Temperature Dependence of Platelet Metabolism.

Temperature plays a fundamental role in biology, influencing cellular function, chemical reaction rates, molecular structures, and interactions. While the temperature dependence of many biochemical reactions is well defined in vitro, the effect of temperature on metabolic function at the network level is poorly understood, and it remains an important challenge in optimizing the storage of cells and tissues at lower temperatures. Here, we used time-course metabolomic data and systems biology approaches to characterize the effects of storage temperature on human platelets (PLTs) in a platelet additive solution. We observed that changes to the metabolome with storage time do not simply scale with temperature but instead display complex temperature dependence, with only a small subset of metabolites following an Arrhenius-type relationship. Investigation of PLT energy metabolism through integration with computational modeling revealed that oxidative metabolism is more sensitive to temperature changes than glycolysis. The increased contribution of glycolysis to ATP turnover at lower temperatures indicates a stronger glycolytic phenotype with decreasing storage temperature. More broadly, these results demonstrate that the temperature dependence of the PLT metabolic network is not uniform, suggesting that efforts to improve the health of stored PLTs could be targeted at specific pathways.

Structure-activity relationship for antibacterial chitosan carrying cationic and hydrophobic moieties.

To overcome the problem of antibiotic resistance and toxicity of synthetic polymers, herein we report the synthesis of biocompatible polymers which can serve as broad spectrum antimicrobials. A regioselective synthetic method was developed to synthesize N-functionalized chitosan polymers having similar degree of substitution of cationic and hydrophobic functionality with different lipophilic chains. We obtained optimum antibacterial effect by utilizing the combination of cationic and longer lipophilic chain in the polymer, against four bacterial strains. Inhibition and killing of bacteria were more pronounced in Gram positive bacteria than in Gram negative bacteria. Growth kinetics and scanning electron microscopy imaging of the polymer treated bacterial cells confirmed the inhibition of bacterial growth, morphological changes in the structure and membrane disruption in the cells as compared to the growth control for each strain. Further investigation into the toxicity and selectivity of the polymers guided us to develop a structure-activity relationship for this class of biocompatible polymers.

Recommendations for in vitro evaluation of blood components collected, prepared and stored in non-DEHP medical devices.

BACKGROUND AND OBJECTIVES: DEHP, di(2-ethylhexyl) phthalate, is the most common member of the class of ortho-phthalates, which are used as plasticizers. The Medical Device Regulation has restricted the use of phthalates in medical devices. Also DEHP has been added to the Annex XIV of REACH, "Registration, Evaluation, Authorisation and Restriction of Chemicals" due to its endocrine disrupting properties to the environment. As such, the sunset date for commercialisation of DEHP-containing blood bags is May 27th 2025. There are major concerns in meeting this deadline as these systems have not yet been fully validated and/or CE-marked. Also, since DEHP is known to affect red cell quality during storage, it is imperative to transit to non-DEHP without affecting blood product quality. Here, EBA members aim to establish common grounds on the evaluation and assessment of blood components collected, prepared and stored in non-DEHP devices. MATERIALS AND METHODS: Based on data as well as the input of relevant stakeholders a rationale for the validation of each component was composed. RESULTS: The red cell components will require the most extensive validation as their quality is directly affected by the absence of DEHP, as opposed to platelet and plasma components. CONCLUSION: Studies in the scope of evaluating the quality of blood products obtained with non-DEHP devices, under the condition that they are carried out according to these recommendations, could be used by all members of the EBA to serve as scientific support in the authorization process specific to their jurisdiction or for their internal validation use.

Transdermal Drug Delivery of Tazarotene: Determining Tazarotene's Potential in Local Transdermal Therapy.

Retinoid-based drugs, while effective, are associated with systemic toxicity. Topical alternatives offer a safer option, and tazarotene, a third-generation synthetic retinoid, holds promise. This study investigates tazarotene's transdermal delivery potential, focusing on its application for joint-related conditions. The aim of this study was to investigate the suitability of tazarotene as a candidate for transdermal delivery into joints. In vitro permeation studies, using porcine skin, assessed tazarotene's transdermal drug delivery from solution and gel formulations. A tape-stripping analysis determined stratum corneum retention and a pilot study using porcine joints assessed tazarotene's ability to reach articular cartilage. Ultra Performance Liquid Chromatography coupled with a mass detector method was used to quantify tazarotene and tazarotenic acid permeation. The results validate that tazarotene can permeate porcine skin and accumulate in articular cartilage in detectable amounts. The detection of tazarotene and tazarotenic acid in both the in vitro permeation studies and the pilot study on porcine joints validate the drug's potential therapeutic use for hand osteoarthritis. This study lays the groundwork for future research, contributing insights into tazarotene's potential for transdermal drug delivery and guiding further exploration in topical retinoid applications.

