Artifacts Introduced by Sample Handling in Chemiluminescence Assays of Nitric Oxide Metabolites.

We recently developed a combination of four chemiluminescence-based assays for selective detection of different nitric oxide (NO) metabolites, including nitrite, S-nitrosothiols (SNOs), heme-nitrosyl (heme-NO), and dinitrosyl iron complexes (DNICs). However, these NO species (NOx) may be under dynamic equilibria during sample handling, which affects the final determination made from the readout of assays. Using fetal and maternal sheep from low and high altitudes (300 and 3801 m, respectively) as models of different NOx levels and compositions, we tested the hypothesis that sample handling introduces artifacts in chemiluminescence assays of NOx. Here, we demonstrate the following: (1) room temperature placement is associated with an increase and decrease in NOx in plasma and whole blood samples, respectively; (2) snap freezing and thawing lead to the interconversion of different NOx in plasma; (3) snap freezing and homogenization in liquid nitrogen eliminate a significant fraction of NOx in the aorta of stressed animals; (4) A "stop solution" commonly used to preserve nitrite and SNOs leads to the interconversion of different NOx in blood, while deproteinization results in a significant increase in detectable NOx; (5) some reagents widely used in sample pretreatments, such as mercury chloride, acid sulfanilamide, N-ethylmaleimide, ferricyanide, and anticoagulant ethylenediaminetetraacetic acid, have unintended effects that destabilize SNO, DNICs, and/or heme-NO; (6) blood, including the residual blood clot left in the washed purge vessel, quenches the signal of nitrite when using ascorbic acid and acetic acid as the purge vessel reagent; and (7) new limitations to the four chemiluminescence-based assays. This study points out the need for re-evaluation of previous chemiluminescence measurements of NOx, and calls for special attention to be paid to sample handling, as it can introduce significant artifacts into NOx assays.

Chronic High-Altitude Hypoxia Alters Iron and Nitric Oxide Homeostasis in Fetal and Maternal Sheep Blood and Aorta.

The mammalian fetus thrives at oxygen tensions much lower than those of adults. Gestation at high altitude superimposes hypoxic stresses on the fetus resulting in increased erythropoiesis. We hypothesized that chronic hypoxia at high altitude alters the homeostasis of iron and bioactive nitric oxide metabolites (NOx) in gestation. To test for this, electron paramagnetic resonance was used to provide unique measurements of iron, metalloproteins, and free radicals in the blood and aorta of fetal and maternal sheep from either high or low altitudes (3801 or 300 m). Using ozone-based chemiluminescence with selectivity for various NOx species, we determined the NOx levels in these samples immediately after collection. These experiments demonstrated a systemic redistribution of iron in high altitude fetuses as manifested by a decrease in both chelatable and total iron in the aorta and an increase in non-transferrin bound iron and total iron in plasma. Likewise, high altitude altered the redox status diversely in fetal blood and aorta. This study also found significant increases in blood and aortic tissue NOx in fetuses and mothers at high altitude. In addition, gradients in NOx concentrations observed between fetus and mother, umbilical artery and vein, and plasma and RBCs demonstrated complex dynamic homeostasis of NOx among these circulatory compartments, such as placental generation and efflux as well as fetal consumption of iron-nitrosyls in RBCs, probably HbNO. In conclusion, these results may suggest the utilization of iron from non-hematopoietic tissues iron for erythropoiesis in the fetus and increased NO bioavailability in response to chronic hypoxic stress at high altitude during gestation.

A physiologically relevant role for NO stored in vascular smooth muscle cells: A novel theory of vascular NO signaling.

