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BackgroundThis study examines the biophysical and preclinical pulmonary activity of synthetic lung surfactants containing novel phospholipase-resistant phosphonolipids or synthetic glycerophospholipids combined with Super Mini-B (S-MB) DATK and/or SP-Css ion-lock 1 peptides that replicate the functional biophysics of surfactant proteins (SP)-B and SP-C. Phospholipase-resistant phosphonolipids used in synthetic surfactants are DEPN-8 and PG-1, molecular analogs of dipalmitoyl phosphatidylcholine (DPPC) and palmitoyl-oleoyl phosphatidylglycerol (POPG), while glycerophospholipids used are active lipid components of native surfactant (DPPC:POPC:POPG 5:3:2 by weight). The objective of the work is to test whether these novel lipid/peptide synthetic surfactants have favorable preclinical activity (biophysical, pulmonary) for therapeutic use in reversing surfactant deficiency or dysfunction in lung disease or injury.MethodsSurface activity of synthetic lipid/peptide surfactants was assessedin vitroat 37 °C by measuring adsorption in a stirred subphase apparatus and dynamic surface tension lowering in pulsating and captive bubble surfactometers. Shear viscosity was measured as a function of shear rate on a Wells-Brookfield micro-viscometer.In vivopulmonary activity was determined by measuring lung function (arterial oxygenation, dynamic lung compliance) in ventilated rats and rabbits with surfactant deficiency/dysfunction induced by saline lavage to lower arterial PO2to ResultsSynthetic surfactants containing 5:3:2 DPPC:POPC:POPG or 9:1 DEPN-8:PG-1 combined with 3% (by wt) of S-MB DATK, 3% SP-Css ion-lock 1, or 1.5% each of both peptides all adsorbed rapidly to low equilibrium surface tensions and also reduced surface tension to ≤1 mN/m under dynamic compression at 37 °C. However, dual-peptide surfactants containing 1.5% S-MB DATK + 1.5% SP-Css ion-lock 1 combined with 9:1 DEPN-8:PG-1 or 5:3:2 DPPC:POPC:POPG had the greatestin vivoactivity in improving arterial oxygenation and dynamic lung compliance in ventilated animals with ARDS. Saline dispersions of these dual-peptide synthetic surfactants were also found to have shear viscosities comparable to or below those of current animal-derived surfactant drugs, supporting their potential ease of deliverability by instillation in future clinical applications.DiscussionOur findings support the potential of dual-peptide synthetic lipid/peptide surfactants containing S-MB DATK + SP-Css ion-lock 1 for treating diseases of surfactant deficiency or dysfunction. Moreover, phospholipase-resistant dual-peptide surfactants containing DEPN-8/PG-1 may have particular applications in treating direct forms of ARDS where endogenous phospholipases are present in the lungs.

Background/objectives.This study examines the surface activity, resistance to biophysical inhibition, and pulmonary efficacy of a synthetic lung surfactant containing glycerophospholipids combined with Super Mini-B (S-MB) DATK, a novel and stable molecular mimic of lung surfactant protein (SP)-B. The objective of the work is to test whether S-MB DATK synthetic surfactant has favorable biophysical and physiological activity for future use in treating surfactant deficiency or dysfunction in lung disease or injury.Methods.The structure of S-MB DATK peptide was analyzed by homology modeling and by FTIR spectroscopy. Thein vitrosurface activity and inhibition resistance of synthetic S-MB DATK surfactant was assessed in the presence and absence of albumin, lysophosphatidylcholine (lyso-PC), and free fatty acids (palmitoleic and oleic acid). Adsorption and dynamic surface tension lowering were measured with a stirred subphase dish apparatus and a pulsating bubble surfactometer (20 cycles/min, 50% area compression, 37 °C).In vivopulmonary activity of S-MB DATK surfactant was measured in ventilated rabbits with surfactant deficiency/dysfunction induced by repeated lung lavages that resulted in arterial PO2values Results.S-MB DATK surfactant had very high surface activity in all assessments. The preparation adsorbed rapidly to surface pressures of 46–48 mN/m at 37 °C (low equilibrium surface tensions of 22–24 mN/m), and reduced surface tension to Conclusions.S-MB DATK is an active mimic of native SP-B. Synthetic surfactants containing S-MB DATK (or related peptides) combined with lipids appear to have significant future potential for treating clinical states of surfactant deficiency or dysfunction, such as neonatal and acute respiratory distress syndromes.

Animal-derived drugs are currently widely-used to treat clinical lung surfactant deficiency, but synthetic surfactants have significant advantages as pharmaceutical agents. This article examines exogenous surfactants containing novel synthetic phospholipase-resistant lipids of extremely high surface activity. Mixtures of these lipid analogs with purified native surfactant apoproteins are detailed as a proof of concept for related fully-synthetic surfactants containing laboratory- produced peptides. The chemistry and biophysics of relevant lipid analogs and peptides are reviewed in the context of developing new synthetic drugs of utility for patients with surfactant deficiency or lung injury-related surfactant dysfunction.

The synthesis of three phosphoglycerols is described, one of which contains the previously unknown phosphonoglycerol headgroup. The surface tension-lowering capabilities of synthetic lung surfactant mixtures containing the PG analogs were measured on the pulsating bubble surfactometer and compared to known controls. The PG-containing mixtures exhibited superior surface tension-lowering properties indicating the significant potential of these analogs as components in synthetic exogenous lung surfactants.

Two novel C16:0 sulfur-linked phosphonolipids (S-lipid and SO(2)-lipid) and two ether-linked phosphonolipids (C16:0 DEPN-8 and C16:1 UnDEPN-8) were studied for surface behavior alone and in mixtures with purified bovine lung surfactant proteins (SP)-B and/or SP-C. Synthetic C16:0 phosphonolipids all had improved adsorption and film respreading compared to dipalmitoyl phosphatidylcholine, and SO(2)-lipid and DEPN-8 reached maximum surface pressures of 72mN/m (minimum surface tensions of

Epifluorescence microscopy was used to study the structure and phase behavior of phospholipid films containing a human-sequence monomeric SP-B1–25 synthetic peptide (mSP-B1–25). Measurements were done directly at the air–water (A/W) interface on films in a Langmuir–Whilhelmy balance coupled to a fluorescence microscope and real-time detection system to yield an approximate optical resolution of 1 Am. Fluorescence was achieved by laser excitation of 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3dodecanoyl)-1-hexadecanoyl-sn-glycero-3-PC (BODIPY-PC, concentration V1 mol%). The presence of mSP-B1–25 in films of 4:1 (mol/mol) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) had a substantial effect on lipid morphology and phase behavior that depended on both surface pressure and peptide concentration (10, 5, and 1 wt.%). The mSP-B1–25 peptide tended to fluidize phospholipid monolayers based on expanded molecular areas and reduced collapse pressures. In addition, epifluorescence measurements revealed the formation of solid-phase domains apparent as three-armed counterclockwise spirals separated from regions of fluid liquid-expanded phase domains in compressed phospholipid–peptide films. The appearance of these separated solid-phase domains resembled pure L-DPPC rather than the ensemble-type solid domains found in films of DPPC/DOPG alone and were most apparent when 10 wt.% mSP-B1–25 was present. In contrast, films containing lower, more physiological mSP-B1–25 contents of 5 and 1 wt.% exhibited a prominent intermediate ddendriticT phase that increased in extent as surface pressure was raised. This phase was characterized by branching structures that formed a lattice-like mesh network with fluorescence intensities between a dye-depleted solid domain and a dye-enriched liquid phase. These results indicate that mSP-B1–25 at near-physiological levels produces morphological changes in phospholipid monolayers analogous to those observed for native SP-B1–79. D 2004 Elsevier B.V. All rights reserved.

Background. Surfactant protein C (SP-C; 35 residues) in lungs has a cationic N-terminal domain with two cysteines covalently linked to palmitoyls and a C-terminal region enriched in Val, Leu and Ile. Native SP-C shows high surface activity, due to SP-C inserting in the bilayer with its cationic N-terminus binding to the polar headgroup and its hydrophobic C-terminus embedded as a tilted, transmembrane α-helix. The palmitoylcysteines in SP-C act as 'helical adjuvants' to maintain activity by overriding the β-sheet propensities of the native sequences. Objective. We studied SP-C peptides lacking palmitoyls, but containing glutamate and lysine at 4-residue intervals, to assess whether SP-C peptides with salt-bridges ("ion-locks") promote surface activity by mimicking the α-helix and membrane topography of native SP-C. Methods. SP-C mimics were synthesized that reproduce native sequences, but without palmitoyls (i.e., SP-Css or SP-Cff, with serines or phenylalanines replacing the two cysteines). Ion-lock SP-C molecules were prepared by incorporating single or double Glu(-)-Lys(+) into the parent SP-C's. The secondary structures of SP-C mimics were studied with Fourier transform infrared (FTIR) spectroscopy and PASTA, an algorithm that predicts β-sheet propensities based on the energies of the various β-sheet pairings. The membrane topography of SP-C mimics was investigated with orientated and hydrogen/deuterium (H/D) exchange FTIR, and also Membrane Protein Explorer (MPEx) hydropathy analysis. In vitro surface activity was determined using adsorption surface pressure isotherms and captive bubble surfactometry, and in vivo surface activity from lung function measures in a rabbit model of surfactant deficiency. Results. PASTA calculations predicted that the SP-Css and SP-Cff peptides should each form parallel β-sheet aggregates, with FTIR spectroscopy confirming high parallel β-sheet with 'amyloid-like' properties. The enhanced β-sheet properties for SP-Css and SP-Cff are likely responsible for their low surfactant activities in the in vitro and in vivo assays. Although standard (12)C-FTIR study showed that the α-helicity of these SP-C sequences in lipids was uniformly increased with Glu(-)-Lys(+) insertions, elevated surfactant activity was only selectively observed. Additional results from oriented and H/D exchange FTIR experiments indicated that the high surfactant activities depend on the SP-C ion-locks recapitulating both the α-helicity and the membrane topography of native SP-C. SP-Css ion-lock 1, an SP-Css with a salt-bridge for a Glu(-)-Lys(+) ion-pair predicted from MPEx hydropathy calculations, demonstrated enhanced surfactant activity and a transmembrane helix simulating those of native SP-C. Conclusion. Highly active SP-C mimics were developed that replace the palmitoyls of SP-C with intrapeptide salt-bridges and represent a new class of synthetic surfactants with therapeutic interest.

