Gender-specific elimination of continuous-infusional 5-fluorouracil in patients with gastrointestinal malignancies: results from a prospective population pharmacokinetic study.

BACKGROUND: This study was initiated to assess the quantitative impact of patient anthropometrics and dihydropyrimidine dehydrogenase (DPYD) mutations on the pharmacokinetics (PK) of 5-fluorouracil (5FU) and to explore limited sampling strategies of 5FU. PATIENTS AND METHODS: We included 32 patients with gastrointestinal malignancies, receiving 46-h continuous-infusional 5FU and performed PK-sampling at baseline, 15, 30, 45 min, 1 and 2 h after the start of infusion and at the end of infusion, for 2 subsequent cycles. Plasma concentrations of 5FU, 5-fluorodihydrouracil (5FUH2), uracil (U) and 5,6-dihydrouracil (UH2) were determined using LC-MS/MS and submitted to population PK analysis using nonlinear mixed-effects modeling. Broad genotyping of DPYD was performed, and the potential impact of the DPYD genotype on the elimination of 5FU was assessed. Limited sampling strategies were evaluated for their accuracy to predict steady-state concentrations of 5FU (CSS(5FU)), using data simulations based on the final PK-model. RESULTS: The area-under-the concentration-time curve of 5FU (AUC(5FU)) was found to be <20 mg h/L in 33 occasions (58 %), between 20 and 30 mg h/L in 17 occasions (30 %) and >30 mg h/L in 7 occasions (12 %). Men had a 26 % higher elimination of 5FU and a 18 % higher apparent elimination of 5FUH2. Accordingly, women had a higher AUC(5FU) compared to men (22 vs. 18 mg h/L, p = 0.04). No DPYD risk variants were found, and the DPYD variants detected (c.496A>G, c.1601G>A, c.1627A>G) were not significantly associated with the elimination of 5FU. Individual baseline UH(2)/U ratio was significantly associated with AUC(5FU) (R = -0.49, p < 0.001). Limited sampling strategies with time-points <3 h after the start of infusion were not adequate to predict CSS(5FU). Female gender was the only predictor of nausea/emesis in the multivariate model. CONCLUSIONS: Gender-specific elimination of 5FU is supported by the present data and may partly explain the gender-specific association between DPYD risk variants and 5FU-specific toxicity.

The origin and colonization history of the barley scald pathogen Rhynchosporium secalis.

The origins of pathogens and their past and present migration patterns are often unknown. We used phylogenetic haplotype clustering in conjunction with model-based coalescent approaches to reconstruct the genetic history of the barley leaf pathogen Rhynchosporium secalis using the avirulence gene NIP1 and its flanking regions. Our results falsify the hypothesis that R. secalis emerged in association with its host during the domestication of barley 10,000 to 15,000 years ago in the Fertile Crescent and was introduced into Europe through the migration of Neolithic farmers. Estimates of time since most recent common ancestor (2500-5000 BP) placed the emergence of R. secalis clearly after the domestication of barley. We propose that modern populations of R. secalis originated in northern Europe following a host switch, most probably from a wild grass onto cultivated barley shortly after barley was introduced into northern Europe. R. secalis subsequently spread southwards into already established European barley-growing areas.

Structures of pulmonary surfactant films adsorbed to an air-liquid interface in vitro.

Phospholipid films can be preserved in vitro when adsorbed to a solidifiable hypophase. Suspensions of natural surfactant, lipid extract surfactants, and artificial surfactants were added to a sodium alginate solution and filled into a captive bubble surfactometer (CBS). Surfactant film was formed by adsorption to the bubble of the CBS for functional tests. There were no discernible differences in adsorption, film compressibility or minimal surface tension on quasi-static or dynamic compression for films formed in the presence or absence of alginate in the subphase of the bubble. The hypophase-film complex was solidified by adding calcium ions to the suspension with the alginate. The preparations were stained with osmium tetroxide and uranyl acetate for transmission electron microscopy. The most noteworthy findings are: (1) Surfactants do adsorb to the surface of the bubble and form osmiophilic lining layers. Pure DPPC films could not be visualized. (2) A distinct structure of a particular surfactant film depends on the composition and the concentration of surfactant in the bulk phase, and on whether or not the films are compressed after their formation. The films appear heterogeneous, and frequent vesicular and multi-lamellar film segments are seen associated with the interfacial films. These features are seen already upon film formation by adsorption, but multi-lamellar segments are more frequent after film compression. (3) The rate of film formation, its compressibility, and the minimum surface tension achieved on film compression appear to be related to the film structure formed on adsorption, which in turn is related to the concentration of the surfactant suspension from which the film is formed. The osmiophilic surface associated surfactant material seen is likely important for the surface properties and the mechanical stability of the surfactant film at the air-fluid interface.