S-nitrosothiols (SNO), dinitrosyl iron complexes (DNIC), and nitroglycerine (NTG) dilate vessels via activation of soluble guanylyl cyclase (sGC) in vascular smooth muscle cells. Although these compounds are often considered to be nitric oxide (NO) donors, attempts to ascribe their vasodilatory activity to NO-donating properties have failed. Even more puzzling, many of these compounds have vasodilatory potency comparable to or even greater than that of NO itself, despite low membrane permeability. This raises the question: How do these NO adducts activate cytosolic sGC when their NO moiety is still outside the cell? In this review, we classify these compounds as 'nitrodilators', defined by their potent NO-mimetic vasoactivities despite not releasing requisite amounts of free NO. We propose that nitrodilators activate sGC via a preformed nitrodilator-activated NO store (NANOS) found within the vascular smooth muscle cell. We reinterpret vascular NO handling in the framework of this NANOS paradigm, and describe the knowledge gaps and perspectives of this novel model.

Evidence for placental-derived iron-nitrosyls in the circulation of the fetal lamb and against a role for nitrite in mediating the cardiovascular transition at birth.

Circulating metabolites of nitric oxide, such as nitrite, iron nitrosyls (FeNO), and nitrosothiols, have vasodilatory bioactivity. In both human and sheep neonates, plasma concentrations of these NO metabolite (NOx) concentrations fall >50% within minutes after birth, raising the possibility that circulating NOx plays a role in maintaining low fetal vascular resistance and in the cardiovascular transition at birth. To test whether the fall in plasma NOx concentrations at birth is due to either ligation of the umbilical cord or oxygenation of the fetus to newborn levels, plasma NOx concentrations were measured during stepwise delivery of near-term fetal lambs. When fetal lambs were intubated and mechanically ventilated with 100% O2 to oxygenate the arterial blood while still in utero with the umbilical circulation still intact, there was no change in plasma NOx levels. In contrast, when the umbilical cord was ligated while fetal lambs were mechanically ventilated with O2 levels that maintained fetal arterial blood gases, plasma NOx levels decreased by nearly 50%. Characterization of the individual NOx species in plasma revealed that the overall fall in NOx at birth was attributable mainly to FeNO compounds. Finally, when the typical fall in NOx after birth was prevented by intravenous nitrite infusion, birth-related changes in blood pressure, heart rate, and carotid flow changes were little affected, suggesting the cardiovascular transition at birth is not dependent on a fall in plasma NOx. In conclusion, this study shows FeNO is released from the placenta and that its decline accounts for most of the measured fall in plasma NOx at birth.

Deferoxamine produces nitric oxide under ferricyanide oxidation, blood incubation, and UV-irradiation.

Deferoxamine (DFO), an iron chelator, is used therapeutically for the removal of excess iron in multiple clinical conditions such as beta thalassemia and intracerebral hemorrhage. DFO is also used as an iron chelator and hypoxia-mimetic agent in in vivo and in vitro basic research. Here we unexpectedly discover DFO to be a nitric oxide (NO) precursor in experiments where it was intended to act as an iron chelator. Production of NO from aqueous solutions of DFO was directly observed by ozone-based chemiluminescence using a ferricyanide-based assay and was confirmed by electron paramagnetic resonance (EPR). DFO also produced NO following exposure to ultraviolet light, and its incubation with sheep adult and fetal blood resulted in considerable formation of iron nitrosyl hemoglobin, as confirmed by both visible spectroscopy and EPR. These results suggest that experiments using DFO can be confounded by concomitant production of NO, and offer new insight into some of DFO's unexplained clinical side effects such as hypotension.

Nitric oxide metabolism in the human placenta during aberrant maternal inflammation.

