37 thereby displacing glycolytic enzymes, otherwise inhibited by competitive binding to the very same domain of band 3.32,33 By promoting glucose oxidation through glycolysis and the Rapoport-Luebering shunt, S1P supplementation promotes the synthesis of ATP and DPG. Exposure to hypoxia (in vivo or in vitro) promotes S1P synthesis by RBC sphingosine kinase 1 (Sphk1), which is critical to metabolic adaptations to high-altitude or pathological hypoxia (for example, chronic kidney disease).37,38 However, previous studies have shown that storage is accompanied by declines in S1P levels (down to 19% of fresh values in day-30 units39), suggesting that exogenous supplementation of S1P could be leveraged as a metabolic intervention in stored RBC units. Other studies, however, have either shown no storage-dependent change40 or additive solution-dependent increases/decreases in S1P levels.41 In light of this, first of all, we leveraged the largest sample set amenable for S1P assessment from the REDS RBC Omics study,42 a longitudinal study on ~2000 samples from 643 blood donors. After confirming that storage causes a decline in S1P levels in RBC units, especially those donated by donors characterized by extreme hemolytic propensity from the REDS RBC Omics cohort,42 we hypothesized that supplementation of S1P to human and murine-packed RBCs would improve storage quality, whereas genetic ablation of Sphk1 would impair S1P biosynthesis and, consequently, exacerbate the storage lesion by negatively affecting glycolysis (and thus ATP and DPG synthesis).

Methods

REDS RBC Omics study participants and samples RBC Omics was conducted under regulations applicable to all human subject research supported by federal agencies as well as the requirements for blood product manipulation specified and approved by the FDA. The data coordinating center (RTI International) of REDS was responsible for the overall compliance of human participant regulatory protocols including institutional review board approval from each participating blood center, from the REDS Central Laboratory (Vitalant Research Institute), and from the data coordinating center, as previously detailed.8,41 Donors were enrolled at the 4 participating REDS US blood centers. Overall, 13 403 individuals aged ≥18 years provided informed consent to participate in the study. Hemolysis parameters (spontaneous, oxidative, or osmotic) were evaluated on stored RBCs from these donors after ~39 to 42 days of storage. Extreme hemolyzed (5th and 95th percentile) donors tested for end-of-storage oxidative hemolysis were asked to donate a second unit of blood. These units were sterilely sampled for metabolomics analysis (n = 643, storage days 10, 23, and 42). Blood collection, sample processing, and other aspects of the screening and recall phases of the RBC Omics Study have been extensively described.9,14.

Human RBC incubation with D7-S1P

To validate S1P uptake by human erythrocytes,43,44 RBCs (n = 8; 4 males and 4 females) were incubated at 37◦C for 0, 3, 6, and 24 hours in AS-3 additive solution supplemented with the stable isotope–labeled D7-S1P (product #860659, Avanti Lipids) at 0, 1,3, 6, or 12 μM.

RBC storage with S1P

Whole blood units were donated by 10 healthy donor volunteers in CP2D (Haemonetics, Boston, MA). Two pools of 5 units were made, upon leukofifiltration, plasma removal, and suspension in AS-3 additive solution, with or without supplementation of S1P (product #MFCD00270077; CAS #26993-30-6; Sigma Aldrich, St. Louis, MO) at 1, 5, or 10 μM.

Mouse blood collection, storage under hypoxic and normoxic conditions, and PTR All murine experimental protocols were approved by the University of Virginia IACUC on 22 April 2019 (protocol n: 4269). Mouse strains C57BL6/J, FVB and Sphk1 knockout (KO) have been previously described.45,46 Murine RBC storage (for 3 and 6 days for FVB mice, and 12 days for C57BL6/J mice), transfusion, and PTR determinations were carried out as previously described, with minor modifications.46 Whole blood was drawn by cardiac puncture under sterile conditions into CPDA-1, centrifuged, and the hematocrit was adjusted to 75% by removing the supernatant. In a subset of experiments, units were supplemented with 5 μM S1P. For hypoxic storage, murine RBCs were bubbled with nitrogen gas in a glove box until SO2 <50% before storage, whereas normoxic counterparts reached SO2 >95% by the end of the storage period. Sealed “units” were preserved at 37◦C for 1 hour and stored at 4◦C for 7 and 12 days. RBCs from C57BL6/J mice were used as the test population and subjected to different storage conditions. UBC-GFP mice (stock #004353) were used as recipients to allow visualization of the test cells in the nonfluorescent gate. To control for differences in transfusion and phlebotomy, RBCs from ROSA26-LCB-mCHERRY mice (mCHERRY) were used as a tracer RBC population (never stored) and were added to stored RBCs immediately before transfusion, as recently described.47 PTR was calculated by dividing the posttransfusion ratio (test: tracer) by the pretransfusion ratio (test: tracer).48 A single PTR value >100% was calculated across all replicates in all experiments, which was explained by the tracer population being damaged and/or outperformed by the test population. Because PTR >100% is not biologically meaningful, this value was set to an absolute value of 100%, as standard practice in the field. At the time of transfusion, blood samples were frozen in liquid nitrogen and stored at −80◦C until subsequent analysis.

