Christian J. Hunter

Nitrite Infusion in Humans and Nonhuman Primates Endocrine Effects, Pharmacokinetics, and Tolerance Formation

Background— The recent discovery that nitrite is an intrinsic vasodilator and signaling molecule at near-physiological concentrations has raised the possibility that nitrite contributes to hypoxic vasodilation and to the bioactivity of nitroglycerin and mediates the cardiovascular protective effects of nitrate in the Mediterranean diet. However, important questions of potency, kinetics, mechanism of action, and possible induction of tolerance remain unanswered. Methods and Results— In the present study, we performed biochemical, physiological, and pharmacological studies using nitrite infusion protocols in 20 normal human volunteers and in nonhuman primates to answer these questions, and we specifically tested 3 proposed mechanisms of bioactivation: reduction to nitric oxide by xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin. We found that (1) nitrite is a relatively potent and fast vasodilator at near-physiological concentrations; (2) nitrite functions as an endocrine reservoir of nitric oxide, producing remote vasodilation during first-pass perfusion of the opposite limb; (3) nitrite is reduced to nitric oxide by intravascular reactions with hemoglobin and with intravascular reductants (ie, ascorbate); (4) inhibition of xanthine oxidoreductase with oxypurinol does not inhibit nitrite-dependent vasodilation but potentiates it; and (5) nitrite does not induce tolerance as observed with the organic nitrates. Conclusions— We propose that nitrite functions as a physiological regulator of vascular function and endocrine nitric oxide homeostasis and suggest that it is an active metabolite of the organic nitrates that can be used therapeutically to bypass enzymatic tolerance.

Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator

The blood anion nitrite contributes to hypoxic vasodilation through a heme-based, nitric oxide (NO)–generating reaction with deoxyhemoglobin and potentially other heme proteins. We hypothesized that this biochemical reaction could be harnessed for the treatment of neonatal pulmonary hypertension, an NO-deficient state characterized by pulmonary vasoconstriction, right-to-left shunt pathophysiology and systemic hypoxemia. To test this, we delivered inhaled sodium nitrite by aerosol to newborn lambs with hypoxic and normoxic pulmonary hypertension. Inhaled nitrite elicited a rapid and sustained reduction (∼65%) in hypoxia-induced pulmonary hypertension, with a magnitude approaching that of the effects of 20 p.p.m. NO gas inhalation. This reduction was associated with the immediate appearance of NO in expiratory gas. Pulmonary vasodilation elicited by aerosolized nitrite was deoxyhemoglobin- and pH-dependent and was associated with increased blood levels of iron-nitrosyl-hemoglobin. Notably, from a therapeutic standpoint, short-term delivery of nitrite dissolved in saline through nebulization produced selective, sustained pulmonary vasodilation with no clinically significant increase in blood methemoglobin levels. These data support the concept that nitrite is a vasodilator acting through conversion to NO, a process coupled to hemoglobin deoxygenation and protonation, and evince a new, simple and inexpensive potential therapy for neonatal pulmonary hypertension.

Sildenafil therapy in patients with sickle cell disease and pulmonary hypertension

Pulmonary hypertension is a frequent complication of sickle cell disease that is associated with haemolysis, impaired nitric oxide bioavailability and high mortality. We sought to evaluate the safety and efficacy of selective pulmonary vasodilators and antiproliferative agents in this at‐risk population. After optimising sickle cell disease therapy to stabilise haemoglobin and fetal haemoglobin levels, we evaluated the safety and efficacy of sildenafil in 12 patients with sickle cell disease and pulmonary hypertension. Sildenafil therapy (mean duration 6 ± 1 months) decreased the estimated pulmonary artery systolic pressure [50 ± 4 to 41 ± 3 mmHg; difference 9 mmHg, 95% confidence interval (CI): 0·3–17, P = 0·043] and increased the 6‐min walk distance (384 ± 30 to 462 ± 28 m; difference 78 m, 95% CI: 40–117, P = 0·0012). Transient headaches occurred in two patients and transient eye‐lid oedema in four patients. No episodes of priapism occurred in the three men in the study; two of them were on chronic exchange transfusions and one had erectile dysfunction. In conclusion: (1) sickle cell disease patients with anaemia and pulmonary hypertension have significant exercise limitation; (2) the 6‐min walk distance may be a valid endpoint in this population; (3) therapy with sildenafil appears safe and improves pulmonary hypertension and exercise capacity. Additional phase I studies in males with sickle cell disease followed by phase II/III placebo controlled trials evaluating the safety and efficacy of sildenafil therapy in sickle cell disease patients with pulmonary hypertension are warranted.