KEY POINTS: Nitric oxide (NO) is a gasotransmitter with important physiological and pathophysiological roles in pregnancy. There is limited information available about the sources and metabolism of NO and its bioactive metabolites (NOx) in both normal and complicated pregnancies. The present study characterized and quantified endogenous NOx in human and mouse placenta following determination of the stability of exogenous NOx in placental homogenates. NOx have differential stability in placental homogenates. NO and iron nitrosyl species (FeNOs), are relatively unstable in placental homogenates from normal placentas. Exogenous NO, nitrite and nitrosothiols react with placental homogenates to form iron nitrosyl complexes. FeNOs were also detected endogenously in mouse and human placenta. NOx levels in placental villous tissue are increased in fetal growth restriction vs. placentas from women with normal pregnancies, particularly in fetal growth restriction associated with pre-eclampsia. Villitis was not associated, however, with an increase in NOx levels in either normotensive or pre-eclamptic placentas. The results call for further investigation of FeNOs in normal and complicated pregnancies. ABSTRACT: Nitric oxide (NO) is a gasotransmitter with important roles in pregnancy under both physiological and pathophysiological conditions. Although products of NO metabolism (NOx) also have significant bioactivity, little is known about the role of NO and NOx under conditions of aberrant placental inflammation during pregnancy. An ozone-based chemiluminescence approach was used to investigate the stability and metabolic fate of NOx in human placental homogenates from uncomplicated pregnancies in healthy mothers compared to that in placental tissue from normotensive and pre-eclamptic pregnancies complicated with fetal growth restriction (FGR) with and without villitis of unknown aetiology. We hypothesized that placental NOx would be increased in FGR vs. normal tissue, and be further increased in villitis vs. non-villitis placentas. Findings indicate that nitrate, nitrite and nitrosothiols, but not NO or iron nitrosyl species (FeNOs), are relatively stable in placental homogenates from normal placentas, and that NO, nitrite and nitrosothiols react with placental homogenates to form iron nitrosyl complexes. Furthermore, NOx levels in placental villous tissue are increased in FGR vs. placentas from women with normal pregnancies, particularly in FGR associated with pre-eclampsia. However, in contrast to our hypothesis, villitis was not associated with an increase in NOx levels in either normotensive or pre-eclamptic placentas. Our results also strongly support the involvement of FeNOs in both mouse and human placenta, and call for their further study as a critical mechanistic link between pre-eclampsia and fetal growth restriction.

L-NAME releases nitric oxide and potentiates subsequent nitroglycerin-mediated vasodilation.

L-NG-Nitro arginine methyl ester (L-NAME) has been widely applied for several decades in both basic and clinical research as an antagonist of nitric oxide synthase (NOS). Herein, we show that L-NAME slowly releases NO from its guanidino nitro group. Daily pretreatment of rats with L-NAME potentiated mesenteric vasodilation induced by nitrodilators such as nitroglycerin, but not by NO. Release of NO also occurred with the NOS-inactive enantiomer D-NAME, but not with L-arginine or another NOS inhibitor L-NMMA, consistent with the presence or absence of a nitro group in their structure and their nitrodilator-potentiating effects. Metabolic conversion of the nitro group to NO-related breakdown products was confirmed using isotopically-labeled L-NAME. Consistent with Fenton chemistry, transition metals and reactive oxygen species accelerated the release of NO from L-NAME. Both NO production from L-NAME and its nitrodilator-potentiating effects were augmented under inflammation. NO release by L-NAME can confound its intended NOS-inhibiting effects, possibly by contributing to a putative intracellular NO store in the vasculature.

Detection of dinitrosyl iron complexes by ozone-based chemiluminescence.

Dinitrosyl iron complexes (DNICs) are important intermediates in the metabolism of nitric oxide (NO). They have been considered to be NO storage adducts able to release NO, scavengers of excess NO during inflammatory hypotensive shock, and mediators of apoptosis in cancer cells, among many other functions. Currently, all studies of DNICs in biological matrices use electron paramagnetic resonance (EPR) for both detection and quantification. EPR is limited, however, by its ability to detect only paramagnetic mononuclear DNICs even though EPR-silent binuclear are likely to be prevalent. Furthermore, physiological concentrations of mononuclear DNICs are usually lower than the EPR detection limit (1 µM). We have thus developed a chemiluminescence-based method for the selective detection of both DNIC forms at physiological, pathophysiological, and pharmacologic conditions. We have also demonstrated the use of the new method in detecting DNIC formation in the presence of nitrite and nitrosothiols within biological fluids and tissue. This new method, which can be used alone or in tandem with EPR, has the potential to offer insight about the involvement of DNICs in many NO-dependent pathways.