Protein Concentrations in Stored Pooled Platelet Concentrates Treated with Pathogen Inactivation by Amotosalen Plus Ultraviolet a Illumination.

Platelet granules contain a diverse group of proteins. Upon activation and during storage, platelets release a number of proteins into the circulation or supernatant of stored platelet concentrate (PC). The aim of this work was to investigate the effect of pathogen inactivation (PI) on a selection of proteins released in stored platelets. MATERIALS AND METHODS: PCs in platelet additive solution (PAS) were produced from whole blood donations using the buffy coat (BC) method. PCs in the treatment arm were pathogen inactivated with amotosalen and UVA, while PCs in the second arm were used as an untreated platelet control. Concentrations of 36 proteins were monitored in the PCs during storage. RESULTS: The majority of proteins increased in concentration over the storage period. In addition, 10 of the 29 proteins that showed change had significantly different concentrations between the PI treatment and the control at one or more timepoints. A subset of six proteins displayed a PI-related drop in concentration. CONCLUSIONS: PI has limited effect on protein concentration stored PC supernatant. The protein's changes related to PI treatment with elevated concentration implicate accelerated Platelet storage lesion (PSL); in contrast, there are potential novel benefits to PI related decrease in protein concentration that need further investigation.

SERS Imaging of Mesenchymal Stromal Cell Differentiation.

Understanding the process of mesenchymal stromal cell (MSC) osteogenic differentiation is essential for a wide range of medical applications. However, these primary cells vary significantly from donor to donor, making it difficult to fully exploit their therapeutic potential. Although osteogenic differentiation has been studied extensively, there is still a shortage of standardized methods for the evaluation of the degree of differentiation. Here, we employ noninvasive surface-enhanced Raman scattering (SERS) for studying such cells, offering a better understanding of cellular processes in situ. We present the long-term differentiation of MSCs on biocompatible gold nanoisland SERS substrates, combining imaging of cells with spectroscopic detection of molecular species and chemical events occurring on the cellular membrane adjacent to the surface of the SERS substrate. We detect multiple signs of bone tissue formation, from an early stage to mature osteoblasts, without labeling. We show that the results correlate very well with classical differentiation-detecting assays, indicating that the SERS imaging technique alone is sufficient to study the progress of osteogenic differentiation of such cells, paving a way toward continuous label-free screening of live cells.

A comparison of platelet quality between platelets from healthy donors and hereditary hemochromatosis donors over seven-day storage.

BACKGROUND: Therapeutic phlebotomy is the standard treatment of hereditary hemochromatosis (HH), the most common genetic disease in people of Northern European descent. Red cell concentrates from HH donors have been reported safe for transfusion, but little data is available on the storage properties of platelet concentrates from HH donors. STUDY DESIGN AND METHODS: Whole blood was collected from 10 healthy individuals and 10 newly diagnosed HH patients with elevated serum ferritin. Platelet-rich plasma (PRP) was prepared and split into four 20-mL units. Platelet quality tests were performed on days 0, 1, 3, 5, and 7 of storage, including platelet aggregation (ADP, arachidonic acid, collagen, and epinephrine agonists), blood gas analysis, flow cytometry (CD41, CD42b, and CD62P expression), and ELISA (sCD40L and sCD62p in supernatant). RESULTS: Mean serum ferritin levels were higher in HH patients than in controls (847.5 vs 45.8 ng/mL, P < .001). Overall, no difference in quality test results was observed between the two study groups over 7-day storage (P > .05), including blood gas analysis, platelet aggregation, and expression of surface (CD62p and CD42b) and secreted (sCD62P and sCD40L) activation markers. Expected alterations in metabolic (CO2 and glucose decrease, O2 and lactate increase, P < .001) and platelet activation markers (CD42b decrease, CD62P increase, P < .05) over time were observed in both groups. CONCLUSION: Although these findings indicate that platelets of individuals with HH are comparable to platelets from healthy donors, more extensive studies are needed before definite conclusions can be drawn.

