'Free' surfactant in gastric aspirates and bronchoalveolar lavage in children with and without reflux oesophagitis.

AIM: Dipalmitoylphosphatidycholine (DPPC) is the characteristic and main constituent of surfactant. Adsorption of surfactant to epithelial surfaces may be important in the masking of receptors. The aims of the study were to (i) compare the quantity of free DPPC in the airways and gastric aspirates of children with gastroesophageal reflux disease (GORD) to those without and (ii) describe the association between free DPPC levels with airway cellular profile and capsaicin cough sensitivity. METHODS: Children aged <14 years were defined as 'coughers' if a history of cough in association with their GORD symptoms was elicited before gastric aspirates and nonbronchoscopic bronchoalveolar lavage (BAL) were obtained during elective flexible upper gastrointestinal endoscopy. GORD was defined as histological presence of reflux oesophagitis. Spirometry and capsaicin cough-sensitivity test was carried out in children aged >6 years before the endoscopy. RESULTS: Median age of the 68 children was 9 years (interquartile range (IQR) 7.2). Median DPPC level in BAL of children with cough (72.7 microg/mL) was similar to noncoughers (88.5). There was also no significant difference in DPPC levels in both BAL and gastric aspirates of children classified according to presence of GORD. There was no correlation between DPPC levels and cellular counts or capsaicin cough-sensitivity outcome measures. CONCLUSION: We conclude that free DPPC levels in the airways and gastric aspirate is not influenced by presence of cough or GORD defined by histological presence of reflux oesophagitis. Whether quantification of adsorbed surfactant differs in these groups remain unknown. Free DPPC is unlikely to have a role in masking of airway receptors.

Comparison of surfactant lipids between pleural and pulmonary lining fluids.

Saturated phospholipids (PCs), particularly dipalmitoylphosphatidylcholine (DPPC), predominate in surfactant lining the alveoli, although little is known about the relationship between saturated and unsaturated PCs on the outer surface of the lung, the pleura. Seven healthy cats were anesthetized and a bronchoalveolar lavage (BAL) was performed, immediately followed by a pleural lavage (PL). Lipid was extracted from lavage fluid and then analyzed for saturated, primarily dipalmitoylphosphatidylcholine (DPPC), and unsaturated PC species using high-performance liquid chromatography (HPLC) with combined fluorescence and ultraviolet detection. Dilution of epithelial lining fluid (ELF) in lavage fluids was corrected for using the urea method. The concentration of DPPC in BAL fluid (85.3+/-15.7 microg/mL) was significantly higher (P=0.021) than unsaturated PCs ( approximately 40 microg/mL). However, unsaturated PCs ( approximately 34 microg/mL), particularly stearoyl-linoleoyl-phosphatidylcholine (SLPC; 17.4+/-6.8), were significantly higher (P=0.021) than DPPC (4.3+/-1.8 microg/mL) in PL fluid. These results show that unsaturated PCs appear functionally more important in the pleural cavity, which may have implications for surfactant replenishment following pleural disease or thoracic surgery.

Surface-active phospholipid (surfactant) in equine tendon and tendon sheath fluid.

AIM: To investigate the presence of surface-active phospholipid (SAPL, or surfactant) in equine tendon and tendon sheath fluid. METHODS: The left front flexor tendon and sheath were removed from five Thoroughbred horses. Phospholipid was extracted from tendon sheath fluid using Folch reagent and quantified using spectroscopy. Transmission electron microscopy (TEM) was used to observe the tendon surfaces. RESULTS: The presence of phospholipid (90.6 (SD 4.3) microg/ml) in tendon sheath fluid, plus the appearance of oligolamellar layers and lamellar bodies on the tendon surface were indicative of SAPL. CONCLUSIONS: Evidence of SAPL was found in equine tendon, and may have a similar lubricating function as reported for synovial joints. CLINICAL RELEVANCE: These findings may have important implications for normal tendon function and possible therapeutic adjuncts for tendon and tendon sheath injuries.

Consolidation responses of delipidized articular cartilage.