The bulk shear viscosities of aqueous dispersions of lavaged calf lung surfactant (LS) and its chloroform:methanol extract (CLSE) were measured as a function of concentration, shear rate and temperature. At 10-mg phospholipid per milliliter, dispersions of LS and vortexed CLSE in 0.15 M NaCl (saline) had low viscosities near 1 cp over a range of shear rates from 225 to 1125 s(-1). Lung surfactant viscosity increased with phospholipid concentration and became strongly non-Newtonian with higher values at low shear rates. At 37 degrees C and 40 mg/ml, LS and vortexed CLSE in saline had viscosities of 38 and 34 cp (77 s(-1)) and 12 and 7 cp (770 s(-1)), respectively. Viscosity values for LS and CLSE were dependent on temperature and, at fixed shear, were lower at 23 degrees C than at 37 or 10 degrees C. Hysteresis was also present in viscosity measurements depending on whether shear rate was successively increased or decreased during study. Addition of 5 mM Ca(2+) at 37 degrees C markedly reduced CLSE viscosity at all shear rates and decreased LS viscosity at low shear rates. Dispersion by sonication rather than vortexing increased the viscosity of CLSE at fixed shear, while synthetic phospholipids dispersed by either method had low, relatively Newtonian viscosities. The complex viscous behavior of dispersions of LS and CLSE in saline results from their heterogeneous aggregated microstructure of phospholipids and apoproteins. Viscosity is influenced not only by the aggregate surface area under shear, but also by phospholipid-apoprotein interactions and aggregate structure/deformability. Similar complexities likely affect the viscosities of biologically-derived exogenous surfactant preparations administered to patients in clinical surfactant therapy.

This work compares two reaction schemes for preparing 2,3-bis(fattyalkylthio)-1-propanols for further synthetic adaptation as hydrophobic analogs of lung surfactant phosphatidylcholines. An attempt to prepare 2,3-bis(fattyalkylthio)-1-propanols based on the previously published methods of Bell and co-workers (B.R. Ganong, C.R. Loomis, Y.A. Hannun, R.M. Bell, 1986. Proc. Natl. Acad. Sci. USA 83, 1184-1188; B.R. Ganong, R.M. Bell, 1987. Methods Enzymol. 141, 313-320; J.P. Walsh, L. Fahrner, R.M. Bell, 1990. J. Biol. Chem. 265, 4374-4381) was found to give the rearranged 1,3-bis(fattyalkylthio)-2-propanols as major products. As a reliable alternative, the reaction of ethyl 2,3-dibromopropionate with 2 equivalents of long chain sodium n-alkanethioates gave the corresponding ethyl 2,3-bis(n-alkylthio)propionates, which were then reduced with LiAlH4 to yield the desired 2,3-bis(fattyalkylthio)-1-propanols. Both 13C and 1H NMR spectroscopy were used to differentiate the two possible 1,3- and 2,3-dithio substituted alcohol products and to rigorously assign their structures.

We studied the mechanisms by which C16:0 lysophosphatidylcholine (LPC) and albumin inhibit the surface activity of calf lung surfactant extract (CLSE) by using a pulsating bubble apparatus with a specialized hypophase exchange system, plus adsorption and Wilhelmy balance measurements. In the absence of inhibitors, CLSE (1 mg phospholipid/mL) reached minimum surface tension (gamma(min))1 mN/m within 5 min of bubble pulsation at 20 cycles/min at 37 degrees C. Mixtures of CLSE:LPC had impaired surface activity depending on LPC content: gamma(min) was raised to 5 mN/m by 14 wt % LPC, to 15 mN/m by 25-30 wt% LPC, and to20 mN/m (67 wt % LPC), even at high CLSE concentrations (3 and 6 mg phospholipid/mL). In contrast, inhibition of CLSE by albumin was more easily abolished when surfactant concentration was raised. Mixtures of albumin (3 mg/mL) and CLSE (1 mg phospholipid/mL) had gamma(min)20 mN/m, but normal values of gamma(min)1 mN/m were reached at higher CLSE concentration (3 mg phospholipid/mL) even when albumin concentration was increased 8-fold to 24 mg/mL. In hypophase exchange studies, LPC, but not albumin, was able to penetrate preformed CLSE surface films and raise gamma(min) CLSE surface films with gamma(min)1 mN/m were isolated by an initial hypophase exchange with saline, and a second exchange with an LPC-containing hypophase raised gamma(min) to approximately 10 mN/m. CLSE surface films retained the ability to reach gamma(min)1 mN/m in analogous hypophase exchange studies with albumin. The ability of LPC to penetrate surface films of CLSE, although albumin could not, was also demonstrated in adsorption experiments in a Teflon dish, where diffusion was minimized by subphase stirring. Wilhelmy balance experiments also demonstrated that LPC could mix and interact with CLSE or dipalmitoyl phosphatidylcholine in solvent-spread surface films. The ability of LPC or other cell membrane lipids to penetrate interfacial films and raise gamma(min) even at high surfactant concentration may increase their inhibitory actions during acute lung injury.

This study examined the degree of additivity of several physiologically relevant protein and nonprotein inhibitors in impairing the surface activity of whole and extracted calf lung surfactant (LS and CLSE) on a pulsating bubble apparatus at 37 degrees C. Inhibitors investigated were albumin, hemoglobin, C16:0 and C18:1 lysophosphatidylcholine (LPC), oleic acid (OA), palmitoleic acid (PA), arachidonic acid (AA), and mixed red blood cell membrane lipids (RBCML). In the absence of inhibitors, LS (0.5 mg/ml) and CLSE (0.75 mg/ml) reached minimum surface tensions < 1 mN/m within 5 min of bubble pulsation (20 cycles/min, 50% area compression). Each inhibitor acting alone was able to reduce the surface activity of LS and CLSE, either raising minimum surface tension or increasing the time course of surface tension lowering or both. Several combinations of inhibitors exhibited additivity in impairing LS or CLSE activity at a lower concentration in mixtures than when present alone (albumin plus either C16:0 LPC, C18:1 LPC, or RBCML; hemoglobin plus either C16:0 LPC, C18:1 LPC, RBCML, PA, OA, or AA). The degree of additivity, however, was typically small in terms of the magnitude of reduction in inhibitor concentration or the rise in minimum surface tension relative to the effects of the most severe single inhibitor present. Substantial synergy was not found for any of the combinations of protein and nonprotein inhibitors investigated. Mixtures of albumin with PA or AA actually had a reduced inhibitory effect on LS and CLSE activity compared with the free fatty acids alone, apparently because of albumin binding of these molecules. In all cases, the detrimental effects of mixed inhibitors on LS and CLSE activity were reversed at increased surfactant concentration. These results indicate that surfactant dysfunction in acute respiratory distress syndrome (ARDS) could be increased in severity by interactions between some inhibitory substances, but that supplementation with exogenous CLSE would be effective in reversing inactivation by the mixtures of blood proteins, membrane lipids, and fatty acids studied.

The relative contributions of zwitterionic and anionic phospholipids to the surface-active function of calf lung surfactant extract (CLSE) were assessed by measurements of surface properties in vitro and pressure-volume (P-V) mechanics in excised rat lungs in situ. Surface activity and mechanical effects were compared for chromatographically purified CLSE subfractions containing the complete mix of phospholipids (PPL) or modified phospholipids depleted in anionic components (mPPL), alone or combined with 1.3% (by weight) of hydrophobic surfactant proteins (SP-B and SP-C). Surface pressure-time (pi-t) adsorption isotherms at 37 degrees C were very similar for dispersions of PPL and mPPL in a Teflon dish with a stirred subphase to minimize diffusion resistance. Combination of either PPL or mPPL with hydrophobic SP substantially improved adsorption, but mixtures of PPL:SP and mPPL:SP had only small differences in pi-t isotherms and reached the same final equilibrium pi of approximately 47 mN/m achieved by CLSE. Surface pressure-area (pi-A) isotherms and maximum surface pressures were also very similar for spread films of PPL versus mPPL and PPL:SP versus mPPL:SP on the Wilhelmy balance (23 degrees C and 37 degrees C). Respreading based on pi-A isotherm area calculations was slightly better in surface-excess films of PPL versus mPPL and PPL:SP versus mPPL:SP, but differences were minor and were smaller at 37 degrees C than at 23 degrees C. Overall dynamic surface activity in oscillating bubble studies was not significantly different for PPL versus mPPL or for PPL:SP versus mPPL:SP, and the latter two mixtures both reached minimum surface tensions1 mN/m (37 degrees C, 20 cycles/min, 0.5 mM phospholipid). Dispersions of PPL:SP, mPPL:SP, and CLSE were also not significantly different in improving P-V mechanics almost to normal when instilled in lavaged, excised rat lungs at 37 degrees C (30 mg/2.5 ml saline). These data suggest that zwitterionic phospholipids have a major role over anionic phospholipids in interacting with hydrophobic SP in the adsorption, dynamic surface tension lowering, film respreading, and pulmonary mechanical activity of the hydrophobic components of calf lung surfactant in CLSE.

