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Synthetic surfactant peptides offer an opportunity to standardize the protein composition of surfactant. We tested the effect of phospholipids (PL) with synthetic full-length SP-B1-78 (B), mutant B (Bser), KL4 peptide (UCLA-KL4), and palmitoylated SP-C1-35 (C) on oxygenation and lung function in a surfactant-deficient rat model. Sixty-four adult rats were ventilated with 100% oxygen, a tidal volume of 7.5 mL/kg, and a rate of 60/min. Their lungs were lavaged with saline until the arterial PO2 dropped below 80 torr, when 100 mg/kg surfactant was instilled. Surfactant preparations included: PL (PL surfactant), PL + 3% B (B surfactant), PL + 3% B and 1% C (BC surfactant), PL + 3% UCLA-KL4 (KL4 surfactant), PL + 3% Bser (Bser surfactant), and PL + 3% B and 1% UCLA-KL4 (BKL4 surfactant). Sixty minutes after surfactant instillation, positive end-expiratory pressure was applied for 5 min, and pressure-volume curves were determined in situ. The six surfactant preparations had a minimum surface tensions10 mN/m on a Langmuir/Wilhelmy balance. Instillation of PL, Bser, and BKL4 surfactant increased mean arterial/alveolar PO2 (aADO2) ratios by 50-100% over postlavage values, whereas KL4 surfactant increased aADO2 ratios by 118%, B surfactant by 191%, and BC surfactant by 225%. Lung volumes at 30 cm H2O pressure were highest after treatment with BC surfactant, intermediate after B and KL4 surfactants, and lowest after BKL4, Bser, and PL surfactants. These data suggest that a surfactant preparation with a combination of synthetic B and C peptides surpasses synthetic B and KL4 surfactants in improving oxygenation and lung compliance in surfactant-deficient rats.

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.

Presented in: Poster Session Monday, May 4, 1998, Poster number 195 † 1658 A point mutation in exon 7 of the human surfactant protein B (SP-B) gene, resulting in a cysteine for agrinine substitution (R236C), is associated with partial SP-B deficiency (Ballard et al., Pediatrics 1995). We synthesized, purified, and characterized this mutant SP-B (BR2326C), full length oxidized SP-B1-78(B), and palmitoylated SP-C1-35(C).

Paradoxical Effects of Exogenous Proteins on Lung Function in Surfactant-Deficient Rats 1771

Surfactant aggregate size correlates with function in vivo. We have compared the effects of mechanical ventilation on the aggregate size of synthetic surfactants in the surfactant depleted rat lung model, lavaged before surfactant treatment and after 1 hour of ventilation (FiO2 = 1.0, rate = 60, VT =7.5 mL/Kg). Aggregate size was measured on a Mitech laser beam sample sizer. We used phospholipids and palmitic acid (PL) alone, or PL plus synthetic peptides: PL + 3% B1-78 (B), PL + 3% B1-78& 1% C1-35 (BC), PL + 3% KL4 (KL4), PL + 3%B1-78 & 1% KL4 (BKL4), and PL + 3% of a serine analog of B1-78(Bser) at 25 mg PL/ml, using a dose of 100 mg/kg body weight. PL and sham treatment were used as controls. Surfactants, initial and final wash were assessed for particle size, and total proteins were measured in each lavage. Synthetic surfactant vesicles had an apparent diameter (AD) of 4-6 μm or more that did not change after surface cycling for 3 hours. In all groups, the first wash contained large particles with an AD of 1-6 μm. In the second wash, the sham treated and the BKL4 groups contained primarily small vesicles with an AD less than 0.2 μm. B, KL4, and Bser treated rats had a significant fraction of vesicles with an AD of 1-4 μm. PL had vesicles with an AD of both less than 0.1 and 1-2 μm. Protein content increased 2-20 folds after ventilation, but the increase did not correlate with vesicle size. These data suggest that synthetic surfactant proteins in PL may undergo only partial large-to-small from conversion in an animal model of surfactant depletion during mechanical ventilation.

During its life cycle, surfactant converts from highly surface active, large aggregates to less surface active, smaller aggregates. This process is probably regulated by a serine protease. We tested whether adding a1-antitrypsin (α1-AT), an antiprotease, to surfactant improves its in vivo function. α1-AT was added to Survanta® and a synthetic surfactant (BC: phospholipids with 3% surfactant peptide B1-78 and 1% palmitoylated surfactant peptide C1-35) at a dose of 100 mg/3 ml surfactant. Adding α1-AT to Survanta and BC did not affect surface activity on a Langmuir/Wilhelmy balance. Adult rats were ventilated with 100% oxygen, a tidal volume of 7.5 ml/kg, and a rate of 60/min. Their lungs were lavaged with saline until the PaO2 dropped below 80 torr, at which time 100 mg/kg of surfactant was instilled. After 60 min of ventilation, the rats were killed and their lungs lavaged. Survanta increased mean arterial/Alveolar PO2 ratios by 91% over post-lavage values versus 136% for Survanta plus α1-AT (p

Surfactant protein B (SP-B) is critical to surfactant function. Survanta®, a modified bovine lung extract, contains less SP-B than surfactants derived from lung lavage (Seeger et al., Eur Resp J 1993;6:971). Supplementation of Survanta with native SP-B increases dynamic compliance and deflation lung volume in preterm rabbits (Mizuno et al., Pediatr Res 1995;37:271). We hypothesized that the in vivo function of Survanta can be improved by supplementation with synthetic SP-B and SP-C and tested this hypothesis in a surfactant-deficient rat model. Full length SP-B1-78 and palmitoylated SP-C1-34 were synthesized, purified, derivatized, and analyzed by mass spectrometry and amino acid analysis. Survanta was extracted, unmodified (Surv-1) or enriched with 1% SP-B (Surv-2), 2% SP-B (Surv-3), or 2% SP-B + 1% SP-C (Surv-4), and resuspended. In vitro surface activity was tested on a modified Langmuir/Wilhelmy balance and SP-B and SP-C content were measured by Fourier transform infrared spectroscopy. Adult rats were anesthetized, tracheotomized, had an arterial line placed, and were ventilated with 100% oxygen, a peak inspiratory pressure of 30 cm H2O and a rate of 40/min. The lungs were lavaged with warmed normal saline until the PaO2 dropped below 60 torr, at which time 100 mg/kg of one of the four surfactant preparations was instilled. After 15 min of ventilation, the rats were killed and pressure-volume curves performedin situ. The mean arterial/Alveolar PO2 ratio was 0.08 post-lavage and increased by 74% after treatment with Surv-1 (n=5), by 92% after treatment with Surv-2 (n=6), by 257% after treatment with Surv-3 (n=5), and by 202% after treatment with Surv-4 (n=6). Surv-4 not only improved PaO2, but also reduced PaCO2 values. These data suggest that the addition of 2% synthetic SP-B to Survanta improves its short-term positive effects on oxygenation in this surfactant-deficient rat model.