Inhaled Fasudil Lacks Pulmonary Selectivity in Thromboxane-Induced Acute Pulmonary Hypertension in Newborn Lambs.

INTRODUCTION: Pulmonary hypertension (PH) is a potentially deadly disease for infants and adults with few existing medical interventions and no cure. In PH, increased blood pressure in the pulmonary artery eventually leads to heart failure. Fasudil, an antagonist of Rho-kinase, causes vasodilation leading to decreased systemic artery pressure and pulmonary artery pressure (PAP). This study compared the effects of fasudil administered as either an intravenous infusion or inhaled aerosol in newborn lambs. HYPOTHESIS: Inhaled aerosol delivery of fasudil will provide selective pulmonary vasodilation when compared with intravenous administration. METHODS: Newborn lambs (∼11 days) were surgically instrumented and mechanically ventilated under anesthesia. A pulmonary artery catheter and ultrasonic flow probe were inserted to measure hemodynamics. Acute PH was pharmaceutically induced via continuous intravenous infusion of thromboxane. After achieving a 2- to 3-fold elevation of PAP, fasudil was administered either as intravenous infusion (2.5 mg/kg) or inhaled aerosol (100 mg of fasudil in 2 mL of saline). Changes in PAP, mean systemic arterial pressure (MABP), pulmonary vascular resistance (PVR), systemic vascular resistance (SVR), cardiac output, and heart rate were assessed. In addition, plasma concentrations of fasudil were measured. RESULTS: Both routes of fasudil delivery produced significant decreases in PAP and PVR but also produced similar decreases in MABP and SVR. The Cmax for intravenous fasudil was greater than that for inhaled fasudil. CONCLUSIONS: These results suggest inhaled fasudil lacks pulmonary selectivity when compared with intravenous fasudil.

Hemodynamic Effects of Glutathione-Liganded Binuclear Dinitrosyl Iron Complex: Evidence for Nitroxyl Generation and Modulation by Plasma Albumin.

Glutathione-liganded binuclear dinitrosyl iron complex (glut-BDNIC) has been proposed to be a donor of nitric oxide (NO). This study was undertaken to investigate the mechanisms of vasoactivity, systemic hemodynamic effects, and pharmacokinetics of glut-BDNIC. To test the hypothesis that glut-BDNICs vasodilate by releasing NO in its reduced [nitroxyl (HNO)] state, a bioassay method of isolated, preconstricted ovine mesenteric arterial rings was used in the presence of selective scavengers of HNO or NO free radical (NO•); the vasodilatory effects of glut-BDNIC were found to have characteristics similar to those of an HNO donor and markedly different than an NO• donor. In addition, products of the reaction of glut-BDNIC with CPTIO [2-(4-carboxyphenyl)-4,4,5-tetramethyl imidazoline-1-oxyl-3-oxide] were found to have electron paramagnetic characteristics similar to those of an HNO donor compared with an NO• donor. In contrast to S-nitroso-glutathione, which was vasodilative both in vitro and in vivo, the potency of glut-BDNIC-mediated vasodilation was markedly diminished in both rats and sheep. Wire myography showed that plasma albumin contributed to this loss of hypotensive effects, an effect abolished by modification of the cysteine-thiol residue of albumin. High doses of glut-BDNIC caused long-lasting hypotension in rats that can be at least partially attributed to its long circulating half-life of ∼44 minutes. This study suggests that glut-BDNIC is an HNO donor, and that its vasoactive effects are modulated by binding to the cysteine residue of plasma proteins, such as albumin.

Nitrite potentiates the vasodilatory signaling of S-nitrosothiols.