This study investigates the importance of thioester-linked acyl groups in lung surfactant protein C (SP-C) in facilitating interactions with phospholipids that yield functionally important surface active behaviors. Native SP-C, palmitoylated at cysteine residues at positions 5 and 6, was isolated from bovine lung surfactant by liquid chromatography. Deacylated SP-C (dSP-C), unchanged in composition and sequence from SP-C but having a decreased alpha-helical content in films with dipalmitoyl phosphatidylcholine (DPPC) of 52 versus 70%, was obtained by treatment with 0.1 M sodium carbonate buffer at pH 10. Surface activity was studied for SP-C and dSP-C combined with column-purified phospholipids (PPL) from calf lung surfactant or with synthetic phospholipids (DPPC or a synthetic phospholipid mixture (SPL) containing 50:35:15, DPPC:egg phosphatidylcholine:egg phosphatidylglycerol). Interfacial measurements included surface pressure time adsorption isotherms for dispersed surfactants with diffusion minimized, dynamic surface pressure area isotherms and respreading for films in the Wilhelmy balance, and overall surface tension lowering at physiologic cycling rate in oscillating bubble experiments. Dispersions of PPL:SP-C and SPL:SP-C rapidly adsorbed to high equilibrium surface pressures of 47-48 mN/m, significantly better than corresponding dispersions containing dSP-C. The adsorption of PPL:dSP-C was essentially unchanged from that of PPL alone, and the adsorption of SPL:dSP-C was improved only slightly over SPL alone. In Wilhelmy balance studies, dynamic respreading was significantly improved over phospholipids alone in films of SP-C plus PPL, SPL, or DPPC. Respreading was improved less markedly by dSP-C in corresponding films with SPL or DPPC and not at all in films with PPL. Maximum surface pressures were also higher in cycled films of SP-C versus dSP-C combined with PPL or SPL. In bubble experiments (37 degrees C, 20 cycles/min), dispersions of PPL:SP-C and SPL:SP-C reached low minimum surface tensions of1 and 5 mN/m, respectively, whereas PPL:dSP-C and SPL:dSP-C only reached minima of approximately 20 mN/m as did PPL and SPL alone. Acylation in SP-C is crucial for its interactions with phospholipids over the full spectrum of adsorption and dynamic surface behaviors important for lung surfactant.

This study shows that the hydrophobic lung surfactant proteins (SP)-B and SP-C are not synergistic in enhancing functionally relevant surface behaviors in films and dispersions with phospholipids, and that SP-B is more effective than SP-C in facilitating surface activity. Purified bovine SP-B, SP. C, or SP-B/C (1/1 by wt) were combined with chroma tographically purified calf lung surfactant phospholipids (PPL), or with dipalmitoyl phosphatidylcholine (DPPC) or complex phospholipid mixtures containing 75% or 50% DPPC (75% SPL or 50% SPL). Adsorption was consistently better in corresponding mixtures of phospholipids plus SP-B versus SP-C, but was not improved further by substitution of SP-B/C for SP-B. Interfacial films of DPPC or SPL plus 1.3% SP-B or 1.3% SP-C had improved respreading compared to phospholipids alone (Wilhelmy balance, 23 degrees C and 37 degrees C), but substitution of mixed SP-B/C for either pure apoprotein did not increase respreading further. Surface-excess films of phospholipids plus SP-B had higher maximum surface pressures, or maintained a high maximum pressure through more consecutive compressions, than corresponding films with SP-C. Dispersions of phospholipids plus 1.3% SP-B or mixed (1/1) SP-B/C (2.6%) rapidly lowered surface tension to < 1 mN/m in oscillating bubble studies (20 cpm, 37 degrees C), while corresponding dispersions containing SP-C reduced surface tension more slowly or reached higher minima. Mixtures of 50% SPL with SP-B versus SP-C were also better able to resist inhibition by serum albumin in bubble and adsorption studies, and inhibition resistance was not significantly improved in mixtures containing 2.6% SP-B/C (1/1) versus 1.3% SP-B. The lack of synergy in hydrophobic apoprotein function, coupled with the greater effectiveness of SP-B in improving phospholipid adsorption, dynamic surface activity, and inhibition resistance, suggests that mixtures of phospholipids plus SP-B or related peptides may be particularly relevant its clinical exogenous surfactants.

Surface pressure-area (*-A) isotherms during dy- namic cycling were measured for films of dipalmitoyl phos- phatidylcholine (DPPC) and column-separated fractions of calf lung surfactant extract (CLSE). Emphasis was on defining the relative importance of lung surfactant phospholipids (PPL), neutral lipids (N), and hydrophobic proteins (SP) in facilitating dynamic respreading and surface tension lowering within the in- terfacial film itself. Solvent-spread films in a Wilhelmy balance were studied at 23' and 37OC over a range of cycling rates for initial concentrations giving both monomolecular and surface- excess films, A striking finding was that PPL films containing the complete mix of surfactant phospholipids had greatly im- proved dynamic respreading compared to DPPC, particularly in surface excess films (30 and 15 AVmolecule). Hydrophobic SP gave an additional increase in dynamic respreading in SP&PL comgared to PPL films for initial concentrations of 60, 30, and 15 A Z/molecule. Neutral lipids also improved respreading slightly in N&PL versus PPL films, but maximum surface pres- sures in N&PL films at 37OC were consistently the lowest of any surf9ctant subfraction. Spread films of SP&PL at 60 and 30 AVmolecule had lower maximum pressures than PPL, but maximum pressures were :lightly larger for SP&PL films at high initial concentration (15 A*/moIecuIe). Supplementary oscillat- ing bubble studies involving both adsorption and film dynamics at rapid cycling rate (20 cycles/min) showed that dispersions of CLSE and SP&PL lowered surface tension to

Thermal and lyotropic phase behavior was studied by X-ray diffraction and differential scanning calorimetry for a diether phosphonolipid analog (DEPN-8) of the major lung surfactant glycerophospholipid dipalmitoyl phosphatidylcholine (DPPC). DEPN-8 differs in an ether, rather than an ester, bond at the acyl chain-backbone linkage and a headgroup phosphonate (isosteric methylene substitution) versus phosphate constituent. Analysis of lamellar diffraction maxima demonstrated that at high relative humidity (98%) and temperatures below the liquid crystal phase transition (approximately 45 degrees C), DEPN-8 formed interdigitated bilayers with a characteristic periodicity of 41.9-46.5 A. At low humidity the gel phase DEPN-8 bilayers were characteristic of a normal L beta phase with a periodicity equivalent to DPPC (57-59 A). Above the liquid crystal thermal phase transition, bilayer spacing for both DEPN-8 and DPPC was 51-52 A, characteristic of the L alpha phase. Complete assessments of both lamellar and in-plane X-ray scattering used to construct electron density profiles and structure-factor plots for DEPN-8 defined more fully the interdigitated bilayer state at high humidity and low temperature. Compared to DPPC, it is energetically favorable for DEPN-8 to form interdigitated bilayers under conditions of excess water and low temperature. The flexible character of the ether bonds in DEPN-8 allows increased hydrophobic interactions between acyl chains, without generating a steric penalty from the increased packing density of the molecules. Additionally, the ether bond and the phosphonate moiety may allow for more energetically favorable interactions between the choline portion of the headgroup and water. The DEPN-8 ether linkage may also contribute to the improved adsorption and film respreading found previously for this phosphonolipid compared to DPPC.

This paper reports the chemical synthesis and purification of a novel phospholipase-resistant C16:0, C16:1 diether phosphonoglycerol with structural analogy to ester-linked anionic phosphatidylglycerol (PG) in endogenous pulmonary surfactant. This diether phosphonoglycerol (PG 1) is studied for phospholipase A(2) (PLA(2)) resistance and for surface activity in synthetic exogenous surfactants combined with Super Mini-B (S-MB) peptide and DEPN-8, a previously-reported diether phosphonolipid analog of dipalmitoyl phosphatidylcholine (DPPC, the major zwitterionic phospholipid in native lung surfactant). Activity experiments measured both adsorption and dynamic surface tension lowering due to the known importance of these surface behaviors in lung surfactant function in vivo. Synthetic surfactants containing 9 : 1 DEPN-8:PG 1 + 3% S-MB were resistant to degradation by PLA(2) in chromatographic studies, while calf lung surfactant extract (CLSE, the substance of the bovine clinical surfactant Infasurf®) was significantly degraded by PLA(2). The 9 : 1 DEPN-8:PG 1 + 3% S-MB mixture also had small but consistent increases in both adsorption and dynamic surface tension lowering ability compared to DEPN-8 + 3% S-MB. Consistent with these surface activity increases, molecular dynamics simulations using Protein Modeller, GROMACS force-field, and PyMOL showed that bilayers containing DPPC and palmitoyl-oleoyl-PC (POPC) as surrogates of DEPN-8 and PG 1 were penetrated to a greater extent by S-MB peptide than bilayers of DPPC alone. These results suggest that PG 1 or related anionic phosphono-PG analogs may have functional utility in phospholipase-resistant synthetic surfactants targeting forms of acute pulmonary injury where endogenous surfactant becomes dysfunctional due to phospholipase activity in the innate inflammatory response.