Comparison of inhibitory effects of oxygen radicals and calf serum protein on surfactant activity.

The effects of the reactive oxygen species (ROS) superoxide anion (O2*-) and hydroxyl radical (*OH) on the surface tension lowering properties of bovine lipid extract surfactant (BLES) were compared to the effects of calf serum protein (CSP) in a captive bubble surfactometer (CBS). O2*- was generated from xanthine/xanthine oxidase (X/XO), and *OH was generated by the Fenton reaction. ROS were demonstrated by electron spin resonance (ESR) using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin trap. Lipid peroxidation was measured using the thiobarbituric acid method. *OH had broad inhibitory effects on surface tension parameters, including adsorption, minimum surface tension, percentage film area change and film compressibility. O2*- showed inhibitory effects on adsorption, film area change and film compressibility but had no significant effect on minimum surface tension. Both O2*- and *OH treatment were associated with a large 'squeezeout' plateau around 20-25 mN/m in the surface tension-area relation, indicating poor film organization during the compression phase. At the concentrations used, ROS were associated with lipid peroxidation of BLES, which also demonstrated radical scavenging properties. Calf serum protein produced inhibitory effects on adsorption, minimum surface tension and percentage film area change that were quantitatively similar to those produced by *OH. The effects on film compression were significantly greater and qualitatively different from those seen with either O2*- or *OH. We conclude that the inhibition of BLES surface activity by ROS and inhibitory proteins can be distinguished in the captive bubble surfactometer and, particularly, by changes in the film compressibility modulus.

Effects of fixatives on function of pulmonary surfactant.

The structure of pulmonary surfactant films remains ill defined. Although plausible film fragments have been imaged by electron microscopy, questions about the significance of the findings and even about the true fixability of surfactant films by the usual fixatives glutaraldehyde (GA), osmium tetroxide (OsO(4)), and uranyl acetate (UA) have not been settled. We exposed functioning natural surfactant films to fixatives within a captive bubble surfactometer and analyzed the effect of fixatives on surfactant function. The capacity of surfactant to reach near-zero minimum surface tension on film compression was barely impaired after exposure to GA or OsO(4). Although neither GA nor OsO(4) prevented the surfactant from forming a surface active film, GA increased the equilibrium surface tension to above 30 mN/m, and both GA and OsO(4) decreased film stability as seen in the slowly rising minimum surface tension from 1 to ~5 mN/m in 10 min. In contrast, the effect of UA seriously impaired surface activity in that both adsorption and minimum surface tension were substantially increased. In conclusion, the fixatives tested in this study are not suitable to fix, i.e., to solidify, surfactant films. Evidently, however, OsO(4) and UA may serve as staining agents.

CSTX-9, a toxic peptide from the spider Cupiennius salei: amino acid sequence, disulphide bridge pattern and comparison with other spider toxins containing the cystine knot structure.