Nitrite and S-nitrosothiols (SNOs) are both byproducts of nitric oxide (NO) metabolism and are proposed to cause vasodilation via activation of soluble guanylate cyclase (sGC). We have previously reported that while SNOs are potent vasodilators at physiological concentrations, nitrite itself only produces vasodilation at supraphysiological concentrations. Here, we tested the hypothesis that sub-vasoactive concentrations of nitrite potentiate the vasodilatory effects of SNOs. Multiple exposures of isolated sheep arteries to S-nitroso-glutathione (GSNO) resulted in a tachyphylactic decreased vasodilatory response to GSNO but not to NO, suggesting attenuation of signaling steps upstream from sGC. Exposure of arteries to 1 µM nitrite potentiated the vasodilatory effects of GSNO in naive arteries and abrogated the tachyphylactic response to GSNO in pre-exposed arteries, suggesting that nitrite facilitates GSNO-mediated activation of sGC. In intact anesthetized sheep and rats, inhibition of NO synthases to decrease plasma nitrite levels attenuated vasodilatory responses to exogenous infusions of GSNO, an effect that was reversed by exogenous infusion of nitrite at sub-vasodilating levels. This study suggests nitrite potentiates SNO-mediated vasodilation via a mechanism that lies upstream from activation of sGC.

S-nitrosothiols dilate the mesenteric artery more potently than the femoral artery by a cGMP and L-type calcium channel-dependent mechanism.

S-nitrosothiols (SNOs) are metabolites of NO with potent vasodilatory activity. Our previous studies in sheep indicated that intra-arterially infused SNOs dilate the mesenteric vasculature more than the femoral vasculature. We hypothesized that the mesenteric artery is more responsive to SNO-mediated vasodilation, and investigated various steps along the NO/cGMP pathway to determine the mechanism for this difference. In anesthetized adult sheep, we monitored the conductance of mesenteric and femoral arteries during infusion of S-nitroso-l-cysteine (L-cysNO), and found mesenteric vascular conductance increased (137 ± 3%) significantly more than femoral conductance (26 ± 25%). Similar results were found in wire myography studies of isolated sheep mesenteric and femoral arteries. Vasodilation by SNOs was attenuated in both vessel types by the presence of ODQ (sGC inhibitor), and both YC-1 (sGC agonist) and 8-Br-cGMP (cGMP analog) mediated more potent relaxation in mesenteric arteries than femoral arteries. The vasodilatory difference between mesenteric and femoral arteries was eliminated by antagonists of either protein kinase G or L-type Ca(2+) channels. Western immunoblots showed a larger L-type Ca(2+)/sGC abundance ratio in mesenteric arteries than in femoral arteries. Fetal sheep mesenteric arteries were more responsive to SNOs than adult mesenteric arteries, and had a greater L-Ca(2+)/sGC ratio (p = 0.047 and r = -0.906 for correlation between Emax and L-Ca(2+)/sGC). These results suggest that mesenteric arteries, especially those in fetus, are more responsive to SNO-mediated vasodilation than femoral arteries due to a greater role of the L-type calcium channel in the NO/cGMP pathway.

Postprandial lipids accelerate and redirect nitric oxide consumption in plasma.

Nitric oxide (NO) and O2 are both three-to four-fold more soluble in biological lipids than in aqueous solutions. Their higher concentration within plasma lipids accelerates NO autoxidation to an extent that may be of importance to overall NO bioactivity. This study was undertaken to test the hypothesis that increased plasma lipids after a high-fat meal appreciably accelerate NO metabolism and alter the byproducts formed. We found that plasma collected from subjects after consumption of a single high-fat meal had a higher capacity for NO consumption and consumed NO more rapidly compared to fasting plasma. This increased NO consumption showed a direct correlation with plasma triglyceride concentrations (p = 0.006). The accelerated NO consumption in postprandial plasma was reversed by removal of the lipids from the plasma, was mimicked by the addition of hydrophobic micelles to aqueous buffer, and could not be explained by the presence of either free hemoglobin or ceruloplasmin. The products of NO consumption were shifted in postprandial plasma, with 55% more nitrite (n = 12, p = 0.002) but 50% less SNO (n = 12, p = 0.03) production compared to matched fasted plasma. Modeling calculations indicated that NO autoxidation was accelerated by about 48-fold in the presence of plasma lipids. We conclude that postprandial triglyceride-rich lipoproteins exert a significant influence on NO metabolism in plasma.