OBJECTIVE: To determine the role of articular cartilage lipids in its load-bearing function. DESIGN: Normal and delipidised, bovine articular cartilage specimens were statically loaded and both the hydrostatic excess pore pressure and creep strain were measured. From this the compression stiffness of the skeletal structures of both types of matrices was determined. BACKGROUND: It has been hypothesized that surfactant injection could relief osteoarthritis, but there is no study in the literature relating to the influence of lipids, the main ingredients of such products, on cartilage load-carriage. METHODS: Articular cartilage specimens were obtained from the patellar grooves of 2-3 year old bovine animals. When required specimen delipidization was carried out using chloroform/methanol rinsing. Both normal and delipidised samples were loaded in the consolidometer and the hydrostatic excess pore pressure and strain were measured. RESULTS: The transient patterns of the hydrostatic excess pore pressure were similar for both types of tissue, with a relatively insignificant increase of 2% in the maximum hydrostatic excess pore pressure of the delipidized samples relative to the normal intact specimens. The maximum creep strain of the delipidised specimens decreased by 10% on average relative to their normal intact counterparts, thereby indicating that delipidization causes stiffening of the cartilage matrix. CONCLUSION: The delipidized fluid-saturated articular cartilage is stiffer than its intact counterpart with consequence for cartilage compliance during function. RELEVANCE: Because osteoarthritis can be accompanied by lipid loss in cartilage, this study contributes to the further understanding of the disease with potential benefit for treatment.

Biomechanical responses of normal and delipidized articular cartilage subjected to varying rates of loading.

In alignment with the proposition that a lipid layer overlays the superficial zone of the articular cartilage, this study presents the consequence of the removal of lipids on the load-bearing characteristics of the tissue. Both normal unmodified and delipidized cartilage matrices were loaded at four different strain-rates of 1.3 x 10(-4)/s, 1.3 x 10(-3)/s, 1.3 x 10(-2)/s, and 1.3 x 10(-1)/s to strains of no more than 40%, to compare their stress-strain and stiffness-strain-rate characteristics. Our results demonstrate that at the lowest strain-rate of 1.3 x 10(-4)/s, the stiffness of the delipidized matrix was lower in comparison to that of the normal unmodified tissue. This response was reversed at higher strain-rates of 1.3 x 10(-2)/s and above. We conclude, therefore, that in general, at physiological rates of loading, the depletion of lipids from the articular cartilage reduces its compliance by at least 25%. We infer from the present study that this degenerative stiffening is an important contributing factor in impairing the tissue's load processing function in osteoarthritic joints.

Adsorption of surfactant to bronchial epithelium: possible role of receptor 'unmasking' in asthma.

BACKGROUND: In a recent study in animals it has been shown how surface-active phospholipid (SAPL) in the form of a commercially available micronized (5 micromphi) dry powder (ALECT/PumactantT) was able to reduce afferent neural feedback to the brainstem in response to a methacholine challenge by the same order of magnitude as drugs commonly prescribed for asthma. The underlying theory assumed that adsorption of SAPL to bronchial epithelium masked irritant receptors eliciting the bronchoconstrictor reflex, thus providing a barrier to noxious stimuli entering the lungs. OBJECTIVE: To test the underlying assumption that SAPL was actually adsorbed (i.e., bound to bronchial epithelium), especially the major and most surface-active component of lung surfactant, namely dipalmitoyl phosphatidylcholine (DPPC). A secondary objective was to investigate any role of phosphatidylglycerol (PG) in promoting the adsorption of DPPC. METHODS: Radiolabeled DPPC dispersed ultrasonically in saline was used to incubate excised sections of porcine bronchial epithelium. The adsorbed DPPC was then quantified by rigorously rinsing the tissue of adhering fluid and then digesting it for beta-scintillation counting. Each test (n=8 runs) was repeated for ratios of DPPC:PG of 9:1, 7:3 (as per ALECT/PumactantT) and 1:1 for both dipalmitoyl PG (DPPG) and EggPG (as incorporated in ALECT/PumactantT). RESULTS: Despite rigorous rinsing postincubation, bronchial epithelium was found to adsorb DPPC at a level roughly equivalent to one close-packed monolayer; whereas both DPPG and EggPG promoted the adsorption of DPPC in a dose-dependent manner, reaching an approximate threefold increase for 7:3 DPPC:PG. CONCLUSION: DPPC adsorbs to bronchial epithelium in amounts necessary for the masking of receptors, and this adsorption (probably chemisorption) is quite strongly promoted by PG either in its indigenous state (DPPG) or in the form (EggPG) used in ALECT to suppress the sensitivity of bronchial irritant receptors in our previous study and in clinical trials just completed.

Normal and prosthetic synovial joints are lubricated by surface-active phospholipid: a hypothesis.