Human Embryonic-Derived Mesenchymal Progenitor Cells (hES-MP Cells) are Fully Supported in Culture with Human Platelet Lysates.

Human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells are mesenchymal-like cells, derived from human embryonic stem cells without the aid of feeder cells. They have been suggested as a potential alternative to mesenchymal stromal cells (MSCs) in regenerative medicine due to their mesenchymal-like proliferation and differentiation characteristics. Cells and cell products intended for regenerative medicine in humans should be derived, expanded and differentiated using conditions free of animal-derived products to minimize risk of animal-transmitted disease and immune reactions to foreign proteins. Human platelets are rich in growth factors needed for cell culture and have been used successfully as an animal serum replacement for MSC expansion and differentiation. In this study, we compared the proliferation of hES-MP cells and MSCs; the hES-MP cell growth was sustained for longer than that of MSCs. Growth factors, gene expression, and surface marker expression in hES-MP cells cultured with either human platelet lysate (hPL) or fetal bovine serum (FBS) supplementation were compared, along with differentiation to osteogenic and chondrogenic lineages. Despite some differences between hES-MP cells grown in hPL- and FBS-supplemented media, hPL was found to be a suitable replacement for FBS. In this paper, we demonstrate for the first time that hES-MP cells can be grown using platelet lysates from expired platelet concentrates (hPL).

Current Status and Future Prospects of Genome-Scale Metabolic Modeling to Optimize the Use of Mesenchymal Stem Cells in Regenerative Medicine.

Mesenchymal stem cells are a promising source for externally grown tissue replacements and patient-specific immunomodulatory treatments. This promise has not yet been fulfilled in part due to production scaling issues and the need to maintain the correct phenotype after re-implantation. One aspect of extracorporeal growth that may be manipulated to optimize cell growth and differentiation is metabolism. The metabolism of MSCs changes during and in response to differentiation and immunomodulatory changes. MSC metabolism may be linked to functional differences but how this occurs and influences MSC function remains unclear. Understanding how MSC metabolism relates to cell function is however important as metabolite availability and environmental circumstances in the body may affect the success of implantation. Genome-scale constraint based metabolic modeling can be used as a tool to fill gaps in knowledge of MSC metabolism, acting as a framework to integrate and understand various data types (e.g., genomic, transcriptomic and metabolomic). These approaches have long been used to optimize the growth and productivity of bacterial production systems and are being increasingly used to provide insights into human health research. Production of tissue for implantation using MSCs requires both optimized production of cell mass and the understanding of the patient and phenotype specific metabolic situation. This review considers the current knowledge of MSC metabolism and how it may be optimized along with the current and future uses of genome scale constraint based metabolic modeling to further this aim.

Visualizing metabolic network dynamics through time-series metabolomic data.

BACKGROUND: New technologies have given rise to an abundance of -omics data, particularly metabolomic data. The scale of these data introduces new challenges for the interpretation and extraction of knowledge, requiring the development of innovative computational visualization methodologies. Here, we present GEM-Vis, an original method for the visualization of time-course metabolomic data within the context of metabolic network maps. We demonstrate the utility of the GEM-Vis method by examining previously published data for two cellular systems-the human platelet and erythrocyte under cold storage for use in transfusion medicine. RESULTS: The results comprise two animated videos that allow for new insights into the metabolic state of both cell types. In the case study of the platelet metabolome during storage, the new visualization technique elucidates a nicotinamide accumulation that mirrors that of hypoxanthine and might, therefore, reflect similar pathway usage. This visual analysis provides a possible explanation for why the salvage reactions in purine metabolism exhibit lower activity during the first few days of the storage period. The second case study displays drastic changes in specific erythrocyte metabolite pools at different times during storage at different temperatures. CONCLUSIONS: The new visualization technique GEM-Vis introduced in this article constitutes a well-suitable approach for large-scale network exploration and advances hypothesis generation. This method can be applied to any system with data and a metabolic map to promote visualization and understand physiology at the network level. More broadly, we hope that our approach will provide the blueprints for new visualizations of other longitudinal -omics data types. The supplement includes a comprehensive user's guide and links to a series of tutorial videos that explain how to prepare model and data files, and how to use the software SBMLsimulator in combination with further tools to create similar animations as highlighted in the case studies.

