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.

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.

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.

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.

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.

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.

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.

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 nitrite in regulation of fetal cephalic circulation in sheep.

Nitrite has been postulated to provide a reservoir for conversion to nitric oxide (NO), especially in tissues with reduced oxygen levels as in the fetus. Nitrite would thus provide local vasodilatation and restore a balance between oxygen supply and need, a putative mechanism of importance especially in the brain. The current experiments test the hypothesis that exogenous nitrite acts as a vasodilator in the cephalic vasculature of the intact, near term fetal sheep. Fetuses were first instrumented to measure arterial blood pressure and carotid artery blood flow and then studied 4-5 days later while in utero without anaesthesia. Initially l-nitro-arginine (LNNA) was given to block endogenous NO production. Carotid resistance to flow increased 2-fold from 0.54 ± 0.01 (SEM) to 1.20 ± 0.08 mmHg min ml(-1) (in 13 fetuses, P < 0.001), indicating NO tonically reduces cerebral vascular tone. Sodium nitrite (or saline as control) was then infused in increasing step-doses from 0.01 to 33 µm in half-log increments over a period of 2 h. Carotid artery pressure, blood flow and vascular resistance did not change compared to fetuses receiving saline, even at plasma nitrite concentrations two orders of magnitude above the physiological range. The results indicate that while cephalic vascular tone is controlled by endogenous nitric oxide synthase activity, exogenously administered nitrite is not a vasodilator at physiological concentrations in the vasculature served by the carotid artery of fetal sheep.

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.

Role of ceruloplasmin in nitric oxide metabolism in plasma of humans and sheep: a comparison of adults and fetuses.

Nitric oxide (NO) is metabolized in plasma, in part by the ferroxidase ceruloplasmin (Cp), to form nitrite and nitrosothiols (SNOs), which are proposed to mediate protective responses to hypoxia and ischemia. We hypothesized that NO metabolism would be attenuated in fetal plasma due to low Cp activity. We measured Cp concentrations and activity in plasma samples collected from adults and fetuses of humans and sheep. We then added NO ([NO]: 1.5 or 100 µM) to plasma and aqueous buffer and measured rates of NO disappearance and the production of nitrite and SNO. Cp concentrations in fetal plasma were <15% of adult levels. In aqueous buffer, 1.5 µM NO disappeared with a half-life of 347 ± 64 s (means ± SE) but in plasma of humans the half-life was 19 ± 2 s (adult) and 11 ± 1 s (fetus, P = 0.004) and in sheep it was 31 ± 3 s (adult) and 43 ± 5 s (fetus, P = 0.04). Cp activity was not correlated with the overall elimination half-life of NO or with the amount of SNO ([NO]: 100 µM) or nitrite ([NO]: 1.5 or 100 µM) produced but correlated with SNO yields at 1.5 µM [NO] (r = 0.92, P = 0.04). Our data demonstrate that Cp is not essential to the increased rate of metabolism of NO in plasma relative to aqueous buffers and that it is not essential to the production of nitrite from NO. Cp may be involved in the conversion of NO to SNO in plasma under near-physiological concentrations of NO.

Effect of chronic perinatal hypoxia on the role of rho-kinase in pulmonary artery contraction in newborn lambs.

Exposure to chronic hypoxia during gestation predisposes infants to neonatal pulmonary hypertension, but the underlying mechanisms remain unclear. Here, we test the hypothesis that moderate continuous hypoxia during gestation causes changes in the rho-kinase pathway that persist in the newborn period, altering vessel tone and responsiveness. Lambs kept at 3,801 m above sea level during gestation and the first 2 wk of life were compared with those with gestation at low altitude. In vitro studies of isolated pulmonary arterial rings found a more forceful contraction in response to KCl and 5-HT in high-altitude compared with low-altitude lambs. There was no difference between the effects of blockers of various pathways of extracellular Ca(2+) entry in low- and high-altitude arteries. In contrast, inhibition of rho-kinase resulted in significantly greater attenuation of 5-HT constriction in high-altitude compared with low-altitude arteries. High-altitude lambs had higher baseline pulmonary artery pressures and greater elevations in pulmonary artery pressure during 15 min of acute hypoxia compared with low-altitude lambs. Despite evidence for an increased role for rho-kinase in high-altitude arteries, in vivo studies found no significant difference between the effects of rho-kinase inhibition on hypoxic pulmonary vasoconstriction in intact high-altitude and low-altitude lambs. We conclude that chronic hypoxia in utero results in increased vasopressor response to both acute hypoxia and serotonin, but that rho-kinase is involved only in the increased response to serotonin.

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.

Inhaled nitrite reverses hemolysis-induced pulmonary vasoconstriction in newborn lambs without blood participation.