Much evidence supports the hypothesis that surface-active phospholipid (SAPL), which imparts the thin hydrophobic outermost lining to the normal articular surface, is the boundary lubricant reducing friction to remarkably low levels. We review this evidence and further hypothesize that SAPL produced in type B synoviocytes will also lubricate prostheses after implantation. This could explain why implanted hips display far less wear than hips in simulated wear trials do, even using protein as the lubricant whereas rougher surfaces can be tolerated in vivo. We introduce the concept that a deficiency of SAPL might explain the selective failure of prostheses just as osteoarthritic articular surfaces are deficient. This, in turn, leads to the replenishment of SAPL, as tested in OA, and the concept of prelubricating prostheses before implantation.

The relationship between inflammation and dipalmitoyl phosphatidycholine in induced sputum of children with asthma.

BACKGROUND: Animal studies have shown elevated surfactant production in response to lung injury. In human airways, the contribution of surfactant to the airway epithelial barrier and importance of eosinophilic inflammation is increasingly appreciated. The relationship between blood and sputum inflammatory indices of childhood asthma to surfactant levels is unknown. In this study we hypothesized that the degree of inflammation influences the level of dipalmitoyl phosphatidycholine (DPPC) in airways of children with asthma. METHODS: Sixteen children with asthma (ages 5.5-16 years) underwent venipuncture, skin prick test, spirometry, hypertonic saline challenge, and induced sputum during a nonacute phase. Sputum (sp) and blood (se) markers of inflammation (eosinophils, neutrophils, eosinophilic cationic protein [ECP]), were related to sputum DPPC levels and several markers of asthma severity (airway hyperresponsiveness, quality of life, FEV1). RESULTS: On multiple regression, sp-DPPC significantly correlated to sp-ECP (r=0.53, P=0.0048). Se-ECP, se-Eo, sp-eosinophils, sp-neutrophils, se-neutrophils, and inhaled steroids dose did not significantly influence sp-DPPC. Exposure to smoke did not influence inflammatory markers. FEV1 and quality of life data did not relate to any blood or sputum variable. A significant association between AHR and se-eosinophils, but not between AHR and se-ECP, sp-eosinophils, or sp-ECP was found. CONCLUSION: Elevated DPPC levels occur in the presence of chronic eosinophilic inflammation in airways of children with stable asthma. Whether this represents an inherent lung mechanism for epithelial protection remains to be elucidated.

Surface-active phospholipid: a Pandora's box of clinical applications. Part II. Barrier and lubricating properties.

In Part I, it was described how their configuration renders phospholipid molecules surface active and capable of acting at interfaces in addition to the liquid-air interface to which conventional theory has hitherto confined the study of 'surfactant' in the lung. Surface-active phospholipid (SAPL) appears no different to comparable surfactants studied in the physical sciences for the highly desirable properties that their adsorption (reversible binding) can impart to solid surfaces. In Part II, these properties are considered in sites where there is no air. Highly desirable properties include boundary lubrication (lubricity), release (antistick) and the ability of the strongly adsorbed and strongly cohesive SAPL linings to act as barriers against abrasion, corrosion and, possibly, against invasion by microorganisms. As the 'sealant', it could be the true barrier rather than the cells providing its mechanical support. Evidence is reviewed for SAPL providing the gastric mucosal barrier to acid in the stomach and preventing the digestion of Helicobacter pylori until that barrier is broken by bile in the duodenum, where H. pylori cause ulcers. The concept that SAPL provides effortless sliding of many tissues, including pleura, pericardium and peritoneum is reviewed. Particular attention is paid to the load-bearing joints, where a deficiency has been associated with osteoarthritis. The ability of the same SAPL lining to perform multiple roles is discussed in relation to the peritoneum, where it could provide the lubricant/release agent preventing surgical adhesions, while imparting semipermeability to 'the membrane' vital for peritoneal dialysis. In each site, the prophylactic use of exogenous SAPL is discussed for its potential clinical applications.

Surface-active phospholipid: a Pandora's box of clinical applications. Part I. The lung and air spaces.

Almost everywhere in the body there are phospholipids, not only comprising the lipid bilayer of membranes, but also in the free state. What is seldom appreciated, except in respirology, is that these 'free' phospholipids are unusual in that many are highly surface active. Surface activity is a property of certain substances (surfactants), conferred by their molecular constitution and configuration, which predisposes them to locate at interfaces because, in doing so, they reduce interfacial energy. When adsorbed (reversibly bound) to solid surfaces, surfactants can impart many highly desirable properties that have been widely studied and long accepted in the physical sciences, while their commercial applications have withstood the test of time. These desirable properties include lubricity (boundary lubrication), release (antistick) and dewatering, while providing a barrier to corrosion, abrasion, solute transmission and to biological microorganisms. Many of these offer obvious roles for surface-active phospholipid (SAPL), ranging from a corrosion inhibitor in the stomach to a load-bearing lubricant in the joints. This opens a veritable 'Pandora's box' of potential clinical applications. Part I of this review challenges traditional beliefs in respirology that 'surfactant' is unique to the lung and, moreover, that its actions are confined to the liquid-air interface. Evidence is discussed that, by binding to alveolar epithelium, SAPL imparts semi-permeability needed before channels pumping ions can also pump water vital for maintaining fluid balance. Evidence is also reviewed for a lining to upper airways, sinuses and Eustachian tube where it can act like a standard release agent.