Comparative Metabolic Network Flux Analysis to Identify Differences in Cellular Metabolism.

Metabolic network flux analysis uses genome-scale metabolic reconstructions to integrate transcriptomics, proteomics, and/or metabolomics data to allow for comprehensive interpretation of genotype to metabolic phenotype relationships. The compilation of many Constraint-based model analysis methods into one MATLAB package, the COBRAtoolbox, has opened the possibility of using these methods to the many biologists with some knowledge of the commonly used statistical program, MATLAB. Here we outline the steps required to take a published genome-scale metabolic reconstruction and interrogate its consistency and biological feasibility. Subsequently, we demonstrate how mRNA expression data and metabolomics data, relating to one or more cell types or biological contexts, can be applied to constrain and generate metabolic models descriptive of metabolic flux phenotypes. Finally, we describe the comparison of the resulting models and model outputs with the aim of identifying metabolic biomarkers and changes in cellular metabolism.

Metabolomics study of platelet concentrates photochemically treated with amotosalen and UVA light for pathogen inactivation.

BACKGROUND: The risk of bacterial contamination and the deterioration of platelet (PLT) quality limit the shelf-life of platelet concentrates (PCs). The INTERCEPT pathogen inactivation system reduces the risk of pathogen transmission by inhibiting nucleic acid replication using a combination of a photo-reactive compound and UVA illumination. The goal of this study was to investigate the effects the INTERCEPT system has on the PLT metabolome and metabolic activity. STUDY DESIGN AND METHODS: Paired units of buffy coat-derived PCs were generated using a pool and split strategy (n = 8). The paired PCs were either treated with the INTERCEPT system or left untreated. Samples were collected on Days 1, 2, 4, and 7 of storage. Ultra-performance chromatography coupled with time-of-flight mass spectrometry was used to analyze the extra- and intracellular metabolomes. Constraint-based metabolic modeling was then used to predict the metabolic activity of the stored PLTs. RESULTS: A relatively large number of metabolites in the extracellular environment were depleted during the processing steps of the INTERCEPT system, in particular, metabolites with hydrophobic functional groups, including acylcarnitines and lysophosphatidylcholines. In the intracellular environment, alterations in glucose and glycerophospholipid metabolism and decreased levels of 2-hydroxyglutarate were observed following the INTERCEPT treatment. Untargeted metabolomics analysis revealed residual amotosalen dimers present in the treated PCs. Systems-level analysis of PLT metabolism indicated that the INTERCEPT system does not have a significant impact on the PLT energy metabolism and nutrient utilization. CONCLUSIONS: The INTERCEPT system significantly alters the metabolome of the stored PCs without significantly influencing PLT energy metabolism.

Gold nanoisland substrates for SERS characterization of cultured cells.

We demonstrate a simple approach for fabricating cell-compatible SERS substrates, using repeated gold deposition and thermal annealing. The substrates exhibit SERS enhancement up to six orders of magnitude and high uniformity. We have carried out Raman imaging of fixed mesenchymal stromal cells cultured directly on the substrates. Results of viability assays confirm that the substrates are highly biocompatible and Raman imaging confirms that cell attachment to the substrates is sufficient to realize significant SERS enhancement of cellular components. Using the SERS substrates as an in vitro sensing platform allowed us to identify multiple characteristic molecular fingerprints of the cells, providing a promising avenue towards non-invasive chemical characterization of biological samples.

Pathogen inactivation with amotosalen plus UVA illumination minimally impacts microRNA expression in platelets during storage under standard blood banking conditions.

BACKGROUND: To reduce the risk of transfusion transmission infection, nucleic acid targeted methods have been developed to inactivate pathogens in PCs. miRNAs have been shown to play an important role in platelet function, and changes in the abundance of specific miRNAs during storage have been observed, as have perturbation effects related to pathogen inactivation (PI) methods. The aim of this work was to investigate the effects of PI on selected miRNAs during storage. STUDY DESIGN AND METHODS: Using a pool and split strategy, 3 identical buffy coat PC units were generated from a pool of 24 whole blood donors. Each unit received a different treatment: 1) Untreated platelet control in platelet additive solution (C-PAS); 2) Amotosalen-UVA-treated platelets in PAS (PI-PAS); and 3) untreated platelets in donor plasma (U-PL). PCs were stored for 7 days under standard blood banking conditions. Standard platelet quality control (QC) parameters and 25 selected miRNAs were analyzed. RESULTS: During the 7-day storage period, differences were found in several QC parameters relating to PI treatment and storage in plasma, but overall the three treatments were comparable. Out of 25 miRNA tested changes in regulation of 5 miRNA in PI-PAS and 3 miRNA U-PL where detected compared to C-PAS. A statistically significant difference was observed in down regulations miR-96-5p on Days 2 and 4, 61.9% and 61.8%, respectively, in the PI-PAS treatment. CONCLUSION: Amotosalen-UVA treatment does not significantly alter the miRNA profile of platelet concentrates generated and stored using standard blood banking conditions.

