Higher lung antioxidant enzyme activity persists after single dose of corticosteroids in preterm lambs.

Although administration of exogenous corticosteroids accelerates the late gestational rise in fetal rat and lamb lung antioxidant enzyme activity, the effect of dosing intervals on these responses remains uncertain. We studied the persistence and efficacy of the antioxidant response in fetal lamb lung to a single fetal dose of corticosteroids injected between 121 and 127 days gestational age. Fetal lambs received 0.5 mg/kg of betamethasone (n = 35) or saline (n = 26) by fetal intramuscular injection 24 h, 48 h, 4 days, or 7 days before preterm delivery at 128 days gestation (term = 150 days). After delivery, the lambs were ventilated for 40 min and killed. Total superoxide dismutase, catalase, glutathione peroxidase activities, and lipid hydroperoxide levels were measured, using homogenized lung. The saline-injected controls were similar at all time points. Lung antioxidant enzyme activity was consistently higher and lipid hydroperoxide presence was lower in the betamethasone-treated groups. We conclude that the positive effect of a single fetal dose of betamethasone on lung antioxidant enzyme activity occurs within 24 h after exposure, persists over a period of 7 days without a major change in the magnitude of the response, and leads to a reduction in lipid hydroperoxide formation during immediate postdelivery oxygen exposure.

A synthetic segment of surfactant protein A: structure, in vitro surface activity, and in vivo efficacy.

Surfactant protein A (SP-A) is a 248-residue, water-soluble, lipid-associating protein found in lung surfactant. Analysis of the amino acid sequence using the Eisenberg hydrophobic moment algorithm predicts that the SP-A segment spanning residues 114-144 has high hydrophobic moments, typical of lipid-associating amphipathic domains. The secondary structure, in vitro surface activity and in vivo lung activity of this SP-A sequence were studied with a 31-residue synthetic peptide analog (A114-144). Analysis of the secondary structure using circular dichroism and Fourier transform infrared spectroscopy indicated association with lipid dispersions and a dominant helical content. Surface activity measurements of A114-144 with surfactant lipid dispersions and the hydrophobic surfactant proteins B and C (SP-B/C) showed that A114-144 enhances surface activity under conditions of dynamic compression and respreading on a Langmuir/Wilhelmy surface balance. Synthetic surfactant dispersions containing A114-144 improved lung compliance in spontaneously breathing, 28-d premature rabbits to a greater degree than surfactant dispersions with synthetic SP-B/C and synthetic surfactant lipids alone. These observations indicate that inclusion of A114-144 may improve synthetic preparations currently used for surfactant replacement therapy.

Inactivation of surfactant in rat lungs.

Although surfactant replacement therapy has dramatically improved the outcome of premature infants with respiratory distress syndrome, approximately 30% of treated infants show a transient or no response. Nonresponse to surfactant replacement therapy may be due to extreme lung immaturity and possibly surfactant inactivation. Surfactant inactivation involves aspecific biophysical events, such as interference with the formation or activity of an alveolar monolayer, and specific interactions with serum proteins, including antibodies, leaking into the alveolar space. As formulations containing surfactant proteins appear to better tolerate serum inactivation, we used an excised rat lung model to compare the susceptibility to serum inactivation of a mixture of synthetic phospholipids selected from surfactant lipid constituents, Exosurf (a protein-free synthetic surfactant), Survanta [containing surfactant proteins B and C (SP-B and -C)], and a porcine surfactant (containing SP-A, -B, and -C). For each of these preparations, we used pressure/volume determinations as an in situ measure of surfactant activity and retested the same preparations after mixing with human serum, a nonspecific surfactant inactivator. Human serum inactivated porcine surfactant to a lesser extent than Survanta, Exosurf, or synthetic phospholipids. Temperature exerted a significant effect on deflation stability, as shown by a greater lung compliance in untreated, normal lungs and a larger improvement in compliance after treating lavaged lungs with synthetic phospholipids at 37 degrees C than at 22 degrees C. We conclude that surfactant containing SP-A, -B, and -C is only moderately susceptible to inactivation with whole serum and may therefore exert a greater clinical response than protein-free surfactants or those containing only SP-B and -C.

Antioxidant-surfactant liposomes mitigate hyperoxic lung injury in premature rabbits.