Role of surfactant in peritoneal dialysis.

Evidence is reviewed that demonstrates how the mesothelial cell in the normal peritoneum and comparable serosal cavities secretes surface-active phospholipid (SAPL) as a means of protecting itself and the membrane it forms with its neighbors. It is shown how SAPL, if adsorbed (reversibly bound) to mesothelium, can impart excellent lubricity, antiwear and release (antistick) properties, while impeding surgical adhesion formation. More-speculative benefits include acting as a deterrent to fibrosis and as a barrier to both protein leakage and pathogen invasion by spanning cell junctions. Such spanning would also "pin down" cell corners, impeding peeling as the first step in exfoliation encountered in prolonged continuous ambulatory peritoneal dialysis (CAPD). The molecular mechanism underlying each of these possible functions is adsorption. Morphological and hydrophobicity studies are discussed as validation for such an adsorbed lining and how it can be fortified by administering exogenous SAPL. Any role for SAPL in ultrafiltration is much more controversial. However, a surfactant lining can explain the very high permeability of the membrane to lipid-soluble drugs, implying that it is a barrier to water-soluble solutes. The clinical and animal evidence is conflicting but would seem to be best explained by a role for the barrier in promoting semipermeability, and hence the osmotic driving force for water transmission. Thus, adsorption of exogenous SAPL in CAPD patients with low ultrafiltration seems to restore this barrier function. The future direction for surfactant in CAPD would seem to rest with the physical chemists in producing formulations that optimize adsorption, probably involving a compromise between water solubility and surface activity of the phospholipids selected. It might even warrant using the interdialytic interval for readsorbing SAPL without the problem of dilution by a large volume of dialysate.

Evidence for surfactant contributing to the gastric mucosal barrier of the horse.

This study was undertaken to determine the hydrophobicity of the luminal surface of the equine stomach and to elucidate the ultrastructure of the lining imparting that property. Gastric and duodenal mucosal samples from 5 horses were collected immediately after euthanasia and subjected to surface contact angle measurement using a goniometer. Gastric mucosal samples from 4 horses and a foal were examined by electron microscopy following a fixation procedure known to preserve phospholipids and oligolamellar structures. Contact angles for the equine gastric glandular mucosal surface (mean +/- s.e. 78.0 +/- 11.0 degrees) were greater than for the duodenum (33.4 +/- 8.7 degrees), (P = 0.003). The contact angles for gastric squamous tissue (50.4 +/- 4.5 degrees) tended to be greater than for duodenum (P = 0.15). Electron microscopy revealed the existence of surfactant as abundant osmiophilic phospholipid material within both squamous and glandular gastric mucosae. These results indicate the hydrophobic nature of the equine gastric mucosae. We propose that the water-repellent nature of the stomach contributes to the 'gastric mucosal barrier' and is imparted by surface-active phospholipid adsorbed to the surface. Phospholipids may also be utilised as a physical barrier to back-diffusion of acid by lining intracellular canaliculi and oxyntic ducts where other defence mechanisms are absent.

Suppression of neural activity of bronchial irritant receptors by surface-active phospholipid in comparison with topical drugs commonly prescribed for asthma.