Increased phospholipase A2 activity demonstrated in some forms of lung injury may contribute to surfactant dysfunction. Phospholipase A2-resistant analogs of dipalmitoylphosphatidylcholine (DPPC) with surfactant properties might therefore be useful lipid components of treatment surfactants for certain lung injuries. The in vivo function of surfactants containing DPPC or the phospholipase-resistant analogs dihexadecylphosphatidylcholine (DEPC) or dihexadecylphosphonotidylcholine (DEPnC), with or without surfactant proteins B and C (SP-B+C), was thus evaluated in preterm rabbits (27 days' gestation). Rabbits randomly received one of seven surfactants (DPPC, DEPC, DEPnC, DPPC+SP-B+C, DEPC+SP-B+C, DEPnC+SP-B+C, or lipid extract surfactant [LES]) or 0.45% NaCl (control) and were ventilated for 30 min. Lipid-only surfactants decreased ventilatory pressures (peak inspiratory pressures minus positive end-expiratory pressure) relative to control (p0.05). Addition of SP-B+C further decreased ventilatory pressures to levels similar to LES (p0.01 versus control, lipid-only surfactants). Lung dynamic compliances and postventilation pressure-volume curves improved in the following order: LES, SP-B+C lipid surfactantslipid-only surfactantscontrol (p0.05). All surfactant preparations decreased intravascular 125I-albumin recoveries in the lungs relative to control (p0.01 for all surfactants versus control). These results indicate that DEPC and DEPnC were as effective as DPPC as lipid components of synthetic surfactants. And like DPPC, the analogs interacted with isolated SP-B+C and improved in vivo function to levels comparable to LES.

This study reports the biochemical separation of the hydrophobic constituents of calf lung surfactant into separate fractions from which specific components are excluded. Gel permeation chromatography on LH-20 with acidified chloroform-methanol separated the constituents of calf lung surfactant extract (CLSE) into fractions containing purified phospholipids (PPL), the neutral lipids and phospholipids (N&PL), or the hydrophobic surfactant proteins (SP)-B and -C together with the phospholipids (SP&PL). Extraction of acid to prevent phospholipid degradation after separation reduced recovery of the apoproteins in SP&PL. This fraction was therefore supplemented with protein purified separately to attain the initial levels present in CLSE. Biochemical analyses confirmed that the resulting preparations had the expected composition not only of protein, neutral lipids and phospholipids, but also of the phospholipid head groups. In addition to these fractions obtained with acidified solvent, elution of CLSE with chloroform-methanol without acid yielded the zwitterionic phospholipids substantially depleted of anionic phosphatidylglycerol and phosphatidylinositol. Limited interfacial measurements also demonstrated that the process of separation did not alter the fundamental surface characteristics of the surfactant constituents. Recombined CLSE (rCLSE) reconstituted from all of the separated components had surface activity indistinguishable from the original CLSE. The individual fractions of surfactant components also had average molecular areas at the air-liquid interface which agreed with predictions based on their biochemical composition. These well defined preparations of the hydrophobic constituents of pulmonary surfactant provide the basis for future studies to establish the role of individual components in the function of this complex surface active material.

Background Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., approximately residues 8-25 and 63-78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1-7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity. Methodology/results FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary alpha-helix and secondary beta-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a "saposin-like" fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B. Conclusion Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.

A homologous series of chiral (R) ether-amide phosphonolipid analogs of naturally occurring (R) glycerophospholipids were synthesized and characterized for their interfacial behaviors. The phosphonolipids possess isoteric ether, amide, and phosphonate functions at positions corresponding to the sn-1, sn-2, and sn-3 ester functions, respectively, of naturally occurring glycerophospholipids. All compounds were synthesized with disaturated C16:0 alkyl/acyl moieties to give structural analogy with dipalmitoyl phosphatidylcholine (DPPC), the major glycerophospholipid component of lung surfactant. Further substitutions at the headgroup nitrogen were also used to generate differences in headgroup size and polarity in the synthetic compounds. The surface activity of the ether-amide phospholipids was investigated in terms of adsorption to the air-water interface, together with studies of dynamic respreading after monolayer collapse and surface tension lowering in dynamically compressed spread films and dispersions. Results showed that several ether-amide phosphonolipids had more rapid adsorption and improved dynamic respreading behavior compared to DPPC, plus the ability to lower surface tension into the range of less than 1 to 4 mN/m in spread films and in dispersions under dynamic conditions. In combination with a series of diether phosphonolipids synthetized in a companion study [1], these ether-amide compounds are useful in the development of molecular structure-surface activity correlates for lung surfactant-related materials, and should assist in investigating the specificity of interactions between phospholipids and other pulmonary biological molecules.

Acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) are characterized by rapid-onset respiratory failure following a variety of direct and indirect insults to the parenchyma or vasculature of the lungs. Mortality from ALI/ARDS is substantial, and current therapy primarily emphasizes mechanical ventilation and judicial fluid management plus standard treatment of the initiating insult and any known underlying disease. Current pharmacotherapy for ALI/ARDS is not optimal, and there is a significant need for more effective medicinal chemical agents for use in these severe and lethal lung injury syndromes. To facilitate future chemical-based drug discovery research on new agent development, this paper reviews present pharmacotherapy for ALI/ARDS in the context of biological and biochemical drug activities. The complex lung injury pathophysiology of ALI/ARDS offers an array of possible targets for drug therapy, including inflammation, cell and tissue injury, vascular dysfunction, surfactant dysfunction, and oxidant injury. Added targets for pharmacotherapy outside the lungs may also be present, since multiorgan or systemic pathology is common in ALI/ARDS. The biological and physiological complexity of ALI/ARDS requires the consideration of combined-agent treatments in addition to single-agent therapies. A number of pharmacologic agents have been studied individually in ALI/ARDS, with limited or minimal success in improving survival. However, many of these agents have complementary biological/biochemical activities with the potential for synergy or additivity in combination therapy as discussed in this article.

The effects of surfactant protein (SP)-A on the dynamic surface tension lowering and resistance to inhibition of dispersions of calf lung surfactant extract (CLSE) and mixtures of synthetic phospholipids combined with SP-B,C hydrophobic apoproteins were studied at 37 degrees C and rapid cycling rate (20 cycles/min). Addition of SP-A to CLSE, which already contains SP-B and -C, gave a slight improvement in the time course of surface tension lowering on an oscillating bubble apparatus in the absence of inhibitory protein molecules such as albumin or hemoglobin. However, when these proteins were present at concentrations of 10-50 mg/ml, SP-A substantially improved the resistance of CLSE to their inhibitory effects. The beneficial effect of SP-A required the presence of Ca2+ ions, and disappeared when EDTA was substituted for this divalent cation in the subphase. The effect was also retained when SP-A was heated to 50 degrees C prior to addition to CLSE, but was abolished by heating SP-A to 99 degrees C. Additional studies showed that similar improvements in resistance to inhibition were found when SP-A was added to synthetic mixtures of dipalmitoyl phosphatidylcholine (DPPC):egg phosphatidylglycerol (PG) (80:20 by weight) reconstituted with 1% SP-B or SP-B and -C, but not to phospholipid mixtures containing only SP-C. The requirements for SP-B and calcium for the beneficial effects of SP-A on surface activity suggest that the formation of ordered, larger phospholipid-apoprotein aggregates may be involved in the process. The finding that SP-A enhances the ability of CLSE and other surfactant mixtures containing SP-B to resist inhibition is an advantage that will need to be weighed against other factors such as increased antigenicity and heat sensitivity in therapeutic applications in surfactant replacement therapy.

The biophysical activity and inhibition of a series of synthetic surfactant mixtures was studied and correlated with physiological effectiveness in restoring pressure-volume (P-V) mechanics of excised lungs. Results showed that several simple mixtures of dipalmitoyl phosphatidylcholine (DPPC) with fatty acids or diacylglycerols could be formulated to give good adsorption facility and dynamic surface tension lowering to less than 1 mN/m in pulsating bubble measurements at 37 degrees C. However, although biophysical activity approached that of natural lung surfactant (LS) and a related surfactant extract (CLSE) under normal conditions, surface properties were sharply inhibited by relatively small amounts of the plasma protein albumin (2 mg/ml) with minimum surface tensions greater than 30 nM/m even at high surfactant concentrations (5-20 mg lipids/ml). This sensitivity to biophysical inhibition was markedly increased compared to LS and CLSE, and had direct consequences for physiological efficacy: in spite of initially high activity, synthetic surfactants did not exert beneficial effects on P-V mechanics when instilled into surfactant-deficient excised rat lungs. Endogenous protein material was shown to be present upon surfactant recovery by lavage, and bubble measurements confirmed surface activity well below pre-instillation levels. Moreover, full biophysical activity was restored when lavage fluid was extracted to separate the synthetic surfactants from endogenous inhibitors. These results show that it is important to define relative sensitivity to biophysical inhibition in the development of effective lung surfactant substitutes. In addition, the existence of inhibition effects can generate an apparent lack of correspondence between initial biophysical activity and ultimate physiological actions of exogenous surfactant mixtures.