Glucose tracing experiments

RBCs (100 μL) from all the mouse strains investigated in this study were incubated at 37◦C for 1 hour in the presence of 1,2,3-13C3-glucose (5 mM, Cambridge Isotopes, product #CLM- 4673) and stored for 7 and 12 days before determination of lactate isotopologues +2/+3 (as markers of PPP-to-glycolysis fluxes), as described.27

Ultra-high-performance liquid chromatography (UHPLC)-mass spectrometry metabolomics

Frozen RBC aliquots (50 μL) were extracted 1:10 in ice-cold extraction solution (methanol:acetonitrile: water, 5:3:2 v/v/v).26 Samples were vortexed and insoluble material pelleted, as previously described.49 Analyses were performed using a Vanquish UHPLC coupled online to a Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany). Samples were analyzed using a 150 and 5-minute gradient-based method,49 as previously described.51,52 S1P measurements were performed via the same platform described earlier, as validated in prior technical notes52 and RBC-centric studies on S1P,37,38 with the aux ilium of stable isotope–labeled internal standards (D7-S1P, product #860659, Avanti Lipids). Data analysis was performed through the aux ilium of the software MAVEN.53 Graphs and statistical analyses (either two-way analysis of variance [ANOVA] or repeated-measures ANOVA) were prepared with GraphPad Prism 8.0 (GraphPad Software, Inc, La Jolla, CA), GENE E (Broad Institute, Cambridge, MA), and MetaboAnalyst 5.0.54 All raw data are available from the corresponding author upon reasonable request.

Results

RBC S1P declines with storage and is higher in packed RBCs from female and older donors As part of the REDS-III RBC Omics study, we analyzed packed RBC samples from 643 donors, at 3 storage time points (ie, days 10, 23, and 42; Figure 1A). These donors were selected among an original cohort of 13 403 healthy volunteers enrolled at 4 different blood centers across the United States and tested for RBC hemolytic propensity (spontaneous, following oxidant, or osmotic insults). Subjects whose RBCs tested in the 5th and 95th percentile for hemolytic propensity were contacted and invited to donate a second unit of blood, for a total of 1929 samples tested in this study. Measurements of S1P from packed RBC units in this study showed a progressive depletion of this metabolite as a function of storage duration (Figure 1B). Such measurements were performed via UHPLC-mass spectrometry, based on accurate intact mass and retention times validated against unlabeled or deuterium-labeled commercial standards (supplemental Figure 1A-B), which also confirmed RBC capacity to take up exogenous S1P in a dose-response manner (supplemental Figure 1C).

In the REDS-III RBC Omics cohort, S1P was nonnormally distributed across all blood donors at the end of storage (supplemental Figure 2A). Identifification of donors in the bottom or top 5% based on S1P levels (Supplemental Figure 2B) resulted in the identifification of 2 separate groups across storage within the REDS-III RBC Omics cohort. Heat map representation of the top 50 metabolites based on donor S1P levels (supplemental Figure 2C) indicated a positive association between elevated S1P and glycolytic metabolites (fructose bisphosphate, bisphosphoglycerate, phosphoglycerate, phosphoenolpyruvate; supplemental Figure 3), markers of oxidant stress (glutathione disulfifide, methionine sulfoxide), and several acylcarnitines, and a negative association with PPP metabolites (6-phosphogluconate, pentose phosphate). Notably, factors like an additive solution, donor sex, and age were all associated with an effect on RBC S1P levels, independent of storage duration (Figure 1C-E). Specifically, RBCs stored in additive solution 1, from female and older donors were found to have higher levels of S1P (Figure 1C-E).

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Figure 1. RBC S1P decreases during storage and is higher in packed RBCs from female and older donors. (A) An overview of the REDS-IV RBC Omics program is provided in panel A. (B-C) As part of the recalled donor arm of the study, 1929 units from 643 extreme hemolyzing donors were tested for RBC S1P levels (AUs, which were found to decrease during storage (B), and were lower early on during storage in AS-3 packed RBC units (AU) (C). (D-E) Higher levels of S1P were observed in blood donated by female donors (box and whisker plot; median ± ranges[D]), older donors (volcano plot of metabolic correlates to age across all blood donors tested in this study; x-axis indicates log2-fold changes per unit of age for each metabolite and the y-axis indicates the negative log10 of q-values for such correlations [E]), independent storage duration and additive. Asterisks indicate significance (ANOVA with multiple column comparisons; **P < .01, ***P < .001, ****P < .0001). AU, an arbitrary unit.