BACKGROUND: Much indirect evidence has been put forward previously in support of the concept that surface-active phospholipid (SAPL) normally masks irritant receptors in the lungs and upper respiratory tract; but this physical barrier is deficient in asthmatics, imparting hyperresponsiveness of the bronchoconstrictor reflex. OBJECTIVE: To determine whether exogenous SAPL applied to bronchial mucosa reduces the sensitivity of irritant receptors to a standard challenge used clinically to diagnose asthma and to compare the effects with those of corticosteroids and beta-stimulation. METHODS: Nerve fibres in the vagi were monitored to record action potentials from irritant receptors identified in the upper airways of rat lungs in response to a methacholine challenge. SAPL in the form of dipalmitoyl phosphatidylcholine (PC) and phosphatidylglycerol (PG) - 7 : 3 PC:PG - was applied as a fine dry powder to enhance surface activity and, hence, chemisorption to epithelium. Comparison was also made with clinical doses of i.v. hydrocortisone and instilled salbutamol together with liquid or solid controls, as appropriate. RESULTS: Neural activity of irritant receptors was found to be significantly (P = 0.0018) decreased by topical SAPL by 35.8% in response to a methacholine challenge in contrast to an increase of 11.2% in response to a solid (lactose) control. Instilled salbutamol and i.v. hydrocortisone also decreased responses to the same challenge by 43.4% and 14.7%, respectively, in contrast to a liquid (saline) control which increased by 24.5%. CONCLUSIONS: Surface-active phospholipid has an appreciable effect upon irritant receptors in rat airways, reducing neural response to a methacholine challenge by an amount comparable to that of Salbutamol. These results support the concept of SAPL masking bronchial irritant receptors and warrant placebo-controlled clinical trials of this dry powder as a means of controlling asthma without the side-effects of current medication. Other possible roles discussed for the SAPL epithelial barrier include the exclusion of viruses and allergens.

Surgical adhesions: evidence for adsorption of surfactant to peritoneal mesothelium.

BACKGROUND: It has been speculated that the formation of surgical adhesions must be preceded by physical adhesion of the two surfaces, a process normally prevented by a lining of adsorbed surface-active phospholipid (surfactant) acting as both a superb boundary (solid-to-solid) lubricant and a release (antistick) agent. Animal trials administering exogenous surfactant as a dry powder (ALEC) have previously demonstrated a reduction of 80% in abdominal adhesions. METHODS: Incubation of rat peritoneum (both live and excised) with radiolabelled dipalmitoyl phosphatidylcholine (DPPC) has been used to demonstrate adsorption; while the normal lining of surfactant in the human abdominal cavity has been confirmed by epifluorescence microscopy using Phosphin E as the hydrophobic probe. AIMS: The overall aim is to confirm that peritoneal mesothelium has a lining of surfactant known for its lubricating and release properties, and that this lining can be enhanced by the adsorption of exogenous material. RESULTS: Adsorption of DPPC to peritoneal mesothelium was 470 ng/cm2 (n = 8) ex vivo and 598 ng/cm2 (n = 18) in vivo, these rates being enhanced by EggPG by 62% ex vivo and 47% in vivo to reach the equivalent of almost three close-packed monolayers. CONCLUSIONS: These results can explain the reduction in surgical adhesions previously reported in animals by administering ALEC (7:3 DPPC:EggPG) as a highly surface-active dry powder, although it is now used in saline suspension to treat respiratory distress syndrome in newborns, in whom it has no side-effects. These findings would appear to justify clinical trials for dry ALEC in suppressing surgical adhesions with minimal risk of an adverse reaction. The results of these trials are also discussed and found to be compatible with the known ability of surfactant to resist physical adhesion by fibronectin, the tacky 'glue' by which fibroblasts attach to surfaces as the first step in formation of fibrinous adhesions.

Boundary lubrication in vivo.

Evidence is reviewed for the concept that the body employs essentially the same lubrication system in many sites in the body where tissues slide over each other with such ease. This system consists of fluid adjacent to surfaces coated with an oligolamellar lining of surface-active phospholipid (SAPL) acting as a back-up boundary lubricant wherever the fluid film fails to support the load--a likely event at physiological velocities. Particular attention is paid to the load-bearing joints, where the issue of identifying the vital active ingredient in synovial fluid is reviewed, coming down--perhaps predictably--in favour of SAPL. It is also explained how Lubricin and hyaluronic acid (HA) could have 'carrier' functions for the highly insoluble SAPL, while HA has good wetting properties needed to promote hydrodynamic lubrication of a very hydrophobic articular surface by an aqueous fluid wherever the load permits. In addition to friction and wear, release is included as another major role of boundary lubricants, especially relevant in environments where proteins are found, many having adhesive properties. The discussion is extended to a mention of the lubrication of prosthetic implants and to disease states where a deficiency of boundary lubricant is implicated, particular attention being paid to osteoarthritis.

An alternative view of the role(s) of surfactant and the alveolar model.

Currently, the study of surfactant proteins is much in vogue, but, in the early days, the physics underlying surfactant function was treated somewhat superficially, leaving assumptions that have become culturally embedded, such as the "bubble" model of the alveolus. This review selectively reexamines these assumptions, comparing each combination of alveolar model and role of surfactant for compatibility with the major features of pulmonary mechanics and alveolar stability, morphology, and fluid balance.