Background. This study examines the surface activity and resistance to phospholipase degradation of a fully-synthetic lung surfactant containing a novel diether phosphonolipid (DEPN-8) plus a 34 amino acid peptide (Mini-B) related to native surfactant protein (SP)-B. Activity studies used adsorption, pulsating bubble, and captive bubble methods to assess a range of surface behaviors, supplemented by molecular studies using Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD), and plasmon resonance. Calf lung surfactant extract (CLSE) was used as a positive control. Results. DEPN-8+1.5% (by wt.) Mini-B was fully resistant to degradation by phospholipase A2 (PLA2) in vitro, while CLSE was severely degraded by this enzyme. Mini-B interacted with DEPN-8 at the molecular level based on FTIR spectroscopy, and had significant plasmon resonance binding affinity for DEPN-8. DEPN-8+1.5% Mini-B had greatly increased adsorption compared to DEPN-8 alone, but did not fully equal the very high adsorption of CLSE. In pulsating bubble studies at a low phospholipid concentration of 0.5 mg/ml, DEPN-8+1.5% Mini-B and CLSE both reached minimum surface tensions ,1 mN/m after 10 min of cycling. DEPN-8 (2.5 mg/ml)+1.5% Mini-B and CLSE (2.5 mg/ml) also reached minimum surface tensions ,1 mN/m at 10 min of pulsation in the presence of serum albumin (3 mg/ml) on the pulsating bubble. In captive bubble studies, DEPN-8+1.5% Mini-B and CLSE both generated minimum surface tensions ,1 mN/m on 10 successive cycles of compression/expansion at quasi-static and dynamic rates. Conclusions. These results show that DEPN-8 and 1.5% Mini-B form an interactive binary molecular mixture with very high surface activity and the ability to resist degradation by phospholipases in inflammatory lung injury. These characteristics are promising for the development of related fully-synthetic lipid/peptide exogenous surfactants for treating diseases of surfactant deficiency or dysfunction.

This study investigates the activity and inhibition resistance in excised rat lungs of a novel synthetic surfactant containing the phospholipase-resistant diether phosphonolipid DEPN-8 plus 1.5% bovine surfactant protein (SP)-B/C compared to calf lung surfactant extract (CLSE). DEPN-8 + 1.5% SP-B/C surpassed CLSE in normalizing surfactant-deficient pressure-volume (P-V) deflation mechanics in lavaged excised lungs in the presence of phospholipase A(2) (PLA(2)) or C18:1 lyso-phosphatidylcholine (LPC). DEPN-8 + 1.5% SP-B/C had activity equal to CLSE in normalizing P-V mechanics in the absence of inhibitors or in the presence of serum albumin. These physiologic activity findings were directly consistent with surface activity measurements on the pulsating bubble surfactometer. In the absence of inhibitors, DEPN-8 + 1.5% SP-B/C and CLSE rapidly reached minimum surface tensions1 mN/m (0.5 and 2.5 mg surfactant phospholipid/ml). DEPN-8 + 1.5% SP-B/C maintained its high surface activity in the presence of PLA(2), while the surface activity of CLSE was significantly inhibited by exposure to this enzyme. DEPN-8 + 1.5% SP-B/C also had greater surface activity than CLSE in the presence of LPC, and the two surfactants had equivalent surface activity in the presence of albumin. DEPN-8 + 1.5% SP-B/C also had slightly greater surface activity than CLSE when exposed to peroxynitrite in pulsating bubble studies. These results support the potential of developing highly active and inhibition-resistant synthetic exogenous surfactants containing DEPN-8 + apoprotein/peptide constituents for use in treating direct pulmonary forms of clinical acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS).

Synthesis methods and initial surface property characterizations are reported for two sulfur-containing phosphonolipids related structurally to dipalmitoyl phosphatidylcholine (DPPC), the major lung surfactant glycerophospholipid. Sulfur linkages in these compounds affect molecular interactions relative to ester linkages, and are structurally resistant to cleavage by phospholipases. The SO2-linked analog synthesized here had increased adsorption and improved film respreading compared to DPPC, while reaching very low surface tensions (1 N/m) in cycled interfacial films on both the Wilhelmy balance and the pulsating bubble surfactometer. This compound appears to have potential utility as a component in future phospholipase-resistant synthetic exogenous surfactants for treating clinical forms of inflammatory lung injury.

Surface activity and sensitivity to inhibition from phospholipase A2(PLA2), lysophosphatidylcholine (LPC), and serum albumin were studied for a synthetic C16:0 diether phosphonolipid (DEPN-8) combined with 1.5% by weight of mixed hydrophobic surfactant proteins (SP)-B/C purified from calf lung surfactant extract (CLSE). Pure DEPN-8 had better adsorption and film respreading than the major lung surfactant phospholipid dipalmitoyl phosphatidylcholine and reached minimum surface tensions 2, and DEPN-8 + 1.5% SP-B/C maintained high adsorption and dynamic surface activity in the presence of this enzyme. Incubation of CLSE with PLA2led to chemical degradation, generation of LPC, and reduced surface activity. DEPN-8 + 1.5% SP-B/C was also more resistant than CLSE to direct biophysical inhibition by LPC, and the two were similar in their sensitivity to biophysical inhibition by serum albumin. These findings indicate that synthetic surfactants containing DEPN-8 combined with surfactant proteins or related synthetic peptides have potential utility for treating surfactant dysfunction in inflammatory lung injury.

The content-dependent activity of surfactant protein (SP)-B was studied in mixtures with dipalmitoyl phosphatidylcholine (DPPC), synthetic lipids (SL), and purified phospholipids (PPL) from calf lung surfactant extract (CLSE). At fixed SP-B content, adsorption and dynamic surface tension lowering were ordered as PPL/SP-B ≈ SL/SP-B > DPPC/SP-B. All mixtures were similar in having increased surface activity as SP-B content was incrementally raised from 0.05 to 0.75% by weight. SP-B had small but measurable effects on interfacial properties even at very low levels ≤0.1% by weight. PPL/SP-B (0.75%) had the highest adsorption and dynamic surface activity, approaching the behavior of CLSE. All mixtures containing 0.75% SP-B reached minimum surface tensions

Composition, surface activity and effects on pressure–volume (P–V) mechanics are examined for lavaged calf lung surfactant (LS) and the clinical exogenous surfactants Infasurf and Survanta. Lavaged LS and Infasurf had closely-matching compositions of phospholipids and neutral lipids. Survanta had higher levels of free fatty acids and triglycerides consistent with its content of added synthetic palmitic acid and tripalmitin. Infasurf and Survanta both contained less total protein than LS because of extraction with hydrophobic solvents, but the total protein content relative to phospholipid in Survanta was about 45% lower than in Infasurf. This difference was primarily due to surfactant protein (SP)-B, which was present by ELISA at a mean weight percent relative to phospholipid of 1.04% in LS, 0.90% in Infasurf, and 0.044% in Survanta. Studies on component fractions separated by gel permeation chromatography showed that SP-B was a major contributor to the adsorption, dynamic surface activity, and P–V mechanical effects of Infasurf, which approached whole LS in magnitude. Survanta had lower adsorption, higher minimum surface tension, and a smaller effect on surfactant-deficient P–V mechanics consistent with minimal contributions from SP-B. Addition of 0.05% by weight of purified bovine SP-B to Survanta did not improve surface or physiological activity, but added 0.7% SP-B improved adsorption, dynamic surface tension lowering, and P–V activity to levels similar to Infasurf. The SP-B content of lung surfactants appears to be a crucial factor in their surface activity and efficacy in improving surfactant-deficient pulmonary P–V mechanics.

