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Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1–7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and – preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo. Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates.

For improvements to be made in long-standing industrial catalytic processes, an understanding of the atomistic mechanisms of the reactions occurring on the surface of the catalyst is required. A variety of experimental techniques can be used to derive information on sorption and reaction processes, but when both the catalyst and reactant mixtures are multicomponent, unambiguous identification of reaction mechanisms is difficult and often controversial. Computational techniques can, however, be used to gain valuable insight and interpret experimental evidence. Herein we show how new methods for modeling surface reactions on oxides can be used to elucidate key steps in a widely studied catalytic processthe conversion of CO 2 to methanol over oxide catalysts.A large quantity of methanol (in excess of 25 million tonnes worldwide) is produced annually using the multicomponent Cu/ZnO/Al 2 O 3 catalyst and CO 2 /CO/H 2 as the feed gas. Many experimental studies of this process have been performed, but no definite reaction mechanism for the production of methanol has been established. However, it has long been acknowledged that the important rate-determining step is the hydrogenation of adsorbed intermediates, for example, the formate ion, at the active sites. Proposed mechanisms for methanol synthesis require the chemisorption of CO 2 before hydrogenation via formate to methanol. Theoretical studies of these systems have been hampered by the difficulty of modeling the catalytically active polar surfaces of ZnO, as well as by problems associated with the restrictions on the size of the system that can be modeled.The nature of the active site for sorption/catalysis of CO 2 still remains unclear; it has been proposed to use clean oxygen-terminated surfaces of zincite as a test system or model catalyst. Temperature-programmed desorption (TPD) studies have shown that the processes that occur at that double couplings and a large excess of building blocks. The application of the A-Tag and the F-Tag to the automated synthesis of more complex oligosaccharides and other biopolymers is currently being explored. Experimental SectionGeneral Procedure for the automated synthesis of oligosaccharides incorporating cap-tags: Octenediol-functionalized resin 12 was loaded into a reaction vessel equipped with a cooling jacket and inserted into a modified ABI-433A peptide synthesizer. The resin was glycosylated with donor 13 or 15 (5 equiv) in CH 2 Cl 2 (3 mL) with TMSOTf as activator. The suspension was mixed (10 s vortex, 50 s rest) for 15 min. The resin was then washed with CH 2 Cl 2 (6 Â 4 mL), and the unglycosylated sites were capped with A-Tag anhydride 2 or F-Tag triflate 11. The resin was subjected to the appropriate deprotection conditions followed by the washing cycle. The deprotected polymer-bound monosaccharide was then elongated by reiteration of the above glycosylation/capping/deprotection protocol. The final trisaccharide was not deprotected, so that analysis of the products was simplified. For A-Tag, the crude material was t...

The erythrocyte membrane is a newly appreciated platform for thiol-based circulatory signaling, and it requires robust free thiol maintenance. We sought to define physiological constraints on erythrocyte antioxidant defense. Hemoglobin (Hb) conformation gates glycolytic flux through the hexose monophosphate pathway (HMP), the sole source of nicotinamide adenine dinucleotide phosphate (NADPH) in erythrocytes. We hypothesized elevated intraerythrocytic deoxyHb would limit resilience to oxidative stress. Human erythrocytes were subjected to controlled oxidant (superoxide) loading following independent manipulation of oxygen tension, Hb conformation, and glycolytic pathway dominance. Sufficiency of antioxidant defense was determined by serial quantification of GSH, NADPH, NADH redox couples. Hypoxic erythrocytes demonstrated greater loss of reduction potential [Delta GSH E(hc) (mV): 123.4+/-9.7 vs. 57.2+/-11.1] and reduced membrane thiol (47.7+/-5.7 vs. 20.1+/-4.3%) (hypoxia vs. normoxia, respectively; P<0.01), a finding mimicked in normoxic erythrocytes after HMP blockade. Rebalancing HMP flux during hypoxia restored resilience to oxidative stress at all stages of the system. Cell-free studies assured oxidative loading was not altered by oxygen tension, heme ligation, or the inhibitors employed. These data indicate that Hb conformation controls coupled glucose and thiol metabolism in erythrocytes, and implicate hypoxemia in the pathobiology of erythrocyte-based vascular signaling.

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