Thomas N. Hansen

Oxidant stress responses in premature infants during exposure to hyperoxia.

ABSTRACT: To assess oxidant stress responses in newborn infants treated with elevated concentrations of oxygen, we measured plasma concentrations of glutathione (GSH) and glutathione disulfide (CSSG) in newborn infants ranging from 23 to 42 wk gestational age. All infants recruited into the study were mechanically ventilated and had catheters placed in their umbilical arteries as part of their normal clinical management. Blood samples were obtained on d 1, 3, and 5 and weekly thereafter or until the catheters were removed. We observed plasma concentrations of GSSG in these infants that were frequently an order of magnitude higher than the 0.1 to 0.3 μM we find in adults. Interestingly, plasma GSSG concentrations were inversely correlated to the inspired oxygen tensions. This effect appeared to arise from the patient selection criteria whereby, of the infants studied, those breathing the lowest partial pressures of oxygen were the smallest and gestationally youngest. A second observation was that plasma concentrations of GSH in the premature infants were substantially, indeed often dramatically, lower than we have observed in adult humans (6 to 10 μM). Finally, we found that in patients with both umbilical arterial and umbilical venous catheters arterial GSSG concentrations were consistently higher than venous concentrations; conversely, arterial GSH concentrations were lower than venous concentrations. The elevated GSSG concentrations we observed in these infants indicate marked oxidant stress responses in prematurely born infants, even in those infants exposed only to room air. The positive arteriovenous gradients of GSSG concentrations across the lungs of these infants suggest that at least some of the increased plasma GSSG originates in the lung. The low plasma GSH concentrations we observed in these same infants suggest deficiencies in an antioxidant that has been shown in numerous animal studies to be critical for prevention of hyperoxia-induced lung injury. Finally, the negative arteriovenous gradients of GSH concentrations across the lung provide the first evidence in humans for pulmonary uptake of GSH.

The pediatric pulmonary and cardiovascular complications of vertically transmitted human immunodeficiency virus (P2C2 HIV) infection study: Design and methods

The P2C2 HIV Study is a prospective natural history study initiated by the National Heart, Lung, and Blood Institute in order to describe the types and incidence of cardiovascular and pulmonary disorders that occur in children with vertically transmitted HIV infection (i.e., transmitted from mother to child in utero or perinatally). This article describes the study design and methods. Patients were recruited from five clinical centers in the United States. The cohort is composed of 205 infants and children enrolled after 28 days of age (Group I) and 612 fetuses and infants of HIV-infected mothers, enrolled prenatally (73%) or postnatally at age < 28 days (Group II). The maternal-to-infant transmission rate in Group II was 17%. The HIV-negative infants in Group II (Group IIb) serves as a control group for the HIV-infected children (Group IIa). The cohort is followed at specified intervals for clinical examination, cardiac, pulmonary, immunologic, and infectious studies and for intercurrent illnesses. In Group IIa, the cumulative loss-to-follow-up rate at 3 years was 10.5%, and the 3-year cumulative mortality rate was 24.9%. The findings will be relevant to clinical and epidemiologic aspects of HIV infection in children.

The effects of dopamine infusion on regional blood flow in newborn lambs

ABSTRACT: The purpose of this project was to investigate the effects of high rates of dopamine infusion on cardiac output and regional blood flow in the Iamb. We studied eight unanesthetized newborn lambs (mean age 7 ± 2 days) during a 15-inin baseline period and while infusing dopamine at 5-, 20-, 80-, and 160 μg/kg/min. We measured cardiac output and mean aortic, pulmonary arterial and left atrial pressures, and organ blood flow using radionuclidelabeled microspheres at each rate of dopamine infusion. Cardiac output increased significantly with increasing rates of infusion up to 80 μg/kg/min but decreased at 160 μg/ kg/ min. Aortic, pulmonary arterial, and left atrial pressures increased at rates of infusion above 5 μg/ kg/ min. Blood flow to all organs was unchanged at the 5 μg/ kg/ min rate of infusion of dopamine while blood flow to the brain and heart increased at the 80 μg/ kg/ min rate of infusion and blood flow to the gut and kidney decreased. We conclude that dopamine is an effective inotropic agent in the newborn lamb but that an inotropic:afterload mismatch exists at high infusion rates. Despite an increase in cardiac output at low rates of infusion, dopamine did not selectively vasodilate the vascular bed of any organs tested. Furthermore, at high rates of infusion dopamine actually impaired blood flow to the gut and kidney.