The content of dipalmitoyl phosphatidylcholine (DPPC) in the phosphatidylcholine (PC) fraction of calf lung surfactant extract (CLSE) is measured by gas chromatography (GC) and estimated from the widely used osmium tetroxide assay for disaturated phosphatidylcholine (DSPC). The surface-active properties of model phospholipid/apoprotein surfactants with varying DPPC content are also defined and compared relative to CLSE. GC analysis of fatty acids in PC isolated from CLSE indicated a possible range of 30 to 65% for DPPC content depending on C16:0 fatty chain mismatching, and further studies using phospholipase A2 treatment indicated an actual DPPC content≤41%. The osmium tetroxide assay gave a very high value of 70% for the DSPC content of surfactant PC, and experiments with synthetic phospholipids demonstrated that this assay responded inappropriately in the presence of monounsaturated PC, leading to falsely elevated DSPC values. The influence of DPPC content on adsorption and film behavior was investigated in model surfactants containing 40, 60, and 80% DPPC (DPPC/egg PC/egg PG, 40:50:10, 60:30:10, and 80:10:10 by mol) combined with 1.3% hydrophobic surfactant protein (SP)-B and -C. The biophysical properties of the model surfactant with 40% DPPC were found to be closer to CLSE than those of mixtures with 60 or 80% DPPC. The adsorption of dispersions containing 40% DPPC with 1.3% SP-B, C was almost identical to CLSE and was improved in rate and magnitude compared with the mixtures with higher DPPC content (60 or 80%). In Wilhelmy balance studies of cycled films, respreading was increased and maximum surface pressure was decreased for the 40% versus higher DPPC content mixtures, again approaching CLSE in behavior. All synthetic phospholipid (SPL):SP mixtures lowered surface tension to

Rabbits treated with intravenous free fatty acid suffer an acute lung injury. Material obtained from these lungs by bronchoalveolar lavage (BAL) has dramatically impaired ability to lower surface tension in vitro despite normal levels of surfactant phospholipids. Although large quantities of surface-active inhibitors are present in BAL, their effects are not sufficient to explain the magnitude of surfactant inactivation. This study determines if alterations in the surfactant aggregates can explain the loss of surfactant function in fatty acid lung injury. In injured animals, the larger, most active surfactant particles recovered by centrifugal pelleting were found to be decreased in amount. The remaining large particles had reduced surface activity compared with control aggregates. In addition, large particles in injured animals had a higher density than control animals on sucrose gradients following equilibrium centrifugation. Interaction with serum components present in the injured BAL could explain these higher densities. The ability of the injured BAL to lower surface tension was improved by supplementation with normal levels of particles from injured lungs. Supplementation of injured BAL with control large aggregates improved activity further and restored the ability to lower surface tension to1 mN/m. Therefore both the decreased amount and activity of large surfactant aggregates in injured animals contributed significantly to the observed inactivation of surfactant. Diminished surfactant function from alteration in surfactant aggregates is a mechanism common to other forms of acute lung injury, and the design of therapies with exogenous surfactants in injured lungs will need to consider strategies that restore surfactant function towards normal.

The inhibitory effects of oleic acid (OA) on the surface activity of pulmonary surfactant were characterized by use of the oscillating bubble surfactometer, the Wilhelmy balance, and excised rat lungs. Oscillating bubble studies showed that OA prevented lavaged calf surfactant [0.5 mM phospholipid (PL)] from lowering surface tension below 15 mN/m at or above a molar ratio of OA/PL = 0.5. In contrast to inhibition of surfactant by plasma proteins, increasing the surfactant concentration did not eliminate inhibition by oleic acid, which occurred at OA/PL greater than 0.67 on the oscillating bubble even at surfactant concentrations of 1.5 and 12 mM PL. Studies of surfactant adsorption showed that preformed films of OA had little effect on the adsorption of pulmonary surfactant. Wilhelmy balance studies showed that OA did interfere with the ability of spread films of surfactant to reach low surface tensions during dynamic compression. Further balance experiments with binary films of OA and dipalmitoyl phosphatidylcholine showed that these compounds were miscible in surface films. Together these findings suggested that OA inhibited pulmonary surfactant activity by disrupting the rigid interfacial film responsible for the generation of very low surface tension during dynamic compression. Mechanical studies in excised rat lungs showed that instillation of OA gave altered deflation pressure-volume characteristics with decreased quasi-static compliance, indicating disruption of pulmonary surfactant function in situ. This alteration of mechanics occurred without major changes in the composition of lavaged PLs or in the tissue compliance of the lungs defined by mechanical measurements during inflation-deflation with saline.(ABSTRACT TRUNCATED AT 250 WORDS)

The biophysical properties and physiologic effects of a series of clinical exogenous pulmonary surfactants was compared to determine the importance of the hydrophobic apoproteins (SP-B and C) as constituents of these preparations. The three exogenous surfactants studied, calf lung surfactant extract (CLSE), Survanta (Surfactant-TA), and Exosurf, all contain dipalmitoyl phosphatidylcholine (DPPC) as their major constituent. CLSE and Survanta also contain 1 to 2% of SP-B,C but Exosurf has the additives hexadecanol and tyloxapol instead to enhance the activity of DPPC. In adsorption experiments, CLSE reached a final surface tension of 22 mN/m, and Survanta and Exosurf reached 28 and 38 mN/m, respectively. Addition of 1% by weight of an apoprotein isolate containing both SP-B and C to Exosurf slightly improved its adsorption. In oscillating bubble studies, CLSE and Survanta decreased surface tension to low values of less than 1 and 2 mN/m, respectively, but Exosurf achieved a minimum value of only 29 mN/m. Addition of SP-B,C to Exosurf improved this minimum to 1 mN/m and approached the behavior of mixtures of synthetic DPPC with SP-B,C. In both adsorption and pulsating bubble experiments, the minimum surface tensions found for Exosurf were almost identical to those generated by tyloxapol alone. In studies of physiologic activity, 20 mg of CLSE or Survanta restored the pressure-volume mechanics of lavaged, surfactant-deficient excised rat lungs to 95 and 50%, respectively, of normal prelavage levels. Instillation of Exosurf (37.5 mg) produced a minimal improvement of only 10% compared to 70% for mixtures containing 1% SP-B,C with either Exosurf or DPPC.(ABSTRACT TRUNCATED AT 250 WORDS)

Biophysical activity and inhibition characteristics were studied for a series of synthetic surfactants composed of purified bovine lung surfactant proteins (SP)-B or -C combined with dipalmitoyl phosphatidylcholine (DPPC) and egg-phosphatidylglycerol (PG) in a weight ratio of 80:20:1 DPPC/PG/protein. Surfactant protein preparations included two isolates of SP-B, confirmed by amino terminal sequence analysis, three isolates of SP-C which were free of SP-B by ELISA and Western blot analysis, and two isolates containing both SP-B and -C. In oscillating bubble studies, mixtures of phospholipids with isolates containing pure SP-B or both SP-B and -C lowered surface tension to less than 1 mN/m within 2 min of pulsation at both 1.25 mg/ml and 2.5 mg/ml. Phospholipids combined with isolates of pure SP-C also had substantial activity, lowering surface tension to between 1 mN/m and 3 mN/m depending on the SP-C preparation used. The surface activity of synthetic phospholipid-apoprotein admixtures was decreased significantly by the plasma proteins albumin and fibrinogen, but with varying characteristics depending on the apoprotein preparation involved. At 1.25 mg/ml phospholipid, admixtures containing either isolate of SP-B were inhibited by 10 mg/ml of plasma proteins, as were those with one of the SP-C preparations. However, admixtures containing the two other pure SP-C preparations were not inhibited until 20-50 mg/ml of albumin or fibrinogen were present. The highest resistance to inhibition was shown by admixtures containing one of the apoprotein isolates having both SP-B and -C.(ABSTRACT TRUNCATED AT 250 WORDS)

Lung development is associated with increases in specific phospholipids and proteins that function as critical pulmonary surfactant components. Attempts to characterize the pattern of surfactant development in fetal rat lungs have been hampered by the lack of a micromethod which will permit quantitative isolation of surface active components from small tissue specimens. As part of studies designed to elucidate the metabolic regulation of lung development in the rat, we developed sucrose density gradient centrifugation procedures to separate pulmonary phospholipids and proteins into a presumed surfactant (S) fraction and a residual (R) fraction. Electron microscopy of S pellets from mature fetuses identified predominant lamellar bodies and minimal contamination; incubation with 5 mM CaCl2 induced the appearance of tubular myelin figures, implying functional potential. This was confirmed by demonstrating low surface tension (less than 1 dyn/cm) in S, but not R, fractions at term gestation (21.5 days) and in 1-day-old neonatal lung isolates, based on dynamic measurements using the oscillating bubble technique. Surface activity was also high in the S pellets from fetuses at 20.5 days of gestation; however, at 19.5 days, minimum surface tension values of at least 19 dyne/cm were seen. These results correlated directly with biochemical analyses which indicated striking increases in three surfactant-associated proteins (SP-A, SP-B, and SP-C) after 19.5 days of gestation; a finding in agreement with previously reported data on the developmental increase of disaturated phosphatidylcholine in fetal rat lung. We conclude that isolation of S fraction components is valuable for demonstrating maturation of the fetal rat lung and may provide a useful tool for the study of regulatory mechanisms influencing surfactant production and function.

The effects of homopolymeric amino acids (molecular weight 2300 to 14,000) on the surface activity of dipalmitoyl phosphatidylcholine (DPPC) and DPPC/egg-phosphatidylglycerol (PG) were characterized by adsorption and dynamic surface tension lowering measurements at 37°C. Homopolyamino acids studied included poly l -leucine (poly-Leu) and poly- l -valine (poly-Val), since Leu and Val are known to be prominent in the structure of hydrophobic lung surfactant apoprotein SP-B and SP-C. In addition, several other homopolyamino acids with varying hydrophobicity index were also investigated, including poly- l -phenylalanine (poly-Phe), poly- l -serine (poly-Ser), poly- l -lysine (poly-Lys) and poly- l -glutamic acid (poly-Glu). Results showed that hydrophobic poly-Leu and poly-Phe at 1 and 10 weight percent greatly increased the adsorption facility of DPPC and DPPC/PG mixtures, with maximum surface pressures (up to 49 mN/m) near the equilibrium limit for phospholipid systems. In oscillating bubble studies, 1% mixture of poly-Leu or poly-Phe with DPPC or 8:2 DPPC/PG lowered surface tension into the range (near 1 mN/m) associated with active lung surfactant. In contrast, mixtures of DPPC and DPPC/PG with the more hydrophilic peptides poly-Ser, poly-Lys and poly-Glu showed little or no enhancement of surface activity over the phospholipids alone. Mixtures of poly-Val and phospholipids did not combine well with the simple co-sonication procedure used, and also exhibited little improvement in surface activity. Taken together, these results suggest that dydrophobicity is an important determinant for interactions of proteins with lung surfactant phospholipids, and that significant biophysical activity effects are not limited to highly specific amino acid sequences of the surfactant apoproteins, SP-B and SP-C. Simple homoamino acid polymers may provide useful model systems for investigating the specificity and character of some of these interactions, as well as the effectiveness of protein- phospholipidcombination in synthetic surfactant mixtures.