CSTX-9 (68 residues, 7530.9 Da) is one of the most abundant toxic polypeptides in the venom of the wandering spider Cupiennius salei. The amino acid sequence was determined by Edman degradation using reduced and alkylated CSTX-9 and peptides generated by cleavages with endoproteinase Asp-N and trypsin, respectively. Sequence comparison with CSTX-1, the most abundant and the most toxic polypeptide in the crude spider venom, revealed a high degree of similarity (53% identity). By means of limited proteolysis with immobilised trypsin and RP-HPLC, the cystine-containing peptides of CSTX-9 were isolated and the disulphide bridges were assigned by amino acid analysis, Edman degradation and nanospray tandem mass spectrometry. The four disulphide bonds present in CSTX-9 are arranged in the following pattern: 1-4, 2-5, 3-8 and 6-7 (Cys6-Cys21, Cys13-Cys30, Cys20-Cys48, Cys32-Cys46). Sequence comparison of CSTX-1 with CSTX-9 clearly indicates the same disulphide bridge pattern, which is also found in other spider polypeptide toxins, e.g. agatoxins (omega-AGA-IVA, omega-AGA-IVB, mu-AGA-I and mu-AGA-VI) from Agelenopsis aperta, SNX-325 from Segestria florentina and curtatoxins (CT-I, CT-II and CT-III) from Hololena curta. CSTX-1/CSTX-9 belong to the family of ion channel toxins containing the inhibitor cystine knot structural motif. CSTX-9, lacking the lysine-rich C-terminal tail of CSTX-1, exhibits a ninefold lower toxicity to Drosophila melanogaster than CSTX-1. This is in accordance with previous observations of CSTX-2a and CSTX-2b, two truncated forms of CSTX-1 which, like CSTX-9, also lack the C-terminal lysine-rich tail.

Surfactant protein interactions with neutral and acidic phospholipid films.

The captive bubble tensiometer was employed to study interactions of phospholipid (PL) mixtures of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) at 50 microg/ml with physiological levels of the surfactant protein (SP) A SP-B, and SP-C alone and in combination at 37 degrees C. All surfactant proteins enhanced lipid adsorption to equilibrium surface tension (gamma), with SP-C being most effective. Kinetics were consistent with the presence of two adsorption phases. Under the conditions employed, SP-A did not affect the rate of film formation in the presence of SP-B or SP-C. Little difference in gamma(min) was observed between the acidic POPG and the neutral POPC systems with SP-B or SP-C with and without SP-A. However, gamma(max) was lower with the acidic POPG system during dynamic, but not during quasi-static, cycling. Considerably lower compression ratios were required to generate low gamma(min) values with SP-B than SP-C. DPPC-POPG-SP-B was superior to the neutral POPC-SP-B system. Although SP-A had little effect on film formation with SP-B, surface activity during compression was enhanced with both PL systems. In the presence of SP-C, lower compression ratios were required with the acidic system, and with this mixture, SP-A addition adversely affected surface activity. The results suggest specific interactions between SP-B and phosphatidylglycerol, and between SP-B and SP-A. These observations are consistent with the presence of a surface-associated surfactant reservoir which is involved in generating low gamma during film compression and lipid respreading during film expansion.

Alveolar surface forces and lung architecture.

The entire alveolar surface is lined by a thin fluid continuum. As a consequence, surface forces at the air-liquid interface are operative, which in part are transmitted to the delicate lung tissue. Morphologic and morphometric analyses of lungs show that the alveolar surface forces exert a moulding effect on alveolar tissue elements. In particular, in lungs at low degrees of inflation, equivalent to the volume range of normal breathing, there is a derecruitment of alveolar surface area with increasing surface tensions which reflects equilibrium configurations of peripheral air spaces where the sum of tissue energy and surface energy is minimum. Thus, changes in surface tension alter the recoil pressure of the lung directly and indirectly by deforming lung tissue and hence changing tissue tensions. However, the interplay between tissue and surface forces is rather complex, and there is a marked volume dependence of the shaping influence of surface forces. With increasing lung volumes the tissue forces transmitted by the fiber scaffold of the lung become the predominant factor of alveolar micromechanics: at lung volumes of 80% total lung capacity or more, the alveolar surface area-volume relation is largely independent of surface tension. Most important, within the range of normal breathing, the surface tension, its variations and the associated variations in surface area are small. The moulding power of surface forces also affects the configuration of capillaries, and hence the microcirculation, of free cellular elements such as the alveolar macrophages beneath the surface lining layer, and of the surfaces of the peripheral airways. Still enigmatic is the coupling mechanism between the fluid continua of alveoli and airways which might also be of importance for alveolar clearance. As to the surface active lining layer of peripheral air spaces, which determines alveolar surface tension, its structure and structure-function relationship are still ill-defined owing to persisting problems of film preservation and fixation. Electron micrographs of alveolar tissue, of lining layers of captive bubbles, and scanning force micrographs of surfactant films transferred on mica plates reveal a complex structural pattern which precludes so far the formulation of an unequivocal hypothesis.