Fetal-maternal nitrite exchange in sheep: Experimental data, a computational model and an estimate of placental nitrite permeability.

INTRODUCTION: Nitrite conveys NO-bioactivity that may contribute to the high-flow, low-resistance character of the fetal circulation. Fetal blood nitrite concentrations depend partly on placental permeability which has not been determined experimentally. We aimed to extract the placental permeability-surface (PS) product for nitrite in sheep from a computational model. METHODS: An eight-compartment computational model of the fetal-maternal unit was constructed (Matlab(®) (R2013b (8.2.0.701), MathWorks Inc., Natick, MA). Taking into account fetal and maternal body weights, four variables (PS, the rate of nitrite metabolism within red cells, and two nitrite distribution volumes, one with and one without nitrite metabolism), were varied to obtain optimal fits to the experimental plasma nitrite profiles observed following the infusion of nitrite into either the fetus (n = 7) or the ewe (n = 8). RESULTS: The model was able to replicate the average and individual nitrite-time profiles (r(2) > 0.93) following both fetal and maternal nitrite infusions with reasonable variation of the four fitting parameters. Simulated transplacental nitrite fluxes were able to predict umbilical arterial-venous nitrite concentration differences that agreed with experimental values. The predicted PS values for a 3 kg sheep fetus were 0.024 ± 0.005 l∙min(-1) in the fetal-maternal direction and 0.025 ± 0.003 l∙min(-1) in the maternal-fetal direction (mean ± SEM). These values are many-fold higher than the reported PS product for chloride anions across the sheep placenta. CONCLUSION: The result suggests a transfer of nitrite across the sheep placenta that is not exclusively by simple diffusion through water-filled channels.

Changes in plasma and urinary nitrite after birth in premature infants at risk for necrotizing enterocolitis.

BACKGROUND: Plasma nitrite serves as a reservoir of nitric oxide (NO) bioactivity. Because nitrite ingestion is markedly lower in newborns than adults, we hypothesized plasma nitrite levels would be lower in newborns than in adults, and that infants diagnosed with necrotizing enterocolitis (NEC), a disease characterized by ischemia and bacterial invasion of intestinal walls, would have lower levels of circulating nitrite in the days prior to diagnosis. METHODS: Single blood and urine samples were collected from 9 term infants and 12 adults, 72 preterm infants every 5 d for 3 wk, and from 13 lambs before and after cord occlusion. RESULTS: Nitrite fell 50% relative to cord levels in the first day after birth; and within 15 min after cord occlusion in lambs. Urinary nitrite was higher in infants than adults. Plasma and urinary nitrite levels in infants who developed NEC were similar to those of preterm control infants on days 1 and 5, but significantly elevated at 15 and 20 d after birth. CONCLUSION: Plasma nitrite falls dramatically at birth while newborn urinary nitrite levels are significantly greater than adults. Acute NEC is associated with elevated plasma and urinary nitrite levels.

Local and systemic vasodilatory effects of low molecular weight S-nitrosothiols.