This paper studies the dynamic surface pressure-area (π-A) behavior of phosphatidylglycerol (PG) with a mixed fatty acid distribution (bacterial) in pure and binary mixed films with dipalmitoyl phosphatidylcholine (DPL). At 23°C, bacterial PG films generate maximum dynamic surface pressures of only 48–49 dyn/cm on a 0.15 M sodium chloride subphase for both dilute and surface excess initial conditions. By contrast, binary mixed films of 90:10 DPL/PG reach maximum π values of the order of 70 dyn/cm at similar conditions, the same as for pure DPL films. A collapse plateau ratio criterion is used to show that respreading after dynamic compression past film collapse is enhanced in 90:10 DPL/PG films as compared to pure DPL for a dilute surface initial condition, but not for the surface excess condition, at room temperature. Respreading in pure bacterial PG films is also slightly improved over corresponding pure DPL films for some initial conditions at 23°C, but the magnitude of this effect is not as large as might be expected from the significant unsaturated and cyclopropane fatty acid percentage present in bacterial PG. Differential Scanning Calorimetry (DSC) measurements on DPL/PG mixtures show decreasing T c with peak broadening as the percentage of bacterial PG is increased. The experiments here do not establish a clearly required functional role for 10% PG in pulmonary surfactant surface behavior. Further surface studies are suggested before long-term clinical trials of PG containing mixtures for exogenous replacement therapy in Neonatal Respiratory Distress Syndrome (RDS) are initiated on a widespread basis.

This work examines the dynamic surface pressure-area behavior of films of unconjugated bilirubin spread from chloroform solution at 22 degrees C on 0.15 M NaCl and buffered phosphate subphases. Film behavior is examined at pH 5.5, 7.4 and 8.0. The interactions of bilirubin in mixed films with dipalmitoyl phosphatidylcholine and with 9:1 dipalmitoyl phosphatidylcholine:dioleolyl phosphatidylcholine are examined at similar temperature and pH values. It is found that unconjugated bilirubin modifies the dynamic surface pressure-area behavior of phospholipid films in both the high and low surface pressure regimes, with bilirubin exerting its greatest effect at low pH where its subphase solubility is low. Because many premature infants suffering from the Respiratory Distress Syndrome (RDS) have accompanying hyperbilirubinemia, with possible bilirubin transport to the alveolar space, the interactions of bilirubin with phospholipid films are discussed in terms of potential effects on pulmonary surfactant in vivo.

Previous studies have shown that cerebral oxygen consumption (CMRO2) increases by nearly 50% at birth. The perinatal factors responsible for this increase are unknown; however, one possibility is that fetal CMRO2 is constrained by the normal intrauterine arterial PO2 (PaO2) of approximately 20 mmHg. We investigated this possibility in seven near-term chronically instrumented fetal sheep (131-138 days gestation) in which we inserted vascular catheters and an endotracheal tube. After 1-3 days recovery, we measured cerebral blood flow (CBF) with radiolabeled microspheres and calculated CMRO2. Measurements were made in utero under three conditions for each fetus: 1) nonventilated control; 2) ventilation with 3% O2-5% CO2-92% N2; and 3) ventilation with an inspired oxygen concentration sufficient to raise fetal PaO2 to normal newborn levels (mean 73 mmHg). A calf lung surfactant extract (CLSE) was instilled into the endotracheal tube of the fetus before ventilation to ensure adequate levels of alveolar surfactant and to maintain stable pH and arterial PCO2. The results showed that increasing fetal arterial PO2 to postnatal levels did not consistently increase CMRO2. CBF decreased as arterial O2 content (CaO2) rose, with an inverse hyperbolic response similar to that previously found to relate CBF to CaO2 during fetal hypoxic hypoxia. This indicates that the normally low intrauterine PaO2 does not intrinsically limit CMRO2 and implies that the rapid increase in CMRO2 at birth reflects the activation of specific cellular and physiological processes at (or near) this unique developmental event.

The lipophilic cation triphenylmethylphosphonium (TPMP+) and the potassium analog Rb+, were used to monitor the membrane potential (delta psi) of freshly isolated rabbit type II alveolar epithelial cells. Type II cells were found to accumulate TPMP+ rapidly at 37 degrees C in Hanks' balanced-salt solution with 5 microM tetraphenyl boron, but this accumulation was partially due to non-membrane potential dependent binding of TPMP+ to the cell. Lysophosphatidylcholine (lysoPC) was found to abolish delta psi and permitted correction for bound TPMP+ or Rb+. TPMP+ remaining in the cell following correction for binding represents the sum of mitochondrial and plasma membrane potential dependent accumulation. The accumulation of Rb+ by the type II cell was found to be independent of the mitochondrial membrane potential and indicated a trans-plasma membrane Rb+ distribution potential of -62.9 +/- 4 mV. A similar value was obtained by estimating the plasma membrane potential dependent accumulation of TPMP+ in type II cells whose mitochondria were depolarized with carbonylcyanide m-chlorophenylhydrazone (CCCP). The release of TPMP+ due to CCCP treatment also permitted an estimation for the trans-mitochondrial membrane potential of -141.8 +/- 10 mV. These techniques of membrane potential measurements were found to be sensitive to changes in delta psi induced by a number of inhibitors and ionophores. The ability to measure the membrane potential of the type II pneumocyte, and the changes caused by various agents, should be useful in characterizing the functional responses of this pulmonary surfactant producing cell.

This research studies the biophysical surface activity of synthetic phospholipids combined in vitro with purified lung surfactant apoprotein, having an Mr of 6000. Hydrophobic surfactant-associated protein (SAP-6) was delipidated and purified from both bovine and canine lung lavage, and was combined in vitro with a synthetic phospholipid mixture (SM) of similar composition to natural lung surfactant phospholipids. SM phospholipids were also combined and studied biophysically with another purified surfactant-associated protein, SAP-35. The biophysical activity of synthetic phospholipid-apoprotein combinants was assessed by measurements of adsorption facility and dynamic surface tension lowering ability at 37 degrees C. The SM-SAP-6 combinants had adsorption facility equivalent to natural lung surfactant, and to the surfactant extract preparations CLSE and surfactant-TA used in exogenous surfactant replacement therapy for the neonatal Respiratory Distress Syndrome (RDS). The synthetic phospholipid-SAP-6 combinants also lowered surface tension to less than 1 dyne/cm under dynamic compression in an oscillating bubble apparatus at concentrations as low as 0.5 mg phospholipid/ml. A striking finding was that this excellent dynamic surface activity was preserved as SAP-6 composition was reduced to values as low as 5 micrograms/5 mg SM phospholipid (0.1% SAP-6 protein), an order of magnitude less than the 1% protein content of CLSE and surfactant-TA. Mixtures of SM phospholipids plus SAP-35, the major surfactant glycoprotein, had significantly lower biophysical activity, which did not approach that of a functional lung surfactant. These results suggest that synthetic exogenous surfactants of potential utility for replacement therapy in RDS can be formulated by combining synthetic phospholipids in vitro with specifically purified, hydrophobic surfactant-associated protein, SAP-6.

Adsorption to the air-water interface of natural lung surfactant obtained by bovine lung lavage is compared and contrasted with the adsorption of mixtures of synthetic phospholipids and of extracted mixed lung lipids containing minimal protein. Surface pressure-time (pi-t) adsorption isotherms are measured at 35 degrees C for the surfactant mixtures as a function of the presence or absence of divalent metal cations (Ca2+ and Mg2+) and of heating to 45 degrees C or 90 degrees C. The effect of aqueous dispersion technique (sonication or mechanical vortexing) on the adsorption process is also studied for the extracted or synthetic phospholipid mixtures. The results imply that the protein component is necessary for the optimal adsorption of natural lung surfactant. However, by taking advantage of different methods available for phospholipid dispersion in an aqueous phase in vitro, it is possible to formulate dispersions of extracted lung phospholipids containing of order 1% protein which adsorb as well as the complete surfactant system. These results suggest that protein concentrations in surfactant mixtures can be minimized for applications such as exogenous lung surfactant replacement for the neonatal Respiratory Distress Syndrome (RDS). However, for situations which may involve alterations in endogenous surfactant function such as in lung injury, effects involving pulmonary surfactant protein and protein-lipid interactions may be of functional significance.