Reversal of Venous Blood Flow with Atrial Tachycardia and Hydrops in Fetal Sheep

ABSTRACT: The purpose of this project was to characterize the reversal of blood flow in the proximal inferior vena cava (IVC) seen in fetal sheep with pacing-induced atrial tachycardia and hydrops. We successfully operated on seven pregnant ewes at 118–130 d gestation to attach ECG and pacing wires, insert vascular catheters, and place Doppler flow probes around the common aortic trunk and the IVC. We also performed two-dimensional and Doppler ultrasonographic exams at baseline, after initiation of pacing, and daily thereafter. All fetuses developed hydrops. Ultrasonographic appearance of ascites and pleural effusion occurred within 4 h in four fetuses and within 48 h in all fetuses. Atrial pacing did not affect arterial pH or arterial O2 tension, but arterial CO2 tension increased by a small amount. Mean IVC pressure increased 75%, whereas mean aortic pressure remained the same. Concentrations of plasma protein and albumin and the hematocrit did not change with atrial pacing. Doppler ultrasound examination and Doppler IVC flow tracings showed that flow reversal began immediately with atrial pacing and disappeared immediately with cessation of pacing. Reversed flow was 21% of forward flow. Inspection of simultaneous recordings of ECG, Doppler aortic and IVC flows, and aortic and IVC pressure tracings revealed that the reversed blood flow occurred in diastole in conjunction with atrial contraction and, therefore, could not be due to tricuspid insufficiency. Our findings of increased venous pressure and reversed venous blood flow suggest that ventricular function is impaired and further suggest that oxygen supply to the ventricles may not be sufficient for the increased demand.

Hyperoxic lung injury in Fischer-344 and Sprague-Dawley rats in vivo☆

Supplemental oxygen remains an important therapy for pulmonary insufficiency, despite the potential adverse effects of hyperoxic exposures. Recently, He et al. reported that hyperoxic ventilation more readily damaged isolated perfused lungs from Fischer-344 rats than from Sprague-Dawley rats (Am. J. Physiol. 259:L451), which correlates with the previously reported strain differences in hepatic responses to diquat-induced oxidant stress in vivo (J. Pharmacol. Exp. Ther. 235:172). We therefore examined the differences in hyperoxic lung injury in Fischer-344 and Sprague-Dawley rats in vivo. Adult male rats were exposed to > 95% O2 and were sacrificed after 24, 48, or 60 h. Control animals were maintained in room air. Dramatically greater increases in pleural effusions and bronchoalveolar lavage protein concentrations in response to hyperoxia were observed in the Fischer-344 rats than in the Sprague-Dawley rats (p < .05 at both 48 and 60 h for both measurements). Additionally, the glutathione concentrations in alveolar lining fluid decreased from 800 microM to 115 microM in Fischer-344 rats after 60 h of > 95% O2, but did not change in Sprague-Dawley rats. We conclude that the greater susceptibility of Fischer-344 than of Sprague-Dawley rats to hyperoxic lung injury in vitro reported previously also is observed in vivo and that this strain difference offers unique opportunities to study mechanisms of hyperoxic lung injury.

Effects of oxygen and constant positive pressure breathing on aADCO2 in hyaline membrane disease.