Surface activity in situ, in vivo, and in the captive bubble surfactometer.

For studies of the mechanical effects of lung surfactants, the captive bubble surfactometer (CBS) combines the advantages of the continuous film of Pattle's bubbles with the feasibility of the Langmuir-Wilhelmy balance to produce surface tension-area hysteresis loops. The CBS allows the compression of films to very low and stable surface tensions of 1-2 mN/m. Such low and stable surface tensions are in line with results obtained from pressure-volume studies on excised lungs. In addition, the CBS is useful to test other essential physical properties of the surfactant system, including: (1) rapid film formation (within seconds) through adsorption from the hypophase; (2) low film compressibility with a fall in surface tension to very low (<2 mN/m) values during surface compression; and (3) effective replenishment of the surface film on expansion by the incorporation of surfactant material from material associated with the surface (the surface associated surfactant reservoir). Morphological observations of films fixed in situ or in vitro reveal frequently their multilayered structure, which is consistent with the concept of the surface reservoir. The deviation of the bubbles from a Laplacian shape at very low surface tension and the morphological observations suggest that the surfactant film cannot be considered a simple monolayer.

Surface activity in vitro: role of surfactant proteins.

Pattle, who provided some of the initial direct evidence for the presence of pulmonary surfactant in the lung, was also the first to show surfactant was susceptible to proteases such as trypsin. Pattle concluded surfactant was a lipoprotein. Our group has investigated the roles of the surfactant proteins (SP-) SP-A, SP-B, and SP-C using a captive bubble tensiometer. These studies show that SP-C>SP-B>SP-A in enhancing surfactant lipid adsorption (film formation) to the equilibrium surface tension of approximately 22-25 mN/m from the 70 mN/m of saline at 37 degrees C. In addition to enhancing adsorption, surfactant proteins can stabilize surfactant films so that lateral compression induced through surface area reduction results in the lowering of surface tension (gamma) from approximately 25 mN/m (equilibrium) to values near 0 mN/m. These low tensions, which are required to stabilize alveoli during expiration, are thought to arise through exclusion of fluid phospholipids from the surface monolayer, resulting in an enrichment in the gel phase component dipalmitoylphosphatidylcholine (DPPC). The results are consistent with DPPC enrichment occurring through two mechanisms, selective DPPC adsorption and preferential squeeze-out of fluid components such as unsaturated phosphatidylcholine (PC) and phosphatidylglycerol (PG) from the monolayer. Evidence for selective DPPC adsorption arises from experiments showing that the surface area reductions required to achieve gamma near 0 mN/m with DPPC/PG samples containing SP-B or SP-A plus SP-B films were less than those predicted for a pure squeeze-out mechanism. Surface activity improves during quasi-static or dynamic compression-expansion cycles, indicating the squeeze-out mechanism also occurs. Although SP-C was not as effective as SP-B in promoting selective DPPC adsorption, this protein is more effective in promoting the reinsertion of lipids forced out of the surface monolayer following overcompression at low gamma values. Addition of SP-A to samples containing SP-B but not SP-C limits the increase in gamma(max) during expansion. It is concluded that the surfactant apoproteins possess distinct overlapping functions. SP-B is effective in selective DPPC insertion during monolayer formation and in PG squeeze-out during monolayer compression. SP-A can promote adsorption during film formation, particularly in the presence of SP-B. SP-C appears to have a superior role to SP-B in formation of the surfactant reservoir and in reinsertion of collapse phase lipids.

Surface activity and film formation from the surface associated material of artificial surfactant preparations.