To Wash or Not to Wash? Comparison of Patient Outcome after Infusion of Cryopreserved Autologous Hematopoietic Stem Cells before and after the Replacement of Manual Washing by Bedside Thawing.

BACKGROUND: Prior to infusion, cryopreserved autologous peripheral blood stem cell (auto-PBSC) grafts can either be thawed at the bedside or thawed and washed at the laboratory. At our center, manual washing of grafts prior to infusion was discontinued in April 2012 and bedside thawing was implemented. METHODS: This study compares the outcomes of two patient groups who received auto-PBSC either after post-thaw washing (n = 84) or bedside thawing (n = 83). RESULTS: No life-threatening infusion-related side effects were reported in either group. There was no significant difference in the mean CD34+ cells/kg dose of infused auto-PBSC in the two groups (p = 0.41), nor in the number of days to neutrophils > 0.5 × 10(9)/L (p = 0.14), days to platelets > 20 × 10(9)/L (p = 0.64), or days to platelets > 50 × 10(9)/L (p = 0.62) after transplant. There was also no difference in the number of days on total parenteral nutrition (p = 0.69), days on G-CSF therapy (p = 0.48), or days with fever (p = 0.73). Finally, there was no significant difference in the number of red cell units transfused (p = 0.32), or platelet units transfused (p = 0.94) after the transplant. One-hundred-day mortality was identical in the two groups (2.4%). CONCLUSION: Both thawing procedures are safe and result in acceptable engraftment and patient outcomes.

POGZ Is Required for Silencing Mouse Embryonic ß-like Hemoglobin and Human Fetal Hemoglobin Expression.

Fetal globin genes are transcriptionally silenced during embryogenesis through hemoglobin switching. Strategies to derepress fetal globin expression in the adult could alleviate symptoms in sickle cell disease and ß-thalassemia. We identified a zinc-finger protein, pogo transposable element with zinc-finger domain (POGZ), expressed in hematopoietic progenitor cells. Targeted deletion of Pogz in adult hematopoietic cells in vivo results in persistence of embryonic ß-like globin expression without affecting erythroid development. POGZ binds to the Bcl11a promoter and erythroid-specific intragenic regulatory regions. Pogz+/- mice show elevated embryonic ß-like globin expression, suggesting that partial reduction of Pogz expression results in persistence of embryonic ß-like globin expression. Knockdown of POGZ in primary human CD34+ progenitor cell-derived erythroblasts reduces BCL11A expression, a known repressor of embryonic ß-like globin expression, and increases fetal hemoglobin expression. These findings are significant, since new therapeutic targets and strategies are needed to treat ß-globin disorders.

Systems analysis of metabolism in platelet concentrates during storage in platelet additive solution.

Platelets (PLTs) deteriorate over time when stored within blood banks through a biological process known as PLT storage lesion (PSL). Here, we describe the refinement of the biochemical model of PLT metabolism, iAT-PLT-636, and its application to describe and investigate changes in metabolism during PLT storage. Changes in extracellular acetate and citrate were measured in buffy coat and apheresis PLT units over 10 days of storage in the PLT additive solution T-Sol. Metabolic network analysis of these data was performed alongside our prior metabolomics data to describe the metabolism of fresh (days 1-3), intermediate (days 4-6), and expired (days 7-10) PLTs. Changes in metabolism were studied by comparing metabolic model flux predictions of iAT-PLT-636 between stages and between collection methods. Extracellular acetate and glucose contribute most to central carbon metabolism in PLTs. The anticoagulant citrate is metabolized in apheresis-stored PLTs and is converted into aconitate and, to a lesser degree, malate. The consumption of nutrients changes during storage and reflects altered PLT activation profiles following their collection. Irrespective of the collection method, a slowdown in oxidative phosphorylation takes place, consistent with mitochondrial dysfunction during PSL. Finally, the main contributors to intracellular ammonium and NADPH are highlighted. Future optimization of flux through these pathways provides opportunities to address intracellular pH changes and reactive oxygen species, which are both of importance to PSL. The metabolic models provide descriptions of PLT metabolism at steady state and represent a platform for future PLT metabolic research.