Surfactant liposomes, encapsulating CuZn-superoxide dismutase (SOD) and catalase, increase alveolar type II cell antioxidant activity and protect cells against oxidant stress. We examined whether intratracheal instillation of antioxidant-surfactant liposomes increases lung antioxidant activity in premature rabbits. Pregnant New Zealand White rabbits were delivered by cesarean section on day 28 or 29 of gestation or allowed to deliver spontaneously. After premature birth or at 2 days of age in the term rabbits, the pups from each litter were divided into four groups. One group received 0.1 ml/15 g birth wt of antioxidant-surfactant liposomes by intratracheal injection and was then exposed to hyperoxia (> 95% oxygen) for 24 h and killed. The second group received an equal amount of surfactant liposomes without antioxidant enzymes and was exposed to hyperoxia for 24 h. The third group received air placebo and was exposed to hyperoxia for 24 h, and the fourth group was killed after birth if premature or at 2 days of age if term. After the pups were killed, lung homogenates were investigated for total SOD and catalase activity and DNA content. Each treatment group consisted of 12-15 rabbit pups. Lung antioxidant enzyme activity increased with advancing maturity. Among the premature rabbits, total lung SOD and catalase activity were lowest in the pups killed before hyperoxia and the air placebo controls exposed to hyperoxia, intermediate in the pups treated with liposomes without antioxidant enzymes and hyperoxia, and highest in the pups that received antioxidant-surfactant liposomes and hyperoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Lysis of red blood cells and alveolar epithelial toxicity by therapeutic pulmonary surfactants.

The risk of pulmonary hemorrhage is increased in extremely low birth weight infants treated with surfactant. The pathogenesis of this increased risk is far from clear. We tested whether exposure of cell membranes to surfactant may lead to increased membrane permeability, hypothesizing that this process may contribute to the occurrence of alveolar hemorrhage after surfactant treatment. Aliquots of washed packed red blood cells (used as membrane model) were suspended in 0.9% NaCl with various concentrations of Survanta or Exosurf for either 2 or 24 h at 37 degrees C. Cytolysis was measured by spectrophotometric determination of free Hb after centrifugation. Red cells suspended in 0.9% NaCl alone, distilled water, or various concentrations of melittin were used as negative and positive controls. Both surfactants were associated with increased hemolysis to 35% of maximum at concentrations of 1.25 mg/2 mL. Above these concentrations, Survanta was associated with no increase in hemolysis, whereas Exosurf increased hemolysis to 60% of maximum at concentrations of 12.5 mg/2 mL. In additional experiments, primary cultures of alveolar type II cells from adult rats were treated with Survanta, Exosurf, the Exosurf components tyloxapol and hexadecanol, melittin, or culture medium alone. After 24 h of incubation, lactate dehydrogenase release into the media was measured as a percent of total lactate dehydrogenase activity to indicate cytotoxicity. Lactate dehydrogenase release was < 10% for control experiments but increased sharply with Exosurf and its components tyloxapol and hexadecanol. These results indicate that surfactant may be associated with in vitro cytotoxicity and that this property differs for different surfactants and different dosages.

Plasma xanthine oxidase activity and lipid hydroperoxide levels in preterm infants.

Ischemia-reperfusion injury may affect morbidity and mortality in preterm and asphyxiated term infants. Reoxygenation of hypoxic tissues leads to the formation of free oxygen radicals by xanthine oxidase that may induce lipid peroxidation, enzyme inhibition, and DNA strand breakage. We measured arterial cord blood samples from 36 healthy term infants for baseline values and arterial blood sampled at 1 and 4 h after birth from 45 preterm infants admitted for intensive care for serial estimates of plasma xanthine oxidase activity and lipid hydroperoxide levels. Mean +/- SEM plasma xanthine oxidase activity in cord blood of term infants was 2.3 +/- 0.4 mU/mL and lipid hydroperoxide levels were 2.6 +/- 0.3 nmol/mL. Eighteen of the 45 preterm infants met the criteria defining poor outcome (poor outcome group) and had lower umbilical arterial pH and base excess than the 27 preterm infants in the control group. Mean plasma xanthine oxidase activity increased from 2.7 +/- 0.4 at 1 h to 4.7 +/- 0.6 mU/mL at 4 h of age (p < 0.001) in the poor outcome group and decreased from 2.1 +/- 0.3 to 1.1 +/- 0.2 mU/mL (p = 0.004) in the control group. Lipid hydroperoxide levels in the poor outcome group increased from 2.8 +/- 0.6 nmol/mL at 1 h to 4.3 +/- 0.6 nmol/mL at 4 h of age (p < 0.001) and decreased from 2.1 +/- 0.6 to 1.6 +/- 0.2 nmol/mL (p = 0.008) in the control group. At 4 h of age, xanthine oxidase activity and lipid hydroperoxide levels were significantly higher in the poor outcome group than in the controls (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Uptake of antioxidants in surfactant liposomes by cultured alveolar type II cells is enhanced by SP-A.