Surfactant proteins B and C (SP-B and SP-C) are present in natural derived surfactant preparations used for treatment of respiratory distress syndrome. Herein the surface activity of an SP-C analogue (SP-C(LKS)), a hybrid peptide between SP-C and bacteriorhodopsin (SP-C/BR) and a model peptide (KL(4)) was studied with a captive bubble surfactometer (CBS). The peptides were mixed with either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidylglycerol (PG) (7:3, by weight) or DPPC/PG/palmitic acid (68:22:9, by weight) at a concentration of 1 mg/ml in HEPES buffer, pH 6.9 and a polypeptide/lipid weight ratio of 0.02--0.03. In some lipid/peptide preparations also 2% of SP-B was included. Adsorption, monitored as surface tension vs. time for 10 min after bubble formation did not show discernible differences for the whole set of preparations. Equilibrium surface tensions of approximately 25 mN/m were reached after 5--10 min for all preparations, although those with SP-C/BR appeared not to reach end point of adsorption within 10 min. Area compression needed to reach minimum surface tension of 0.5--2.0 mN/m was least for the KL(4) preparation, about 13% in the first cycle. 3% SP-C(LKS) in DPPC:PG (7:3, by weight) reached minimum surface tension upon 27% compression in the first cycle. If DPPC:PG:PA (68:22:9, by weight) was used instead only 16% area compression was needed and 14% if also 2% SP-B was included. 3% SP-C(LKS) in DPPC:PG (7:3, by weight)+2% SP-B needed 34% compression to reach minimum surface tension. The replenishment of material from a surface associated surfactant reservoir was estimated with subphase depletion experiments. With the 2% KL(4) preparation incorporation of excess material took place at a surface tension of 25--35 mN/m during stepwise bubble expansion and excess material equivalent to 4.3 monolayers was found. When 2% SP-B was added to 3% SP-C(LKS) in DPPC:PG (7:3, by weight) the number of excess monolayers increased from 1.5 to 3.6 and the incorporation took place at 30--40 mN/m. When SP-B was added to 3% SP-C(LKS) in DPPC:PG:PA (68:22:9, by weight) the number of excess monolayers increased from 0.5 to 3.4 and incorporation took place at 40--50 mN/m. With 2% SP-C/BR incorporation took place at 40--45 mN/m, frequent instability clicks were observed and excess material of approximately 1.1 monolayer was estimated.

Semi-preparative gas chromatographic separation of all-trans-perhydrotriphenylene enantiomers on a chiral cyclodextrin stationary phase.

Enantiomers of all-trans-perhydrotriphenylene (PHTP) were separated by gas chromatography using heptakis(6-O-tert.-butyldimethylsilyl-2,3-di-O-methyl)-beta-cyclodextrin (TBDMS-beta-CD) as the chiral selector. Conditions for semi-preparative separations were established using a 2 m x 2 mm I.D. packed column and subsequently extended to a 1.8 m x 4 mm I.D. column which enabled separations on a mg scale. The column packing was TBDMS-beta-CD dissolved in SE-54 coated on Chromosorb P AW-DMCS 80-100 mesh. Optimization of the chromatographic conditions (oven temperature, carrier gas flow, and column load) with respect to better efficiency and peak retention resulted in a system capable of separating up to 10 mg of the racemate per day. Purities of separated enantiomers were determined by capillary gas chromatography. Yields and purities of the fractions obtained by single- and double-step separations are compared. Highly enriched enantiomers with purities of up to 99.6% (99.2% ee) were obtained by a single separation step.

Surfactant phospholipids and surface activity among preterm infants with respiratory distress syndrome who develop bronchopulmonary dysplasia.

Our objective was to determine if preterm infants with respiratory distress syndrome who develop bronchopulmonary dysplasia have abnormalities in surfactant phospholipids and/or function. Tracheal aspirate samples obtained from preterm infants with respiratory distress syndrome on days 1, 3-5, 7-10, 14-17, 21-24 and 27-30 were analyzed for total phospholipids and phospholipids fractions by determination of total phospholipid phosphorus and thin layer chromatography, respectively. Surfactant properties were assessed with captive bubble surfactometer. Sixteen out of 56 (29%) infants died during the first 30 d of life. Infants who died were more immature, required more ventilatory support and had a surfactant with lower surface-tension-reducing properties than infants who survived (p < 0.05). Surviving infants were divided into group I (no bronchopulmonary dysplasia at 27-30 d, n = 25) and group II (with bronchopulmonary dysplasia at 27-30 d, n = 15). No significant differences in concentrations of surfactant phospholipids nor measurements of surface tension were noted among groups of infants. Surfactant therapy after birth was associated with a significant increase in concentrations of total phospholipids, lecithin, phosphatidylinositol and lower surface-tension measurements at 3-5 d of age among surviving infants (p < 0.01). Abnormalities in concentrations of surfactant phospholipids or surfactant function could not be demonstrated during the first month of life among preterm infants with respiratory distress syndrome who develop bronchopulmonary dysplasia.