S-nitrosothiols (SNOs) such as S-nitroso-L-cysteine (L-cysNO) are endogenous compounds with potent vasodilatory activity. During circulation in the blood, the NO moiety can be exchanged among various thiol-containing compounds by S-transnitrosylation, resulting in SNOs with differing capacities to enter the cell (membrane permeability). To determine whether the vasodilating potency of SNOs is dependent upon membrane permeability, membrane-permeable L-cysNO and impermeable S-nitroso-D-cysteine (D-cysNO) and S-nitroso-glutathione (GSNO) were infused into one femoral artery of anesthetized adult sheep while measuring bilateral femoral and systemic vascular conductances. L-cysNO induced vasodilation in the infused hind limb, whereas D-cysNO and GSNO did not. L-cysNO also increased intracellular NO in isolated arterial smooth muscle cells, whereas GSNO did not. The infused SNOs remained predominantly in a low molecular weight form during first-passage through the hind limb vasculature, but were converted into high molecular weight SNOs upon systemic recirculation. At systemic concentrations of ~0.6 µmol/L, all three SNOs reduced mean arterial blood pressure by ~50%, with pronounced vasodilation in the mesenteric bed. Pharmacokinetics of L-cysNO and GSNO were measured in vitro and in vivo and correlated with their hemodynamic effects, membrane permeability, and S-transnitrosylation. These results suggest local vasodilation by SNOs in the hind limb requires membrane permeation, whereas systemic vasodilation does not. The systemic hemodynamic effects of SNOs occur after equilibration of the NO moiety amongst the plasma thiols via S-transnitrosylation.

Dietary intake and bio-activation of nitrite and nitrate in newborn infants.

Nitrate and nitrite are commonly thought of as inert end products of nitric oxide (NO) oxidation, possibly carcinogenic food additives, or well-water contaminants. However, recent studies have shown that nitrate and nitrite play an important role in cardiovascular and gastrointestinal homeostasis through conversion back into NO via a physiological system involving enterosalivary recirculation, bacterial nitrate reductases, and enzyme-catalyzed or acidic reduction of nitrite to NO. The diet is a key source of nitrate in adults; however, infants ingest significantly less nitrate due to low concentrations in breast milk. In the mouth, bacteria convert nitrate to nitrite, which has gastro-protective effects. However, these nitrate-reducing bacteria are relatively inactive in infants. Swallowed nitrite is reduced to NO by acid in the stomach, affecting gastric blood flow, mucus production, and the gastric microbiota. These effects are likely attenuated in the less acidic neonatal stomach. Systemically, nitrite acts as a reservoir of NO bioactivity that can protect against ischemic injury, yet plasma nitrite concentrations are markedly lower in infants than in adults. The physiological importance of the diminished nitrate→nitrite→NO axis in infants and its implications in the etiology and treatment of newborn diseases such as necrotizing enterocolitis and hypoxic/ischemic injury are yet to be determined.

Role of blood and vascular smooth muscle in the vasoactivity of nitrite.

Recent evidence from humans and rats indicates that nitrite is a vasodilator under hypoxic conditions by reacting with metal-containing proteins to produce nitric oxide (NO). We tested the hypothesis that near-physiological concentrations of nitrite would produce vasodilation in a hypoxia- and concentration-dependent manner in the hind limb of sheep. Anesthetized sheep were instrumented to measure arterial blood pressure and femoral blood flows continuously in both hind limbs. Nitrite was infused into one femoral artery to raise the nitrite concentration in the femoral vein by 10 to 15-fold while the sheep breathed 50%, 14% or 12% oxygen in inspired air. In contrast to reports in humans and rats, the nitrite infusion had no measurable effect on mean femoral blood flows or vascular conductances, regardless of inspired O2 levels. In vitro experiments showed no significant difference in the release of NO from nitrite in sheep and human red blood cells. Further experiments demonstrated nitrite is converted to NO in rat artery homogenates faster than sheep arteries, and that this source of NO production is attenuated in the presence of a heme oxidizer. Finally, western blots indicate that concentrations of the heme-containing protein cytoglobin, but not myoglobin, are markedly lower in sheep arteries compared with rats. Overall, the results demonstrate that nitrite is not a physiological vasodilator in sheep. This is likely due to a lack of conversion of nitrite to NO within the vascular smooth muscle, perhaps due to deficient amounts of the heme-containing protein cytoglobin.