Key Neuroprotective Role for Endogenous Adenosine A1 Receptor Activation During Asphyxia in the Fetal Sheep

BACKGROUND AND PURPOSE The fetus is well known to be able to survive prolonged exposure to asphyxia with minimal injury compared with older animals. We and others have observed a rapid suppression of EEG intensity with the onset of asphyxia, suggesting active inhibition that may be a major neuroprotective adaptation to asphyxia. Adenosine is a key regulator of cerebral metabolism in the fetus. METHODS We therefore tested the hypothesis that infusion of the specific adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), given before 10 minutes of profound asphyxia in near-term fetal sheep, would prevent neural inhibition and lead to increased brain damage. RESULTS DPCPX treatment was associated with a transient rise and delayed fall in EEG activity in response to cord occlusion (n=8) in contrast with a rapid and sustained suppression of EEG activity in controls (n=8). DPCPX was also associated with an earlier and greater increase in cortical impedance, reflecting earlier onset of primary cytotoxic edema, and a significantly smaller reduction in calculated cortical heat production after the start of cord occlusion. After reperfusion, DPCPX-treated fetuses but not controls developed delayed onset of seizures, which continued for 24 hours, and sustained greater selective hippocampal, striatal, and parasagittal neuronal loss after 72-hour recovery. CONCLUSIONS These data support the hypothesis that endogenous activation of the adenosine A1 receptor during severe asphyxia mediates the initial suppression of neural activity and is an important mechanism that protects the fetal brain.

Fetal lamb cerebral blood flow (CBF) and oxygen tensions during hypoxia: a comparison of laser Doppler and microsphere measurements of CBF

This study was undertaken to compare microsphere and laser Doppler flowmetry techniques for the measurement of cerebral blood flow, to assess the effect of probe implantation at the tip of the sensing probe and to measure brain tissue PO2 (tPO2) in response to acute hypoxia. Fetal sheep of ≈131 days gestation (n= 8) were chronically instrumented with bilateral laser Doppler probes in the parietal cortices and catheters for injection of fluorescent microspheres. Five days after surgery fetuses were subjected to 1 h periods of baseline control breathing, hypoxia and recovery. Microspheres were injected 10 min prior to and 10, 30, 50 and 120 min after initiation of hypoxia. Microspheres were counted in four 12 mm3 tissue samples from each hemisphere, the tip of the laser Doppler probe being positioned in the centre of one of the cubes. The cube containing the probe tip was also subdivided into 4 mm3 pieces of tissue. In response to hypoxia, fetal arterial PO2 declined from 21 ± 2 to 12 ± 1 Torr and brain tissue PO2 fell from 10 ± 1 to a nadir of 1 ± 1 Torr. Each method detected a significant increase in CBF that reached a maximum after 30‐45 min, although the increase of flow measured by laser Doppler flowmetry was less than that measured by spheres after 10 and 30 min (P < 0.05). Microspheres did not detect altered flow at the probe tip or heterogeneity of flow in surrounding volumes of cortical tissue. In summary, laser Doppler flowmetry is a useful measure of continuous relative changes of CBF in the chronically instrumented fetal sheep. Flow compensations in acute hypoxia are not adequate to sustain O2 delivery, and other compensations, including reduced metabolic rate, are possible.