Effect of budesonide and salbutamol on surfactant properties.

The objective of this study was to evaluate the in vitro effect of budesonide and salbutamol on the surfactant biophysical properties. The surface-tension properties of two bovine lipid extracts [bovine lipid extract surfactant (BLES) and Survanta] and a rat lung lavage natural surfactant were evaluated in vitro by the captive bubble surfactometer. Measurements were obtained before and after the addition of a low and high concentration of budesonide and salbutamol. Whereas salbutamol had no significant effect, budesonide markedly reduced the surface-tension-lowering properties of all surfactant preparations. Surfactant adsorption (decrease in surface tension vs. time) was significantly reduced (P < 0.01) at a high budesonide concentration with BLES, both concentrations with Survanta, and a low concentration with natural surfactant. At both concentrations, budesonide reduced (P < 0.01) Survanta film stability (minimal surface vs. time at minimum bubble volume), whereas no changes were seen with BLES. The minimal surface tension obtained for all surfactant preparations was significantly higher (P < 0.01), and the percentage of film area compression required to reach minimum surface tension was significantly lower after the addition of budesonide. In conclusion, budesonide, at concentrations used therapeutically, adversely affects the surface-tension-lowering properties of surfactant. We speculate that it may have the same adverse effect on the human surfactant.

[The mucociliary system of the lung--role of surfactants]. / Der mukoziliäre Apparat der Lunge--die Rolle des Surfactant.

Many pollution particles enter the organism via the lung. In the lung, on a surface of 140 m2, the blood is separated from the air by a tissue barrier of only 1/1000 mm. The conducting airways (trachea, bronchi, bronchioli) are a very effective aerodynamic filter for inhaled particles. The mucociliary transport system functions like a self-cleaning mechanism within the filter. Inhaled particles and particles deposited in the lungs play a crucial aetiological and therapeutic role. The discussion in health policy on the relationship between the increase in air pollution and lung damage is of great importance at the present time. Epidemiological studies of recent years have shown very clearly that there is a correlation between morbidity and mortality as a consequence of respiratory and cardiogenic problems and the concentration of PM10 particles in ambient air. So far, however, this correlation has not been explained. The intrathoracic airways are coated by a respiratory epithelium. This has an irregular coating of viscous liquid, consisting of a low viscous sol phase and a high viscous gel phase. It seems, however, that those phases are not clearly distinguishable. The gel phase is moved towards the pharynx by the metachronal ciliary beat transporting the particles out of the lungs. Furthermore, at the air-liquid interface, there exists a continuous surfactant film which reduces the surface tension as is the case in the alveoli. When particles are deposited on the airway wall, that is, on the surfactant film, they are wetted by surface forces and displaced into the liquid phases. Thus, the surfaces of the particles are probably changed by the surfactant or by surfactant components. Many of these particles are transported in the liquid (gel phase) towards the pharynx (mucociliary transport), whereas some of them remain in close association with the epithelium (sol phase). Such particles remain in the airways for days or even weeks. They are either phagocytised by macrophages and carried off via the airways or taken up by dendritic cells and transported into the tissue from where they reach the lymph nodes via lymph drainage and are presented to the T-lymphocytes. The displacement of particles into the liquid phases, caused by the surfactant, can be considered as the initial step in a complex cascade of defence processes in the lungs. The surface of the particles is probably modified by surfactant or surfactant components. These modified particles may be directed to that clearance pathway which is most beneficial for our health, that is, out of the lungs or into the lymphatic glands, where an immune reaction can be triggered. We therefore consider surfactant to be a primary immune barrier.

Palmitoylation of a pulmonary surfactant protein C analogue affects the surface associated lipid reservoir and film stability.

