• Washing older blood before transfusion reduces plasma iron, improving outcomes from established infection in canines.• In contrast, washing fresh blood before transfusion increases in vivo plasma CFH release, worsening outcomes.In a randomized controlled blinded trial, 2-year-old purpose-bred beagles (n 5 24), with Staphylococcus aureus pneumonia, were exchanged-transfused with either 7-or 42-dayold washed or unwashed canine universal donor blood (80 mL/kg in 4 divided doses). Washing red cells (RBC) before transfusion had a significantly different effect on canine survival, multiple organ injury, plasma iron, and cell-free hemoglobin (CFH) levels depending on the age of stored blood (all, P < .05 for interactions). Washing older units of blood improved survival rates, shock score, lung injury, cardiac performance and liver function, and reduced levels of non-transferrin bound iron and plasma labile iron. In contrast, washing fresh blood worsened all these same clinical parameters and increased CFH levels. Our data indicate that transfusion of fresh blood, which results in less hemolysis, CFH, and iron release, is less toxic than transfusion of older blood in critically ill infected subjects. However, washing older blood prevented elevations in plasma circulating iron and improved survival and multiple organ injury in animals with an established pulmonary infection. Our data suggest that fresh blood should not be washed routinely because, in a setting of established infection, washed RBC are prone to release CFH and result in worsened clinical outcomes. (Blood. 2014;123(9):1403-1411 IntroductionTransfusion of older stored canine universal donor blood in a canine model of experimental Staphylococcus aureus pneumonia results in markedly increased lung injury and mortality rates.1 Transfusion with older blood is also associated with increased levels of cell-free hemoglobin (CFH), transferrin bound iron (TBI), non-TBI (NTBI) and plasma labile iron (PLI). NTBI represents iron excess bound to proteins that do not normally handle circulating iron, and PLI is the toxic iron moiety in plasma. Whereas increased nitric oxide scavenging by CFH causing vasoconstriction and vascular injury and increased available iron promoting bacterial growth represent 2 candidate mechanisms of injury, multiple other biological changes have been documented with increasing blood storage interval.2,3 Some of these changes involve the release into the plasma of biologically active proteins, microvesicles, potassium, acid, and plasticizer, all of which can be reduced by means of standard red cell (RBC) washing procedures. [4][5][6][7][8][9][10] The clinical effect(s) of washing on the RBC storage lesion has not been studied.RBC washing has long been performed to reduce potassium levels in stored blood transfused to neonates, debris from RBCs recovered during surgery, cryoprotectant glycerol from cryopreserved RBCs, and plasma proteins from blood intended for patients who have been sensitized to those proteins.11-13 Automated cell washers cap...

Background Massive exchange-transfusion of 42-day-old red blood cells (RBCs) in a canine model of S. aureus pneumonia resulted in in vivo hemolysis with increases in cell-free hemoglobin (CFH), transferrin bound iron (TBI), non-transferrin bound iron (NTBI), and mortality. We have previously shown that washing 42-day-old RBCs before transfusion significantly decreased NTBI levels and mortality, but washing 7-day-old RBCs increased mortality and CFH levels. We now report the results of altering volume, washing, and age of RBCs. Study Design and Methods Two-year-old purpose-bred infected beagles were transfused with increasing volumes (5-10, 20-40, or 60-80 mL/kg) of either 42- or 7-day-old RBCs (n=36) or 80 mL/kg of either unwashed or washed RBCs with increasing storage age (14, 21, 28, or 35 days) (n=40). Results All volumes transfused (5-80 mL/kg) of 42-day-old RBCs, resulted in alike (i.e., not significantly different) increases in TBI during transfusion as well as in CFH, lung injury, and mortality rates after transfusion. Transfusion of 80 mL/kg of RBCs stored for 14, 21, 28 and 35 days resulted in increased CFH and NTBI in between levels found at 7 and 42 days of storage. However, washing RBCs of intermediate ages (14-35 days) does not alter NTBI and CFH levels or mortality rates. Conclusions Preclinical data suggest that any volume of 42-day-old blood potentially increases risks during established infection. In contrast, even massive volumes of 7-day-old blood result in minimal CFH and NTBI levels and risks. In contrast to the extremes of storage, washing blood stored for intermediate ages does not alter risks of transfusion or NTBI and CFH clearance.

Nitroxyl (HNO) reacts with thiols and this reactivity requires the use of donors with 1-nitrosocyclohexyl acetate, pivalate and trifluoroacetate forming a new group. These acyloxy nitroso compounds inhibit glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by forming a reduction reversible active site disulfide and a reduction irreversible sulfinic acid or sulfinamide modification at Cys 244. Addition of these acyloxy nitroso compounds to AhpC C165S yields a sulfinic acid and sulfinamide modification. A potential mechanism for these transformations includes nucleophilic addition of the protein thiol to a nitroso compound to yield an N-hydroxysulfenamide, which reacts with thiol to give disulfide or rearranges to sulfinamides. Known HNO donors produce the un-substituted protein sulfinamide as the major product while the acetate and pivalate give substituted sulfinamides that hydrolyze to sulfinic acids. These results suggest that nitroso compounds form a general class of thiol-modifying compounds allowing their further exploration.

Dietary nitrate (NO3−) and nitrite (NO2−) support nitric oxide (·NO) generation and downstream vascular signaling responses. These nitrogen oxides also generate secondary nitrosating and nitrating species that react with low molecular weight thiols, heme centers, proteins and unsaturated fatty acids. To explore the kinetics of NO3− and NO2− metabolism and the impact of dietary lipid on nitrogen oxide metabolism and cardiovascular responses, the stable isotopes Na15NO3 and Na15NO2 were orally administered in the presence or absence of conjugated linoleic acid (cLA). The reduction of 15NO2− to 15NO was indicated by electron paramagnetic resonance spectroscopy detection of hyperfine splitting patterns reflecting 15NO-deoxyhemoglobin complexes. This formation of 15NO also translated to decreased systolic and mean arterial blood pressures and inhibition of platelet function. Upon concurrent administration of cLA, there was a significant increase in plasma cLA nitration products 9- and 12-15NO2-cLA. Co-administration of cLA with 15NO2− also impacted the pharmacokinetics and physiological effects of 15NO2−, with cLA administration suppressing plasma NO3− and NO2− levels, decreasing 15NO-deoxyhemoglobin formation, NO2− inhibition of platelet activation, and the vasodilatory actions of NO2−, while enhancing the formation of 9- and 12-15NO2-cLA. These results indicate that the biochemical reactions and physiologic responses to oral 15NO3− and 15NO2− are significantly impacted by dietary constituents such as unsaturated lipids. This can explain the variable responses to NO3− and NO2− supplementation in clinical trials and reveals dietary strategies for promoting the generation of pleiotropic nitrogen oxide-derived lipid signaling mediators.