Role of Nitric Oxide in Hypoxic Cerebral Vasodilatation in the Ovine Fetus

To investigate the role of nitric oxide (NO) in fetal cerebral circulatory responses to acute hypoxia, near‐term fetal sheep were instrumented with laser Doppler probes placed in the parasagittal parietal cortices and vascular catheters in the sagittal sinus and brachiocephalic artery. After a 3 day recovery period, responses of cortical blood flow (CBF) to hypoxia were compared with and without inhibition of nitric oxide synthase (NOS). After an initial 30 min baseline period, fetuses were given a bolus followed by a continuous infusion of Nω‐nitro‐l‐arginine methyl ester (l‐NAME), or saline vehicle as control. After administration of l‐NAME, CBF decreased by 14 ± 6 % (P < 0.01) despite increases in arterial blood pressure of 15 mmHg, resulting in an ∼60 % increase in cerebrovascular resistance. Thirty minutes following initiation of l‐NAME or vehicle infusion, fetal systemic hypoxia was induced by allowing the ewes to breathe 10–11 % oxygen. In control fetuses CBF increased progressively to 145 ± 9 % of baseline (P < 0.01) after 30 min, while cortical release of cyclic guanylate cyclase (cGMP), an index of NOS activity, increased 26 ± 8 % (P < 0.05). In contrast, CBF in l‐NAME‐treated fetuses increased to only 115 % of the reduced CBF baseline, whereas cortical release of cGMP did not change significantly. In summary, basal levels of NO lower resting cortical vascular resistance by ∼15 % in the fetal sheep. Inhibition of NO synthesis attenuates hypoxic cerebral relaxation but does not completely prevent the characteristic increases in CBF. Hypoxic increases in NO directly increase cortical production of cGMP and inhibition of NO synthesis ablates these changes in cGMP.

Post‐hypoxic hypoperfusion is associated with suppression of cerebral metabolism and increased tissue oxygenation in near‐term fetal sheep

Secondary cerebral hypoperfusion is common following perinatal hypoxia–ischaemia. However, it remains unclear whether this represents a true failure to provide sufficient oxygen and nutrients to tissues, or whether it is simply a consequence of reduced cerebral metabolic demand. We therefore examined the hypothesis that cerebral oxygenation would be reduced during hypoperfusion after severe asphyxia, and further, that the greater neural injury associated with blockade of the adenosine A1 receptor during the insult would be associated with greater hypoperfusion and deoxygenation. Sixteen near‐term fetal sheep received either vehicle or 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) for 1 h, followed by 10 min of severe asphyxia induced by complete occlusion of the umbilical cord. Infusions were discontinued at the end of the occlusion and data were analysed for the following 8 h. A transient, secondary fall in carotid artery blood flow and laser Doppler flow was seen from approximately 1–4 h after occlusion (P < 0.001), with no significant differences between vehicle and DPCPX. Changes in laser Doppler blood flow were highly correlated with carotid blood flow (r2= 0.81, P < 0.001). Cortical metabolism was suppressed, reaching a nadir 1 h after occlusion and then resolving. Cortical tissue P  O 2 was significantly increased at 1, 2 and 3 h after occlusion compared to baseline, and inversely correlated with carotid blood flow (r2= 0.69, P < 0.001). In conclusion, contrary to our initial hypothesis, delayed posthypoxic hypoperfusion was associated with suppression of cerebral metabolism and increased tissue P  O 2, and was not significantly affected by preceding adenosine A1 blockade. These data suggest that posthypoxic hypoperfusion is actively mediated and reflects suppressed cerebral metabolism.

Cerebral Oxygenation during Postasphyxial Seizures in Near-Term Fetal Sheep

After exposure to asphyxia, infants may develop both prolonged, clinically evident seizures and shorter, clinically silent seizures; however, their effect on cerebral tissue oxygenation is unclear. We therefore examined the hypothesis that the increase in oxygen delivery during postasphyxial seizures might be insufficient to meet the needs of increased metabolism, thus causing a fall in tissue oxygenation, in unanesthetized near-term fetal sheep in utero (gestational age 125 ± 1 days). Fetuses were administered an infusion of the specific adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, followed by 10 mins of asphyxia induced by complete umbilical cord occlusion. The fetuses then recovered for 3 days. Sixty-one episodes of electrophysiologically defined seizures were identified in five fetuses. Tissue PO2 (tPO2) did not change significantly during short seizures (<3.5 mins), 5.2 ± 0.2 versus baseline 5.6 ± 0.1 mm Hg (NS), but fell to 2.2 ± 0.2 mm Hg during seizures lasting more than 3.5 mins (P<0.001). During prolonged seizures, cortical blood flow did not begin to increase until tPO2 had begun to fall, and then rose more slowly than the increase in metabolism, with a widening of the brain to blood temperature gradient. In conclusion, in the immature brain, during prolonged, but not short seizures, there is a transient mismatch between cerebral blood flow and metabolism leading to significant cerebral deoxygenation.