Surfactant protein C (SP-C) is a lipopeptide that contains two thioester-linked palmitoyl groups and is considered to be important for formation of the alveolar surface active lipid film. Here, a non- or dipalmitoylated SP-C analogue (SP-C(Leu)), in which all helical Val residues were replaced with Leu and Cys-5 and Cys-6 were replaced with Ser, was tested for surface activity in a captive bubble system (CBS). SP-C(Leu), either palmitoylated at Ser-5 and Ser-6 or non-palmitoylated, was added to mixtures of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidyl glycerol (PG)/palmitic acid (PA), 68:22:9, (by mass) at a concentration of 2 and 5%. With 2% peptide, surface film formation was rapid, reaching a surface tension below 25 mN/m within 5 s, but the samples with 5% SP-C(Leu) required more than 20 s to reach values below 25 mN/m. Minimum surface tension for the samples with dipalmitoylated SP-C(Leu) was below 1.5 mN/m and very stable, as the surface tension increased by less than 0.5 mN/m within 10 min at constant bubble volume. Minimum surface tension for the non-palmitoylated SP-C(Leu) was approximately 2 and 5 mN/m for 2 and 5% peptide, respectively, but the films were less stable as seen by frequent bubble clicking at low surface tensions. Films with dipalmitoylated SP-C(Leu) that were dynamically cycled at 20-30 cycles/min were substantially less compressible at a surface tension of 20 mN/m (0.007 m/mN) than those that contained the non-palmitoylated peptide (0.02 m/mN). After subphase depletion, the incorporation of lipids into the surface active film during initial bubble expansion occurred at a relatively low surface tension (about 35 mN/m) for the samples with dipalmitoylated SP-C(Leu) compared to approximately 45 mN/m for those containing the non-palmitoylated peptide. Furthermore, for samples that contained non-palmitoylated SP-C(Leu), the ability to reach near zero stable surface tension was lost after a few adsorption steps, whereas with the dipalmitoylated peptide the film quality did not deteriorate even after more than 10 expansion steps and the incorporation of reservoir material equivalent to more than two monolayers. It appears that the covalently linked palmitoyl groups of the SP-C analogue studied are important for the mechanical stability of the lipid film, for the capacity to incorporate material from the reservoir into the surface active film upon area expansion, and for the low film compressibility of dynamically cycled films.

SP-B refining of pulmonary surfactant phospholipid films.

Pulmonary surfactant stabilizes the alveoli by lining the air-fluid interface with films that reduce surface tension to near 0 mN/m (gamma(min)). Surfactant protein B (SP-B) enhances the surface activity of surfactant phospholipids. A captive bubble tensiometer (CBT) was used to study the properties of adsorbed films of dipalmitoylphosphatidylcholine (DPPC) with acidic 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) or neutral 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine with (7:3) and without 1% dimeric SP-B. SP-B enhanced the adsorption rate of DPPC-containing neutral or acidic lipid suspensions (1 mg/ml) to a similar extent. Quasi-static cycling of these films revealed that SP-B significantly decreased the film area reduction required to reach gamma(min) for the acidic but not for the neutral system. The results obtained with DPPC-phosphatidylglycerol (PG)-SP-B were consistent with selective DPPC adsorption into the surface monolayer during film formation. Film area reduction required to reach gamma(min) with this system (with and without calcium) approached that of pure DPPC, suggesting selective DPPC insertion and PG squeeze-out. Dynamic cycling of such films showed that larger film area reductions were required to reach gamma(min) for the neutral than for acidic system, even after 20 cycles. Fluorescence microscopy of solvent-spread DPPC-POPG-SP-B planar films revealed highly condensed structures at approximately 25 mN/m, although no specific PG phase-segregated structures could be identified. The study suggests that specific interactions of SP-B with acidic phospholipids of surfactant may be involved in the generation and maintenance of DPPC-rich films in the alveoli.

Alterations in pulmonary surfactant after rapid arousal from torpor in the marsupial Sminthopsis crassicaudata.