Systems biology as an emerging paradigm in transfusion medicine.

Blood transfusions are an important part of modern medicine, delivering approximately 85 million blood units to patients annually. Recently, the field of transfusion medicine has started to benefit from the "omic" data revolution and corresponding systems biology analytics. The red blood cell is the simplest human cell, making it an accessible starting point for the application of systems biology approaches.In this review, we discuss how the use of systems biology has led to significant contributions in transfusion medicine, including the identification of three distinct metabolic states that define the baseline decay process of red blood cells during storage. We then describe how a series of perturbations to the standard storage conditions characterized the underlying metabolic phenotypes. Finally, we show how the analysis of high-dimensional data led to the identification of predictive biomarkers.The transfusion medicine community is in the early stages of a paradigm shift, moving away from the measurement of a handful of chosen variables to embracing systems biology and a cell-scale point of view.

Quantitative time-course metabolomics in human red blood cells reveal the temperature dependence of human metabolic networks.

The temperature dependence of biological processes has been studied at the levels of individual biochemical reactions and organism physiology (e.g. basal metabolic rates) but has not been examined at the metabolic network level. Here, we used a systems biology approach to characterize the temperature dependence of the human red blood cell (RBC) metabolic network between 4 and 37 °C through absolutely quantified exo- and endometabolomics data. We used an Arrhenius-type model (Q10) to describe how the rate of a biochemical process changes with every 10 °C change in temperature. Multivariate statistical analysis of the metabolomics data revealed that the same metabolic network-level trends previously reported for RBCs at 4 °C were conserved but accelerated with increasing temperature. We calculated a median Q10 coefficient of 2.89 ± 1.03, within the expected range of 2-3 for biological processes, for 48 individual metabolite concentrations. We then integrated these metabolomics measurements into a cell-scale metabolic model to study pathway usage, calculating a median Q10 coefficient of 2.73 ± 0.75 for 35 reaction fluxes. The relative fluxes through glycolysis and nucleotide metabolism pathways were consistent across the studied temperature range despite the non-uniform distributions of Q10 coefficients of individual metabolites and reaction fluxes. Together, these results indicate that the rate of change of network-level responses to temperature differences in RBC metabolism is consistent between 4 and 37 °C. More broadly, we provide a baseline characterization of a biochemical network given no transcriptional or translational regulation that can be used to explore the temperature dependence of metabolism.

Mannose and fructose metabolism in red blood cells during cold storage in SAGM.

BACKGROUND: Alternate sugar metabolism during red blood cell (RBC) storage is not well understood. Here we report fructose and mannose metabolism in RBCs during cold storage in SAGM and the impact that these monosaccharides have on metabolic biomarkers of RBC storage lesion. STUDY DESIGN AND METHODS: RBCs were stored in SAGM containing uniformly labeled 13 C-fructose or 13 C-mannose at 9 or 18 mmol/L concentration for 25 days. RBCs and media were sampled at 14 time points during storage and analyzed using ultraperformance liquid chromatography-mass spectrometry. Blood banking quality assurance measurements were performed. RESULTS: Red blood cells incorporated fructose and mannose during cold storage in the presence of glucose. Mannose was metabolized in preference to glucose via glycolysis. Fructose lowered adenosine triphosphate (ATP) levels and contributed little to ATP maintenance when added to SAGM. Both monosaccharides form the advanced glycation end product glycerate. Mannose activates enzymes in the RBC that take part in glycan synthesis. CONCLUSIONS: Fructose or mannose addition to RBC SAGM concentrates may not offset the shift in metabolism of RBCs that occurs after 10 days of storage. Fructose and mannose metabolism at 4°C in SAGM reflects their metabolism at physiologic temperature. Glycerate excretion is a measure of protein deglycosylation activity in stored RBCs. No cytoprotective effect was observed upon the addition of either fructose or mannose to SAGM.