Cerebral Metabolism during Cord Occlusion and Hypoxia in the Fetal Sheep: A Novel Method of Continuous Measurement Based on Heat Production

This study was undertaken to validate a new method of measuring cerebral metabolic rate in the fetal sheep based on heat production in a local region of the brain. Heat production was compared to oxygen use in 20 near‐term fetuses during basal conditions, moderate hypoxia and cord occlusion. Thermocouples were placed to measure core and brain temperature and a composite probe placed in the parietal cortex to measure changes in cortical blood flow (CBF) using laser Doppler flowmetry and tissue PO2 using fluorescent decay. Catheters were inserted in a brachiocephalic artery and sagittal sinus for blood sampling. With moderate hypoxia, induced by administering 10−12 % oxygen to the ewes, fetal arterial PO2 declined from 23 ± 1 to 11 ± 1 Torr and brain tissue PO2 fell from 7.6± 0.7 to a nadir of 0.8 ± 0.4 Torr, while CBF increased to 139 ± 5 % of baseline. Cortical heat production, calculated as the product of CBF, the temperature gain from artery to brain tissue, and the specific heat of blood, decreased by 45 ± 11 % in parallel to similar declines in oxygen uptake. With severe asphyxia induced by complete cord occlusion for 10 min, fetal arterial PO2 declined from 23 ± 1 to 9 ± 2 Torr and brain tissue PO2 fell from 7.0 ± 0.7 to essentially 0 Torr while CBF decreased 40 ± 5 %. Cortical heat production decreased by 78 ± 6 % while oxygen use declined by 90 ± 3 %. Glucose uptake increased significantly relative to oxygen use and lactate concentration increased in sagittal sinus blood. We conclude that local measurements of heat production in the brain provide a useful index of overall metabolic rate, closely reflecting oxygen use in moderate hypoxia and indicating a significant contribution from anaerobic metabolism during severe asphyxia.

Effect of Mild Hypothermia and Hypoxia on Blood Flow and Oxygen Consumption of the Fetal Sheep Brain

This study was undertaken to measure the effects of mild hypothermia on cerebral blood flow and metabolism and cardiovascular responses to hypoxia in the fetal sheep. Near-term fetal sheep were chronically instrumented with laser Doppler flowmetry in the parietal cortex for measurement of relative changes in cerebral blood flow, as well as with arterial and sagittal sinus catheters for measurement of oxygen extraction by the brain and a cooling coil around the fetal thorax. Fetuses were studied during cooling alone, cooling with superimposed maternal hypoxia to achieve a fetal arterial Po2 of 1.33 to 1.60 kPa, or hypoxia alone. In response to cooling alone [1.6° ± 0.1°C (mean ± SEM) decrease in brain temperature], fetal blood pressure and heart rate both increased significantly whereas cerebral blood flow decreased 14 ± 4%, commensurate with a 24 ± 8% decline in cerebral metabolic rate. Administration of moderate hypoxia during cooling resulted in a significant increase in cerebral blood flow, decreased heart rate, and no further increase in blood pressure. In response to hypoxia alone, fetal blood pressure was significantly increased, heart rate was decreased, and cerebral blood flow increased by 24 ± 8%, whereas cerebral metabolic rate decreased by 38 ± 13%. Arteriovenous oxygen extraction was unchanged by cooling alone but increased significantly in response to hypoxia administered during cooling. We therefore conclude that oxygen delivery to the fetal sheep brain remains coupled to metabolic rate during hypothermia and that hypothermia does not impair the compensatory cardiovascular responses of the fetus to acute moderate hypoxia.