The adsorption of aqueous phospholipid dispersions containing dipalmitoyl phosphatidylcholine (DPPC) is investigated at 35-37 degrees C as a function of dispersion preparation technique. Systems studied in terms of surface pressure-time (pi-t) adsorption behavior were pure DPPC, 9:1 DPPC:dipalmitoyl phosphatidylethanolamine, 7:3 DPPC:egg phosphatidylglycerol (PG), and lipids extracted from cow lung lavage. The results show that pi-t characteristics can differ significantly depending on the technique by which the DPPC-containing mixtures are initially dispersed in 0.15 M NaCl solution. Examples of path dependence include the fact that DPPC, which will not adsorb at T = 35 degrees C when placed in powdered crystals on the subphase surface, exhibits measurable pi-t changes after subphase dispersion by sonication or by mechanical vortexing. For 7:3 DPPC:PG, dispersion by sonication on ice or by mechanical vortexing gives faster adsorption than dispersion by sonication without temperature control. The effect of heating to T = 45 degrees C, which is greater than the gel to liquid crystal transition temperature of DPPC (Tc = 41 degrees C), is found to be particularly detrimental to the adsorption of 7:3 DPPC:PG. Of the phospholipid mixtures studied, extracted cow lung lipids exhibited by far the greatest adsorption capability and also showed less path dependence than 7:3 DPPC:PG. Similarly, in terms of dispersion techniques investigated, sonication on ice tended to give the most rapid adsorption for a given phospholipid mixture.

Surfactant-associated protein of Mr = 35,000, SAP-35, is the major glycoprotein present in mammalian pulmonary surfactants. In this study, canine SAP-35 and several of its COOH-terminal peptides were purified and characterized by amino acid composition and NH2-terminal sequencing analysis. These proteins were then studied in terms of their specific lipid-binding characteristics and surface activity when combined with a synthetic phospholipid mixture, SM, chosen as an approximation of lung surfactant phospholipids. Purified, delipidated SAP-35 bound SM strongly. In contrast, SAP-21 (a non-collagenous fragment generated by collagenase digestion) bound phospholipid weakly; SAP-18 (an acidic COOH-terminal fragment comprising residues Gly-118 to Phe-231) did not bind phospholipid, demonstrating the importance of hydrophobic amino acid residues Gly-81 to Val-117 and the NH2-terminal collagenous domain in interaction of the SAP-35 with phospholipids. In surface activity experiments, purified SAP-35 enhanced the adsorption of SM phospholipids in terms of both rate and overall surface tension lowering. However, the adsorption facility of the SM-SAP-35 mixture did not approach that of either whole surfactant or the surfactant extract preparations, calf lung surfactant extract or surfactant-TA, used in exogenous surfactant replacement therapy for the neonatal respiratory distress syndrome. In addition, the dynamic surface activity of the SM-SAP-35 mixture was well below that of natural surfactant or surfactant extracts. This was also true of mixtures of SM phospholipids combined with the SAP-18 and SAP-21 fragments of SAP-35.

These experiments characterize the effects of albumin on the dynamic surface activity of natural lung surfactant (LS), and an extracted mixed lipid fraction (CLL), at physiologic temperature, humidity, and film cycling rate on an oscillating bubble apparatus. Measurements of albumin effects on the surface pressure-time (pi-t) adsorption isotherms of CLL and LS are also reported. Results show that albumin in concentrations greater than or equal to 20 mg/ml increased the minimum dynamic surface tension of LS suspensions (0.4 mg phospholipid/ml) from less than 1 dyne/cm to 21 dynes/cm at 37 degrees C. Albumin in low concentrations (2 mg/ml) had a similar detrimental effect on the dynamic surface activity of extracted surfactant lipids, CLL. In addition, albumin also inhibited the isolated adsorption facility of LS and CLL; instead of adsorbing rapidly to their maximum spreading pressures of 45 dynes/cm, both surfactant mixtures (at 0.063 and 0.125 mg phospholipid/ml) adsorbed more slowly or reached lower final surface pressures in the presence of plasma protein. A striking finding was that albumin inhibition of surface activity was moderated or abolished at high lipid concentrations. For example, minimum dynamic surface tensions less than 1 dyne/cm were reached on the oscillating bubble for natural LS at concentrations greater than 0.75 mg/ml and CLL at concentrations greater than 1.5 mg/ml, even in the presence of very large amounts of albumin (100 mg/ml). Similarly, LS and CLL adsorption facility was protected from albumin inhibition at sufficiently high phospholipid concentrations. Albumin inhibition of natural LS adsorption was also moderated by the presence of 1.4 mM Ca2+ ions. These results show that albumin in plasma transudates has the potential to seriously impair alveolar surfactant activity in vivo. However, the detrimental effect will be mitigated if a critical threshold of phospholipid is present.

The microstructure of aqueous dispersions of pulmonary surfactant lipids extracted from bovine lung lavage was investigated by electron microscopic analysis. Following organic solvent extraction (chloroform/methanol) from natural lung surfactant, the mixed lipids (CLL), with 1% residual protein, were dispersed in water by two techniques, probe sonication at 4 degrees C and mechanical vortexing. Surface pressure-time adsorption isotherms were defined for the CLL dispersions, followed by staining with tannic acid, uranyl acetate, and lead citrate for electron microscopy of microstructure. CLL extract dispersions adsorbed in seconds to surface pressures near 45 dynes/cm (surface tension 25 dynes/cm) at low concentrations less than or equal to 0.25 mg/ml after dispersion by sonication of 4 degrees C and by mechanical vortexing. Ultrastructurally, the CLL dispersions were somewhat heterogeneous, but large thin-walled phospholipid vesicles, both intact and fragmented, predominated. No tubular myelin was formed. By contrast, natural lung surfactant (LS) from bronchoalveolar lavage had characteristic regions of tubular myelin when it adsorbed well at low concentrations (less than or equal to 0.25 mg phospholipid/ml). When divalent cations were removed from solution with 5 mM EDTA, this distinctive microstructure was not present and natural LS adsorbed less rapidly; higher concentrations of 0.63 mg phospholipid/ml were necessary for maximal adsorption of natural LS without tubular myelin. These results suggest that while tubular myelin is associated with optimal adsorption for natural LS, it is not a required configuration for rapid adsorption facility at low phospholipid concentrations in general. Specifically, for dispersions of surfactant extracts, other microstructures allow adsorption facility equivalent to that of natural LS with tubular myelin.

Exposure to high concentrations of oxygen can result in tissue damage, particularly in the lung. Lung pathology induced by hyperoxia includes changes in lung cell populations and morphology. Presumably, alterations in gene expression underlie some of these cellular changes. In order to better understand the molecular basis of these events, a cDNA library was constructed from the mRNA of the lungs of a hyperoxia-exposed rabbit and differentially screened for clones corresponding to hyperoxia-induced messages. This approach has led to the isolation of four clones, three of which are presented in this communication. One clone corresponds to a message whose steady state levels were induced 6-fold and encodes the tissue inhibitor of metalloproteinases, a protein that plays a key role in the regulation of connective tissue turnover in some cells and potentiates erythroid development in others.

The dynamic surface pressure-area properties of pure and binary mixed films of rac-1,2-dipalmitoyl-glycero-3-phosphocholine, 1,2-diocoyl-sn-glycero-3-phosphocholine and cholesterol were investigated at 23 degrees C and 37 degrees C. The pure films and binary mixtures of rac-1,2-dipalmitoyl-glycero-3-phosphocholine: 1,2-dioeoyl-sn-glycero-3-phosphocholine and rac-1,2-dipalmitoyl-glycero-3-phosphocholine:cholesterol were characterized both for dilute surface initial concentrations of 150 Angstrom 2/molecule and for surface excess initial values of 15-50 Angstrom 2/molecule. The results show that the addition of 1,2-dioeoyl-sn-glycero-3-phosphocholine or cholesterol in a binary film with rac-1,2 dipalmitoyl-glycero-3-phosphocholine acts to improve the surface reentry and respreading properties over those of pure rac-1,2-dipalmitoyl-glycero-3-phosphocholine films upon dynamic compression past collapse. This effect is even more pronounced at 37 degrees C than at 23 degrees C, as demonstrated by the application of a collapse plateau ratio criterion. The enhanced dynamic respreading correlates with the effect of 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol in decreasing the gel to liquid crystal transition temperature of rac-1,2-dipalmitoyl-glycero-3-phosphocholine water dispersons, as well as with their facilitation of post-collapse dynamic surface pressure relaxation in binary films with rac-1,2-dipalmitoyl-glycero-3-phosphocholine.

Adult rabbits exposed to 100% O2 for 64 h and then returned to room air for up to 200 h, develop a lung injury characterized by decreased levels of alveolar surfactant followed by a rebound recovery. In the present study we isolated alveolar type II cells from rabbits at various times during hyperoxic exposure and recovery and measured rates of phosphatidylcholine (PC) synthesis, cellular lipid content, and the specific activity of glycerol 3-phosphate (G-3-P) acyltransferase, an enzyme that catalyzes one of the early reactions in phosphoglyceride biosynthesis. These biochemical parameters were compared with measurements of cell size and cell cycle phase by laser flow cytometry. Results showed that alterations in alveolar phospholipid levels in vivo correlated consistently with cellular lipid metabolic changes measured in isolated type II pneumocytes. In particular, alveolar pneumocytes isolated from lungs of rabbits exposed to 100% O2 for 64 h exhibited a 60% decrease in PC synthesis, cell lipid content, and G-3-P acyltransferase activity. All variables then followed a pattern of recovery to normal and ultimately supranormal levels beginning at approximately 3 days postexposure, at which point there was also a measured increase in the number of type II cells in S phase. These findings suggest that O2-induced changes in type II cell surfactant biosynthesis may account, at least in part, for observed changes in lung phospholipid levels in vivo.