Summary: The effects of 100% oxygen breathing and constant positive pressure breathing (CPPB) on venous admixture (Qva/Qt) and arterial-alveolar difference for PCO2 (aADCO2) were examined in seven infants with hyaline membrane disease (HMD). Increasing FIO2 from 0.63-0.99 with CPPB constant at 2 cm H2O resulted in significant decrease in Qva/Qt from 0.67-0.47, but produced no change in aADCO2 (13.0 torr vs. 15.0 torr). Increasing CPPB to 8 cm H2O with FIO2 returned to 0.63 also resulted in decreased Qva/Qt (0.50), but in addition aADCO2 decreased significantly to 7.0 torr. The reduction in Qva/Qt with oxygen breathing and with CPPB is interpreted as a reduction in true right-to-left-shunt and a corresponding increase of effective blood flow through the lung. In 100% oxygen the increase in effective pulmonary perfusion occurred in a poorly ventilated compartment and as such was not reflected in the aADCO2. On the other hand, with CPPB, the increase in perfusion was accompanied by an increase in ventilation and, hence, the aADCO2 decreased. To illustrate these effects we constructed a three compartment model for the lung in HMD, calculated the VA/Qc for the well-ventilated compartment in each circumstance, constructed O2-CO2 diagrams and arrived at predicted values for the aADCO2 for each of the three clinical conditions. These predicted values agree well with those measured, considering the possible errors in our methods and assumptions and considering the absolute changes that may occur with CPPB, namely, increased cardiac output and decreased ventilation. This, in turn, provides strong support for the proposed three compartment model and for the existence of an open, but severely underventilated compartment in HMD.Speculation: In HMD, the aADCO2 seems to be responsive to the effects of CPPB on both ventilation and perfusion, and as such would be valuable clinically in determining optimal levels of CPPB.

The effect of alkalosis on hypoxia-induced pulmonary vasoconstriction in lungs of newborn rabbits.

ABSTRACT: The purpose of this study was to determine whether metabolic and respiratory alkalosis reduce hypoxia- induced pulmonary vasoconstriction in lungs of newborn rabbits. To accomplish this, we isolated and perfused with blood the lungs from 33 newborn rabbits, 3-14 d old. In all pairs of lungs, we first measured the magnitude of hypoxia-induced pulmonary vasoconstriction at a pH of 7.30-7.42. We then measured the effect of alkalosis on the magnitude of hypoxic pulmonary vasoconstruction by following one of two different sequences of exposure to hypoxia and alkalosis. For the first sequence, we exposed 13 lungs to hypoxia, and during the hypoxic exposure we either decreased the inspired PCo, (respiratory alkalosis, n=8) or infused NaHCC>3 (metabolic alkalosis, n=5) to achieve a pH of 7.50-7.65. For the second sequence, we first decreased the inspired Pco2 (n=9) or infused Na- HCO3 («=11) to achieve a pH of 7.50-7.65 and then exposed the lungs to hypoxia. We found that hypoxic pulmonary vasoconstriction was reduced by either respiratory or metabolic alkalosis, when alkalosis was induced during hypoxia. When respiratory or metabolic alkalosis was induced before hypoxia, the magnitude of hypoxiainduced pulmonary vasoconstriction was the same as at the normal pH. We conclude that both metabolic and respiratory alkalosis reduce ongoing hypoxic pulmonary vasoconstriction in lungs of newborn rabbits. However, neither mode of alkalosis blunts pulmonary vasoconstriction in response to subsequent exposures to hypoxia.

Endotoxin Induces Glutathione Reductase Activity in Lungs of Mice

ABSTRACT: Glutathione reductase catalyzes the NADPH-dependent conversion of glutathione disulfide to glutathione and helps protect the lung from injury by reactive oxygen. In animals allowed to breathe nearly 100% oxygen, the activities of other antioxidants in the lung can be induced by treatment with endotoxin, and this induction is associated with increased tolerance to hyperoxia. The purpose of this study was to see whether glutathione reductase activity in the lungs of mice increased with endotoxin treatment alone. We studied 60 FVB mice (20 males and 40 females). Half received endotoxin (500 μg/kg) intraperitoneally at time 0 and 24 h, and the controls received an equal volume of saline. At 48 h we killed the mice and removed their lungs. Treatment of mice with endotoxin increased glutathione reductase activily in the lung 55% (0.035 ± 0.005 to 0.054 ± 0.010 μmo NADPH reduced/min/mg prolein; mean ± SD; endoloxin differenl from conlrol, p < 0.001). The increase in aclivily was Ihe same for male and female mice. We measured Ihe specific prolein for glutathione reductase by Weslern analysis and mRNA for glutathione reductase using a slol-blol analysis and found lhat both increased roughly 2-fold with endoloxin treatmenl. This suggesls lhat endotoxin Ireatment resulted in either increased rale of transcription of glulathione reductase mRNA or increased mRNA slabilily. We conclude that endoloxin treatment increases glutathione reductase aclivily in the lung and thai Ihis increase in activity may play a role in subsequent protection from hyperoxia.