Torpor in the dunnart, Sminthopsis crassicaudata, alters surfactant lipid composition and surface activity. Here we investigated changes in surfactant composition and surface activity over 1 h after rapid arousal from torpor (15-30 degrees C at 1 degrees C/min). We measured total phospholipid (PL), disaturated PL (DSP), and cholesterol (Chol) content of surfactant lavage and surface activity (measured at both 15 and 37 degrees C in the captive bubble surfactometer). Immediately after arousal, Chol decreased (from 4.1 +/- 0.05 to 2.8 +/- 0.3 mg/g dry lung) and reached warm-active levels by 60 min after arousal. The Chol/DSP and Chol/PL ratios both decreased to warm-active levels 5 min after arousal because PL, DSP, and the DSP/PL ratio remained elevated over the 60 min after arousal. Minimal surface tension and film compressibility at 17 mN/m at 37 degrees C both decreased 5 min after arousal, correlating with rapid changes in surfactant Chol. Therefore, changes in lipids matched changes in surface activity during the postarousal period.

Sulfuric acid aerosol induces changes in alveolar surface tension in the guinea pig but not in the rat.

The purpose of this study was to investigate the effects of an acid aerosol, at high concentration, on the surface properties of the extracellular fluid lining the airways and alveolae. Guinea pigs and rats were exposed to 43 mg/m3 and 94 mg/m3 of sulfuric acid aerosol mass median aerodynamic diameter (MMAD) 0.9 micron or water aerosol (control), respectively, for 4 hours in an exposure chamber. Surfactant material was extracted from bronchoalveolar lavage fluid (BAL) by centrifugation, and phospholipid, protein, and cell concentrations measured. The extract was reconstituted to 300 micrograms/mL of phospholipid, and its surface properties assessed with a captive bubble surfactometer. The minimum surface tension for the acid-exposed guinea pig BAL was 12.1 +/- 8.48 (mean +/- SD) mN/m, which was significantly higher than the control group, 2.0 +/- 0.43 (mean +/- SD) or the acid-exposed rats, 1.29 +/- 0.11 (mean +/- SD). The change in film area obtained by compressing the film from equilibrium surface tension (25 mN/m) to its minimum value (gamma min) was 62.9 +/- 13.83 (mean +/- SD)% for acid-exposed guinea pigs, compared to 16.3 +/- 5.77 (mean +/- SD)% for the control guinea pigs. The most sensitive index of surfactant inhibition was found to be the maximum film compressibility (Cmax) of the compression isotherm. This index was 119 times greater for the acid-exposed guinea pigs compared to control animals. These abnormalities were associated with an elevation of total protein (0.95 +/- 0.33 [mean +/- SD] mg/mL compared to 0.13 +/- 0.03 [mean +/- SD] mg/mL in controls) and polymorphonuclear leucocytes in the BAL. There was no change in total phospholipids. By contrast BAL retrieved from rats exposed to approximately twice the concentration of acid aerosol showed no cellular nor biochemical abnormalities and its surface tension properties were normal. We conclude that the abnormalities of surfactant activity in the acid-exposed guinea pigs result from the cellular and humoral responses of acute lung injury rather than a direct effect of acid.

Surfactant-spreading and surface-compression disturbance on a thin viscous film.

Spreading of a new surfactant in the presence of a pre-existing surfactant distribution is investigated both experimentally and theoretically for a thin viscous substrate. The experiments are designed to provide a better understanding of the fundamental interfacial and fluid dynamics for spreading of surfactants instilled into the lung. Quantitative measurements of spreading rates were conducted using a fluorescent new surfactant that was excited by argon laser light as it spread on an air-glycerin interface in a petri dish. It is found that pre-existing surfactant impedes surfactant spreading. However, fluorescent microspheres used as surface markers show that pre-existing surfactant facilitates the propagation of a surface-compression disturbance, which travels faster than the leading edge of the new surfactant. The experimental results compare well with the theory developed using lubrication approximations. An effective diffusivity of the thin film system is found to be Deff = (E*gamma)/(mu/H), which indicates that the surface-compression disturbance propagates faster for larger background surfactant concentration, gamma, larger constant slope of the sigma*-gamma* relation, -E*, and smaller viscous resistance, mu/H. Note that sigma* and gamma* are the dimensional surface tension and concentration, respectively, mu is fluid viscosity, and H is the unperturbed film thickness.