Conjugated Linoleic Acid Modulates Clinical Responses to Oral Nitrite and Nitrate.

Dietary NO3- (nitrate) and NO2- (nitrite) support ˙NO (nitric oxide) 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. Coadministration 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 physiological 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. Clinical Trial Registration- URL: http://www.clinicaltrials.gov . Unique identifier: NCT01681836.

In a canine pneumonia model of exchange transfusion, altering the age but not the volume of older red blood cells markedly alters outcome.

BACKGROUND: Massive exchange transfusion of 42-day-old red blood cells (RBCs) in a canine model of Staphylococcus 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 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.

Detecting and monitoring NO, SNO and nitrite in vivo.

The detection and quantification of nitric oxide and related reactive nitrogen species in vivo is vital to the understanding of the pathology and/or treatment of numerous conditions. To that end, several detection and quantification methods have been developed to study NO, as well as its redox relatives, nitrite and S-nitrosothiols. While no single technique can offer a complete picture of the nitrogen cycle in a given system in vivo, familiarity with the benefits and limitations of several common tools for NOx determination can assist in the development of new diagnostics and therapeutics.

Washing older blood units before transfusion reduces plasma iron and improves outcomes in experimental canine pneumonia.

In a randomized controlled blinded trial, 2-year-old purpose-bred beagles (n = 24), with Staphylococcus aureus pneumonia, were exchanged-transfused with either 7- or 42-day-old 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.

Direct and nitroxyl (HNO)-mediated reactions of acyloxy nitroso compounds with the thiol-containing proteins glyceraldehyde 3-phosphate dehydrogenase and alkyl hydroperoxide reductase subunit C.

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 Cys244. 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 unsubstituted 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.

Nitroxyl accelerates the oxidation of oxyhemoglobin by nitrite.

Angeli's salt (Na2N2O3) decomposes into nitroxyl (HNO) and nitrite (NO2(-)), compounds of physiological and therapeutic interest for their impact on biological signaling both through nitric oxide and nitric oxide independent pathways. Both nitrite and HNO oxidize oxygenated hemoglobin to methemoglobin. Earlier work has shown that HNO catalyzes the reduction of nitrite by deoxygenated hemoglobin. In this work, we have shown that HNO accelerates the oxidation of oxygenated hemoglobin by NO2(-). We have demonstrated this HNO mediated acceleration of the nitrite/oxygenated hemoglobin reaction with oxygenated hemoglobin being in excess to HNO and nitrite (as would be found under physiological conditions) by monitoring the formation of methemoglobin in the presence of Angeli's salt with and without added NO2(-). In addition, this acceleration has been demonstrated using the HNO donor 4-nitrosotetrahydro-2H-pyran-4-yl pivalate, a water-soluble acyloxy nitroso compound that does not release NO2(-) but generates HNO in the presence of esterase. This HNO donor was used both with and without NO2(-) and acceleration of the NO2(-) induced formation of methemoglobin was observed. We found that the acceleration was not substantially affected by catalase, superoxide dismutase, c-PTIO, or IHP, suggesting that it is not due to formation of extramolecular peroxide, NO2 or H2O2, or to modulation of allosteric properties. In addition, we found that the acceleration is not likely to be related to HNO binding to free reduced hemoglobin, as we found HNO binding to reduced hemoglobin to be much weaker than has previously been proposed. We suggest that the mechanism of the acceleration involves local propagation of autocatalysis in the nitrite-oxygenated Hb reaction. This acceleration of the nitrite oxyhemoglobin reaction could affect studies aimed at understanding physiological roles of HNO and perhaps nitrite and use of these agents in therapeutics such as hemolytic anemias, heart failure, and ischemia reperfusion injury.

Angeli's salt counteracts the vasoactive effects of elevated plasma hemoglobin.

Plasma hemoglobin (Hb) released during intravascular hemolysis has been associated with numerous deleterious effects that may stem from increased nitric oxide (NO) scavenging, but has also been associated with reactive oxygen species generation and platelet activation. Therapies that convert plasma oxyHb to metHb, or metHb to iron-nitrosyl Hb, could be beneficial because these species do not scavenge NO. In this study, we investigated the effects of Angeli's salt (AS; sodium α-oxyhyponitrite, Na2N2O3), a nitroxyl (HNO) and nitrite (NO2(-)) donor, on plasma Hb oxidation and formation of iron-nitrosyl Hb from metHb and on the vasoactivity of plasma Hb. We hypothesized that AS could ameliorate hemolysis-associated pathology via its preferential reactivity with plasma Hb, as opposed to red-cell-encapsulated Hb, and through its intrinsic vasodilatory activity. To test this hypothesis, we infused (n=3 per group) (1) cell-free Hb and AS, (2) cell-free Hb+0.9% NaCl, (3) AS+3% albumin, and (4) 3% albumin+0.9% NaCl (colloid controls for Hb and AS, respectively) in a canine model. Co-infusion of AS and cell-free Hb led to preferential conversion of plasma Hb to metHb, but the extent of conversion was lower than anticipated based on the in vivo concentration of AS relative to plasma Hb. This lower metHb yield was probably due to reactions of nitroxyl-derived AS with plasma components such as thiol-containing compounds. From a physiological and therapeutic standpoint, the infusion of Hb alone led to significant increases in mean arterial pressure (p=0.03) and systemic vascular resistance index (p=0.01) compared to controls. Infusion of AS alone led to significant decreases in these parameters and co-infusion of AS along with Hb had an additive effect in reversing the effects of Hb alone on the systemic circulation. Interestingly, in the pulmonary system, the decrease in pressure when AS was added to Hb was significantly less than would have been expected compared to the effects of Hb and AS alone, suggesting that inactivation of scavenging with AS reduced the direct vasodilatory effects of AS on the vasculature. We also found that AS reduced platelet activation when administered to whole blood in vitro. These data suggest that AS-like compounds could serve as therapeutic agents to counteract the negative vasoconstrictive consequences of hemolysis that occur in hemolytic anemias, transfusion of stored blood, and other diseases. Increases in metHb in the red blood cell, the potential of AS for neurotoxicity, and hypotension would need to be carefully monitored in a clinical trial.