Reactive oxygen species (ROS) may play an important role in the chronic pulmonary morbidity of preterm infants. We therefore studied the magnitude and mechanisms of oxidative inactivation of a natural lung surfactant (NLS) and of two surfactants used for treatment of respiratory distress syndrome, beractant and KL4 surfactant (KL4). Incubation with Fenton reagents, 2-4 mM peroxynitrite (ONOO-) or 0.5 mM hypochlorous acid (OCl-), resulted in an increased minimum surface tension (MST) of all surfactants; the order of effect on MST was beractant > KL4 > NLS. After incubation with Fenton reagents, NLS contained a higher concentration of conjugated dienes (p < 0.01) but lower concentration of malondialdehyde (p < 0.001) than beractant. Protein carbonyl concentrations after treatment with Fenton reagents were higher in NLS and KL4 than in beractant (p < 0. 05). Surface area cycling for 24 h with 2 mM ONOO- or 0.5 mM OCl- caused both beractant and KL4 to increase the proportion of light subtypes from 8-10% to 26-29%; with Fenton reagents, there was disappearance of the light subtype and formation of ultraheavy subtype 74-91% with poor MST. Natural and therapeutic surfactants differ markedly in their sensitivity to ROS, which may be important for surfactants in therapeutic use because oxidative inactivation may limit their effect. Oxidation of natural surfactant may result in reduced function and contribute to chronic lung disease.

Studies were conducted to assess the efficacy and safety of a synthetic peptide-containing surfactant in the treatment of respiratory distress syndrome (RDS) in preterm (approximately 80% of normal gestation) infant rhesus monkeys. Surfactant was prepared consisting of the phospholipids dipalmitoylphosphatidyl choline and palmitoyl-oleoyl phosphatidyl glycerol and a synthetic peptide modeled after surfactant protein B (SP-B), "KL4-Surfactant" contained a peptide having the sequence KLLLLKLLLLKLLLLKLLLLK, where "K" is lysine and "L" is leucine. The peptide was selected because it mimics the repeating stretches of hydrophobic residues with intermittent basic hydrophilic residues seen in SP-B. KL4-Surfactant was shown to have biophysical activity assessed as the ability to lower surface tension at an air-liquid interface in a pulsating bubble surfactometer. Thirty premature rhesus monkeys were treated shortly after birth with one dose of KL4-Surfactant. The arterial to alveolar oxygen partial pressure ratio (a/A) was found to rise from a pretreatment level of 0.11 +/- 0.01 (mean +/- SEM), indicative of severe RDS, to 0.40 +/- 0.02 at 12-13 h post-treatment. The improvement in oxygenation persisted throughout the study period, with a mean a/A at 22-23 h of 0.45 +/- 0.07. Chest radiographs and gross and microscopic examination of the lungs all confirmed the reversal of the atelectasis seen before treatment. Animals treated with a dose of 200 mg/kg showed a faster, more consistent, and greater response than did a group treated with an average dose of 127 mg/kg. There was no evidence of toxicity after treatment with the higher dose as demonstrated by physiologic, hematologic, biochemical, and pathologic data. The importance of the peptide in the synthetic surfactant was apparent from the results obtained with a control group of nine premature monkeys treated with a non-peptide-containing surfactant; the a/A of this group was 0.15 +/- 0.03 at nine hours of age as compared with a value of 0.38 +/- 0.02 for 30 comparable animals receiving KL4-Surfactant.

The serum components of C-reactive protein, lysophosphati-dylcholine, fibrinogen, and fibrinogen proteolytic products have been shown to reduce surface tension-lowering abilities of lung surfactant. The inhibitory effects of these serum components were compared among four different surfactants: natural lung surfactant, a phospholipid mixture that had no surfactant proteins, KL4 surfactant which has a synthetic surfactant protein B (SP-B)-like peptide, and beractant (BER) which has both SP-B and SP-C. The pulsating bubble surfactometer was used to measure the surface tension of these surfactants after the addition of inhibitors. Inhibition of BER and KL4 surfactant was observed with some serum components within 1 min of pulsation, but was reversed after 3 min of pulsation for KL4 surfactant and to a lesser extent with BER. The surface tension of phospholipid mixture alone was significantly increased and did not improve with further pulsations. Natural lung surfactant was least inhibited and was affected only at very high fibrinogen concentrations (5 mg/mL). At identical concentrations of these inhibitors, KL4 surfactant was inhibited less compared with BER. We conclude that the response of a lung surfactant to inhibitory agents may depend on the presence or absence of surfactant-related protein(s) in the surfactant and the concentration of exogenous surfactant used. KL4 surfactant, which has a synthetic peptide in lieu of SP-B, resists inhibition to these serum components more than BER at similar phospholipid concentrations.

Intrapulmonary distribution of ;i peptide-phospholipid (KI.,) surfactant administered through ;III adapter permitting maintenance of positive end-expiratory pressure was compared with distribution by instillation with disconnection from mechanical ventilation in 1 0 surpactant-deficient Mucnca trr~tllafu preterm infant\. Animals rcceived KL, surf:ic.tant (200 ni@Iig) when the arterial to alveolar (oxygen ratio) (a!Xo,) was 50.22 (approximately 50 min after birth) on mechanical ventilation. Six rhesus infants received bolus instillation of two half doses of KId4 stlrfactant through an endotracheal tube adapter over 10-15 s while maintaining positive end-expiratory pressurc (0.4 kPa) accompanied by turning to the right and left lateral positions for 60 s. In four rhcsus premature infiiuts KL, surfactant was injected as two half-dose boluses through the endotracheal tube with disconnection from mechanical ventilation while positioning the infant rhesus monkey in the right and left 1;itcral positions for 30 s of mech;inical ventilation between instillation. Acute effects on oxygen s;~turation were nionitored, and physiologic measures of a/Ao,, mean airway pressure, and the ventilatory eBiciency index were monitored over the 13-11 study. Intrapulmonary distribution of KL, surk~ctant was determined using dye-labeled microsphcres or [7~]dipalmitoylpho~phatidylcho-line-labeled KL, surfactant, measured by colorimetry or by scintillalion counting.

We previously demonstrated that 48 h of 100 ppm inhaled nitric oxide (NO) and 90% O2 causes surfactant dysfunction and pulmonary inflammation in mechanically ventilated newborn piglets. Because peroxynitrite (the product of NO and superoxide) is thought to play a major role in the injury process, recombinant human superoxide dismutase (rhSOD, a scavenger of superoxide) might minimize this insult. Four groups of newborn piglets (1-3 days of age) were ventilated with 100 ppm NO and 90% O2 for 48 h. Piglets received no drug, 5 mg/kg rhSOD intratracheally at time 0, 5 mg/kg rhSOD intratracheally at 0 and 24 h, or 10 mg/kg rhSOD by nebulization at time 0. At 48 h, bronchoalveolar lavage (BAL) was performed, and lung tissue was analyzed for markers of inflammation, oxidative injury, acute lung injury, and surfactant function. There were significant differences between rhSOD-treated piglets and untreated controls with respect to BAL neutrophil chemotactic activity, cell counts, and protein concentration as well as lung tissue malondialdehyde concentrations. Minimum surface tension of BAL surfactant from all groups studied was increased, with no differences found among groups. These data suggest that rhSOD, at the doses used, mitigated the inflammatory changes, oxidative damage, and acute lung injury from exposure to 100 ppm NO and 90% O2 but did not appear to improve surfactant function. This has important clinical implications for infants treated with hyperoxia and NO for neonatal lung disorders.