Figure 2. Storage of human RBCs with S1P promotes glycolysis and generation of ATP, at the expense of steady-state levels of metabolites from the PPP and other antioxidant pathways. (A) Human-packed RBCs were stored in AS-3, either untreated or supplemented with 1, 5, or 10 μM of S1P. (B-C) Heat maps from metabolomics

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Figure 2 (continued) analyses of RBCs and supernatants from this experiment are shown in panels B and C, respectively. (D) Line plots (median ± ranges) for representative metabolites in glycolysis (pyruvate), ATP, PPP metabolites (ribose phosphate and pentose phosphate isobars, sedoheptulose phosphate), total glutathione (pools of reduced and oxidized), free fatty acids, and leukotrienes (LTA4 and LTB4). (E) A schematic representation of the results.

steady-state levels of PPP metabolites were observed in a dose-response manner (for example, ribose phosphate and pentose phosphate isomers; sedoheptulose phosphate; Figure 2D). Consistently, we observed decreases in total glutathione (pools of reduced and oxidized), and increases in free long-chain polyunsaturated and highly unsaturated fatty acids (C18:1, 18:3, 20:3, 20:5, 22:5, and 22:6), markers of oxidant stress–induced fatty acid desaturase activity,57 as a function of S1P levels (Figure 2D). Similarly, in S1P-supplemented RBCs, we observed an accumulation of oxylipins, including leukotrienes (LTA4 and LTB4) and hydroxy eicosatetraenoic acids, as well as oxidized purines (for example, hypoxanthine), markers of poor PTR in both mice48 and humans58 (Figure 2D). In brief, storage in the presence of S1P did boost glycolysis and ATP levels, at the expense of the antioxidant system, that is, the PPP and glutathione pools, and purine and lipid oxidation (Figure 2E).

Mouse RBC storage upon supplementation of S1P decreases PTR

To further validate these findings in a tractable animal model, we performed similar storage experiments in mice, in which PTR studies are more easily executed and mechanistic intervention (for example, via genetic ablation of the S1P biosynthetic pathway) are amenable to testing. Cognizant that not all mouse strain RBCs store similarly,46 we performed follow-up studies on multiple mouse strains. Indeed, some mouse strains have been previously46,48 labeled as good storers, because of their elevated PTRs (for example, C57BL6/J), whereas other mouse strains are characterized by an exacerbated metabolic storage lesion and poor PTRs (FVB).46 Therefore, S1P supplementation (5 μM) was performed both in good and poor storer strains (Figure 3A), resulting in lower PTRs in both strain types upon S1P supplementation (Figure 3B-C). An overview of the murine protocol for measurements of PTR with the combined use of UBC-GFP recipient mice and mCHERRY reporter cells is provided in supplemental Figure 4A-C, along with representative scatter plots from fellow cytometry experiments. Poorer storage quality in S1Psupplemented RBCs was associated with a measurable effect on glycolysis, and free and acyl-conjugated carnitines in both good storer C57BL/6 and poor storer FVB mice (heat maps in Figure 3D-E, respectively).

Genetic ablation of S1P biosynthesis in Sphk1 KO mice is associated with improved storage quality

Given the negative effect of S1P supplementation on human and murine RBC storage quality, we hypothesized that an opposite, beneficial effect would be observed in response to genetic ablation of the S1P biosynthetic pathway in Sphk1 KO mice. To test this hypothesis, RBCs from wild-type (WT) C57BL/6 and Sphk1 KO mice were stored (Figure 4A), which resulted in a beneficial effect on storage-induced depletion of acylcarnitines and glutathione pools, and mitigation of free fatty acid and purine oxidation product accumulation (allantoate, heat map in Figure 4B). Confirming Sphk1 genetic ablation, signifificantly lower levels of RBC S1P were observed in these mice (Sphk1 mice had ~30% of S1P levels of WT mice at baseline - Figure 4C). Sphk1 KO stored RBCs were also characterized by lower levels of glycolytic metabolites (glucose-6-phosphate and hexose phosphate isomers, fructose bisphosphate, glyceraldehyde- 3-phosphate, and DPG) and higher levels of PPP metabolites (6-phosphogluconate, sedoheptulose phosphate; Figure 4D). Consistent with the beneficial effect of decreased S1P levels, Sphk1 mice (already with a C57BL6/J good storer background) had higher PTR (P < .05) than WT good storer mice (Figure 4E).

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Figure 4. Improved PTR and antioxidant metabolism in RBCs from Sphk1 KO mice. (A-C) Storage of mouse RBCs from WT C57BL/6 and Sphk1 KO mice (A) resulted in altered metabolism (heat map in panel B), including signifificantly lower levels of RBC S1P (C), as expected. (D) Specifically, Sphk1 KO stored RBCs were characterized by lower levels of glycolytic metabolites (hexose phosphate, fructose bisphosphate, glyceraldehyde-3-phosphate, and DPG) and higher levels of PPP metabolites (6-phosphogluconate, sedoheptulose phosphate). Sphk1 mice had higher PTR (P < .05) than WT good storer mice (median ± ranges) (E). In panel E, 1 data point for PTR in Sphk1 mice was set to 100%, as explained in “Materials and methods.”

Consistent with increased antioxidant capacity and improved metabolic phenotypes and PTRs, Sphk1 mice were characterized by a mitigated storage lesion to the proteome (Figure 5A-B). Specifically, signifificantly lower levels of oxidized proteins (cysteine and methionine oxidation) were observed in Sphk1 KO mice, including the most abundant cytosolic and membrane proteins, hemoglobin subunit beta 1 and band-3 anion transporter, respectively (Figure 5C).

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Sphingosine 1-phosphate Has A Negative Effect On RBC Storage Quality

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