Antioxidant delivery may be targeted toward the alveolar epithelium by encapsulating superoxide dismutase (SOD) and catalase in liposomes made from pulmonary surfactant. We studied whether antioxidant-surfactant liposomes increase cellular antioxidant activity in alveolar type II cells and whether this effect is influenced by the presence of surfactant protein A (SP-A). Cu,Zn SOD and catalase were encapsulated in liposomes made from synthetic phospholipids with or without 5% SP-A or from natural cow surfactant. Alveolar type II cells from adult rats were preincubated for 20 h, and liposome mixtures were added for 24 h, followed by measurement of cellular SOD and catalase activities (U/mg DNA). Antioxidant-surfactant liposomes increased alveolar type II cell antioxidant activity sharply. Uptake of SOD/catalase from liposomes with synthetic phospholipids and SP-A was twice that from liposomes without SP-A and did not further improve in the presence of SP-B and -C. Encapsulation of antioxidants diminished the surface activity of the surfactant liposomes, but this feature was absent in the presence of SP-A. These data suggest that: 1) antioxidant-surfactant liposomes augment alveolar type II cell antioxidant activity, 2) liposomal uptake is facilitated by the presence of SP-A, and 3) inhibition of surface activity of surfactant by encapsulated antioxidants can be reversed by SP-A.

Mitigation of pulmonary oxygen toxicity in rats by intratracheal instillation of polyethylene glycol-conjugated antioxidant enzymes.

Hyperoxic lung injury may be mitigated by increasing alveolar epithelial antioxidant activity. We examined whether intratracheal instillation of superoxide dismutase (SOD) and catalase, conjugated to polyethylene glycol (PEG) to permit cellular access, reduces hyperoxic lung injury. Adult rats, pretreated intratracheally with 1,500 U PEG-SOD and 10,000 U PEG-catalase or with inactivated PEG-SOD/catalase, 1% PEG, or saline (treated controls), were exposed to hyperoxia (fraction of inspired oxygen > 0.95) for 48 h and compared with untreated air controls. Alveolar wash protein values in the treated control groups were significantly higher than in the PEG-SOD/catalase and air control groups, which had comparable values. Lung homogenate and alveolar type II cell SOD and catalase activities were higher after PEG-SOD/catalase treatment and lower after the control treatments when compared with untreated air controls. Lung homogenate dipalmitoyl phosphatidylcholine decreased and alveolar wash dipalmitoyl phosphatidylcholine increased after hyperoxia, but these changes were less after PEG-SOD/catalase treatment. Rats pretreated intratracheally with PEG-SOD/catalase survived significantly longer in hyperoxia than saline controls. These data indicate the potential of intratracheal antioxidant treatment to reduce pulmonary oxygen toxicity.

Phospholipid and surfactant protein A concentrations in tracheal aspirates from infants requiring extracorporeal membrane oxygenation.

To test the hypothesis that infants with severe respiratory failure and the need for extracorporeal membrane oxygenation (ECMO) are surfactant deficient, we measured the amount of surfactant phospholipids, disaturated phosphatidylcholine, surfactant protein A, and protein in tracheal aspirates from 22 infants, who received ECMO therapy for respiratory failure with meconium aspiration syndrome (n = 18) or pneumonia (n = 4). Tracheal suction material was obtained in a standardized way every 4 hours during the period of ECMO treatment and pooled for 24-hour periods. During ECMO, mean total phospholipid, disaturated phosphatidylcholine, and surfactant protein A values in tracheal aspirates increased and protein values decreased significantly, predominantly during the 72-hour period before infants were weaned from ECMO. Of the 22 infants, 14 had an increase in tracheal aspirate phospholipid values of more than 200% and were found to need a shorter period of ECMO support (p less than 0.005) and post-ECMO ventilatory support (p less than 0.025) than did the eight infants with stationary or only moderate increases in tracheal aspirate phospholipid values, three of whom had pneumonia. We conclude that infants with respiratory failure who require ECMO treatment often have surfactant deficiency. We speculate that surfactant treatment might decrease the need for or the duration of ECMO support.