Effects of an Aorticopulmonary Shunt on Lung Fluid Balance in the Young Lamb

ABSTRACT: We studied the effects of increased pulmonary blood flow on lung fluid balance in seven chronically instrumented lambs (18 ± 1 d) with surgically created aorticopulmonary shunts. We measured mean pulmonary arterial and left atrial pressure (LAP), pulmonary blood flow, lung lymph flow, and lymph (CL) and plasma (CP) protein concentration with the shunt closed and opened. With the shunt partially open, a 35% increase in pulmonary blood flow resulted in an increase in pulmonary arterial pressure (15.5 ± 1 to 19.5 ± 1 torr) and LAP (2.0 ± 0.5 to 3.5 ± 0.5 torr). Lung lymph flow nearly doubled (1.53 ± 0.28 to 2.83 ± 0.52 mL/h) whereas the CL decreased (4.1 ± 0.1 to 3.4 ± 0.1 g/dL) resulting in a decrease in the CL/CP ratio (0.67 ± 0.01 to 0.58 ± 0.01). With the shunt fully open, pulmonary blood flow increased 65% over baseline, pulmonary arterial pressure increased from 16.5 ± 2.0 to 26.5 ± 5 torr, and LAP increased from 1.5 ± 0.5 to 6.5 ± 2.0 torr. Lung lymph flow increased (1.1 ± 0.2 to 3.1 ± 0.2 mL/h) whereas CL (4.1 + 0.1 to 3.1 ± 0.3 g/dL) and CL/CP (0.66 ± 0.02 to 0.51 ± 0.05) decreased. All changes were statistically significant (p < 0.05). When the effects of increased LAP on lung fluid balance (partial mitral valve obstruction) were tested in five additional lambs, an identical change in LAP as seen in lambs with partially open shunts (2.5 ± 0.5 to 4.0 ± 0.5 torr) resulted in only a 20% increase in lung lymph flow and a small decrease in the CL/CP ratio (0.61 ± 0.02 to 0.57 ± 0.02), whereas pulmonary blood flow did not change. We conclude that in the newborn lamb increased pulmonary blood flow from a large central shunt increased the rate of transvascular fluid filtration in the lung by increasing filtration pressure. This increase could not be explained simply on the basis of a change in left atrial pressure and implies that resistance downstream from fluid filtering sites is relatively fixed.

Biochemical Manifestations of Oxygen Toxicity in the Newborn Lamb

ABSTRACT: The purpose of this project was to study the role of lipid peroxidation in oxygen-induced lung injury in the newborn lamb. It was our hypothesis that injury to the microvascular bed of the lung by oxygen would coincide with a burst of peroxidative activity and would be accompanied by an increased rate of excretion of ethane and pentane in expired gas. We measured vascular pressures, the rate of lung lymph flow and concentrations of ethane and pentane in exhaled gas in 10 newborn lambs that breathed >95% oxygen continuously. Our marker for oxygen-induced lung injury was an increase in the permeability of the microvascular bed of the lung to protein (an increase in the rate of lung lymph flow accompanied by an increase in the protein concentration in lymph). Although all 10 lambs demonstrated an abrupt increase in microvascular permeability to protein within 48 to 96 h of exposure to >95% oxygen, the rates of ethane and pentane excretion remained unchanged throughout the entire experimental period. Lung tissue concentrations of glutathione decreased by 40% in the oxygen-exposed lambs and the concentrations of glutathione disulfide increased 85% relative to air-breathing controls. Activities of glutathione reductase and superoxide dismutase were lower in the lungs of the oxygen- exposed lambs than in controls, whereas the activities of glutathione peroxidase and catalase were not changed. We conclude that, in the lamb, changes in the rates of excretion of ethane and pentane do not correlate with the timing of injury to the microvascular bed of the lung.