BACKGROUND: Nitrite can be converted to nitric oxide (NO) by a number of different biochemical pathways. In newborn lambs, an aerosol of inhaled nitrite has been found to reduce pulmonary blood pressure, possibly acting via conversion to NO by reaction with intraerythrocytic deoxyhemoglobin. If so, the vasodilating effects of nitrite would be attenuated by free hemoglobin in plasma that would rapidly scavenge NO. METHODS AND RESULTS: Pulmonary vascular pressures and resistances to flow were measured in anesthetized newborn lambs. Plasma hemoglobin concentrations were then elevated, resulting in marked pulmonary hypertension. This effect was attenuated if infused hemoglobin was first oxidized to methemoglobin, which does not scavenge NO. These results further implicate NO as a tonic pulmonary vasodilator. Next, while free hemoglobin continued to be infused, the lambs were given inhaled NO gas (20 ppm), inhaled sodium nitrite aerosol (0.87 mol/L), or an intravascular nitrite infusion (3 mg/h bolus, 5 mg · kg⁻¹ · h⁻¹ infusion). Inhaled NO and inhaled nitrite aerosol both resulted in pulmonary vasodilation. Intravascular infusion of nitrite, however, did not. Increases in exhaled NO gas were observed in lambs while breathing the nitrite aerosol (≈ 20 ppb NO) but not during intravascular infusion of nitrite. CONCLUSIONS: We conclude that the pulmonary vasodilating effect of inhaled nitrite results from its conversion to NO in airway and parenchymal lung tissue and is not dependent on reactions with deoxyhemoglobin in the pulmonary circulation. Inhaled nitrite aerosol remains a promising candidate to reduce pulmonary hypertension in clinical application.

Inhaled nitric oxide therapy increases blood nitrite, nitrate, and s-nitrosohemoglobin concentrations in infants with pulmonary hypertension.

OBJECTIVE: To measure the circulating concentrations of nitric oxide (NO) adducts with NO bioactivity after inhaled NO (iNO) therapy in infants with pulmonary hypertension. STUDY DESIGN: In this single center study, 5 sequential blood samples were collected from infants with pulmonary hypertension before, during, and after therapy with iNO (n = 17). Samples were collected from a control group of hospitalized infants without pulmonary hypertension (n = 16) and from healthy adults for comparison (n = 12). RESULTS: After beginning iNO (20 ppm) whole blood nitrite levels increased approximately two-fold within 2 hours (P<.01). Whole blood nitrate levels increased to 4-fold higher than baseline during treatment with 20 ppm iNO (P<.01). S-nitrosohemoglobin increased measurably after beginning iNO (P<.01), whereas iron nitrosyl hemoglobin and total hemoglobin-bound NO-species compounds did not change. CONCLUSION: Treatment of pulmonary hypertensive infants with iNO results in increases in levels of nitrite, nitrate, and S-nitrosohemoglobin in circulating blood. We speculate that these compounds may be carriers of NO bioactivity throughout the body and account for peripheral effects of iNO in the brain, heart, and other organs.

Nitrate reductase activity of bacteria in saliva of term and preterm infants.

The salivary glands of adults concentrate nitrate from plasma into saliva where it is converted to nitrite by bacterial nitrate reductases. Nitrite can play a beneficial role in adult gastrointestinal and cardiovascular physiology. When nitrite is swallowed, some of it is converted to nitric oxide (NO) in the stomach and may then exert protective effects in the gastrointestinal tract and throughout the body. It has yet to be determined either when newborn infants acquire oral nitrate reducing bacteria or what the effects of antimicrobial therapy or premature birth may be on the bacterial processing of nitrate to nitrite. We measured nitrate and nitrite levels in the saliva of adults and both preterm and term human infants in the early weeks of life. We also measured oral bacterial reductase activity in the saliva of both infants and adults, and characterized the species of nitrate reducing bacteria present. Oral bacterial conversion of nitrate to nitrite in infants was either undetectable or markedly lower than the conversion rates of adults. No measurable reductase activity was found in infants within the first two weeks of life, despite the presence of oral nitrate reducing bacteria such as Actinomyces odontolyticus, Veillonella atypica, and Rothia mucilaginosa. We conclude that relatively little nitrite reaches the infant gastrointestinal tract due to the lack of oral bacterial nitrate reductase activity. Given the importance of the nitrate-nitrite-NO axis in adults, the lack of oral nitrate-reducing bacteria in infants may be relevant to the vulnerability of newborns to hypoxic stress and gastrointestinal tract pathologies.

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.

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.

High fetal plasma adenosine concentration: a role for the fetus in preeclampsia?

OBJECTIVE: Clinical observations suggest a role for the fetus in the maternal manifestations of preeclampsia, but the possible signaling mechanisms remain unclear. This study compares the fetal plasma concentrations of adenosine from normal pregnancies with those from preeclampsia. STUDY DESIGN: This secondary data analysis included normal pregnancies (n = 27) and patients with preeclampsia (n = 39). Patients with preeclampsia were subclassified into patients with (n = 25) and without (n = 14) abnormal uterine artery Doppler velocimetry (UADV). RESULTS: Fetal plasma concentrations of adenosine were significantly higher in patients with preeclampsia (1.35 ± 0.09 µmol/L) than in normal pregnancies (0.52 ± 0.06 µmol/L; P < .0001). Fetal plasma concentrations of adenosine in patients with preeclampsia with abnormal UADV (1.78 ± 0.15 µmol/L), but not with normal UADV (0.58 ± 0.14 µmol/L), were significantly higher than in normal pregnancies (P < .0001). CONCLUSION: Patients with preeclampsia with sonographic evidence of chronic uteroplacental ischemia have high fetal plasma concentrations of adenosine.