Small Aggregate Fractions Research Articles

Phosphorus and Heavy Metal Attachment and Release in Sandy Soil Aggregate Fractions

The presence of P and heavy metals in different forms or in association with different size fractions influences availability and discharge of these elements from watersheds. Understanding the association of P and heavy metals with size fractions can improve evaluation of leaching potential of P and heavy metals from soils. In this study, five aggregate-size fractions, ranged from 1.00 to 0.50 to <0.053 mm, were separated from seven Florida sandy soils by dry sieving. Each aggregate fraction was characterized by phosphate sorption, sequential fractionation of P, total, water- and Mehlich III-extractable concentrations of P and heavy metals. Size differences in sand, silt, and clay aggregates influence the amount and strength of element binding. Elemental attachment (particularly heavy metals) increased with decreasing aggregate sizes. Phosphorus and heavy metals in the sandy soils are readily transported to surface waters with suspended fine particles. Higher percentages of water-extractable, Mehlich III-extractable P, and heavy metals were found in both the 0.50- to 0.25- and 0.25- to 0.125-mm aggregate fractions, suggesting that P and heavy metals in these two fractions had higher release potential. The sequential fractionation of P suggested that the 1.00- to 0.50-mm fraction contained a larger percentage of Ca-bound P, whereas the 0.50- to 0.25-, 0.25- to 0.125-, and 0.125- to 0.053-mm fractions had higher ratios of labile P (H2O-P and NaHCO3–P). Phosphorus release from smaller aggregate fractions is faster with a higher P1/P168 ratio than from larger aggregate fractions because of larger amounts of water soluble P attached in the smaller aggregate fractions.

Characterization of Cytoplasmic α-Synuclein Aggregates: FIBRIL FORMATION IS TIGHTLY LINKED TO THE INCLUSION-FORMING PROCESS IN CELLS

The alpha-synuclein fibrillation process has been associated with the pathogenesis of several neurodegenerative diseases. Here, we have characterized the cytoplasmic alpha-synuclein aggregates using a fractionation procedure with which different aggregate species can be separated. Overexpression of alpha-synuclein in cells produce two distinct types of aggregates: large juxtanuclear inclusion bodies and small punctate aggregates scattered throughout the cytoplasm. Biochemical fractionation results in an inclusion-enriched fraction and two small aggregate fractions. Electron microscopy and thioflavin S reactivity of the fractions show that the juxtanuclear inclusion bodies are filled with amyloid-like alpha-synuclein fibrils, whereas both the small aggregate fractions contain non-fibrillar spherical aggregates with distinct size distributions. These aggregates appear sequentially, with the smallest population appearing the earliest and the fibrillar inclusions the latest. Based on the structural and kinetic properties, we suggest that the small spherical aggregates are the cellular equivalents of the protofibrils. The proteins that co-exist in the Lewy bodies, such as proteasome subunit, ubiquitin, and hsp70 chaperone, are present in the fibrillar inclusions but absent in the protofibrils, suggesting that these proteins may not be directly involved in the early aggregation stage. As predicted in the aggresome model, disruption of microtubules with nocodazole reduced the number of inclusions and increased the size of the protofibrils. Despite the increased size, the protofibrils remained non-fibrillar, suggesting that the deposition of the protofibrils in the juxtanuclear region is important in fibril formation. This study provides evidence that the cellular fibrillation also involves non-fibrillar intermediate species, and the microtubule-dependent inclusion-forming process is required for the protofibril-to-fibril conversion in cells.

Total extracellular surfactant is increased but abnormal in a rat model of gram-negative bacterial pneumonia.

An in vivo rat model was used to evaluate the effects of Escherichia coli pneumonia on lung function and surfactant in bronchoalveolar lavage (BAL). Total extracellular surfactant was increased in infected rats compared with controls. BAL phospholipid content in infected rats correlated with the severity of alveolar-capillary leak as reflected in lavage protein levels (R(2) = 0.908, P < 0.0001). Western blotting showed that levels of surfactant protein (SP)-A and SP-D in BAL were significantly increased in both large and small aggregate fractions at 2 and 6 h postinstillation of E. coli. SP-B was also increased at these times in the large aggregate fraction of BAL, whereas SP-C levels were increased at 2 h and decreased at 6 h relative to controls. The small-to-large (S/L) aggregate ratio (a marker inversely proportional to surfactant function) was increased in infected rats with >50 mg total BAL protein. There was a significant correlation (R(2) = 0.885, P < 0.0001) between increasing S/L ratio in BAL and pulmonary damage assessed by total protein. Pulmonary volumes, compliance, and oxygen exchange were significantly decreased in infected rats with >50 mg of total BAL protein, consistent with surfactant dysfunction. In vitro surface cycling studies with calf lung surfactant extract suggested that bacterially derived factors may have contributed in part to the surfactant alterations seen in vivo.

Analytical laser induced liquid beam desorption mass spectrometry of protonated amino acids and their non-covalently bound aggregates

We have used analytical laser induced liquid beam desorption in combination with high resolution mass spectrometry ( m/Δm≥ 1000) for the study of protonated amino acids (ornithine, citrulline, lysine, arginine) and their non-covalently bound complexes in the gas phase desorbed from water solutions. We report studies in which the desorption mechanism has been investigated. The results imply that biomolecule desorption at our conditions is a single step process involving laser heating of the solvent above its supercritical temperature, a rapid expansion, ion recombination and finally isolation and desorption of only a small fraction of preformed ions and charged aggregates. In addition, we report an investigation of the aqueous solution concentration and pH-dependence of the laser induced desorption of protonated species (monomers and dimers). The experimental findings suggest that the desorption process depends critically upon the proton affinity of the molecules, the concentration of other ions, and of the pH value of the solution. Therefore the ion concentrations measured in the gas phase very likely reflect solution properties (equilibrium concentrations). Arginine self-assembles large non-covalent singly protonated multimers (n = 1...8) when sampled by IR laser induced water beam desorption mass spectrometry. The structures of these aggregates may resemble those of the solid state and may be preformed in solution prior to desorption. A desorption of mixtures of amino acids in water solution enabled us to study (mixed) protonated dimers, one of the various applications of the present technique. Reasons for preferred dimerization - leading to simple cases of molecular recognition - as well as less preferred binding is discussed in terms of the number of specific H-bonds that can be established in the clusters.

Glutathione Replacement Preserves the Functional Surfactant Phospholipid Pool Size and Decreases Sepsis‐Mediated Lung Dysfunction in Ethanol‐Fed Rats

Background Alcohol abuse increases the incidence of acute respiratory distress syndrome (ARDS). Previous evidence from our laboratory links ethanol‐mediated glutathione depletion to impaired surfactant production by alveolar epithelial cells in vitro and to endotoxin‐mediated edematous injury in isolated lungs ex vivo. ARDS patients have an imbalance between the inactive small aggregate (SA) and the bioactive large aggregate (LA) forms of surfactant phospholipid (as reflected by increased SA/LA ratios). Therefore, we hypothesized that ethanol ingestion, via glutathione depletion, could alter surfactant phospholipid distribution between LA and SA forms and thereby exacerbate sepsis‐mediated lung dysfunction in vivo.Methods Rats fed an isocaloric diet with or without ethanol (36% total calories) for 6 weeks were made septic via cecal ligation and perforation. Some ethanol‐fed rats had their diets supplemented with the glutathione precursor procysteine (l‐2‐oxothiaxolidine‐4‐carboxylate). Sepsis physiology was assessed by determining respiratory rates, arterial blood pressures, and plasma lactate levels, and lung dysfunction was assessed by determining lung lavage fluid protein levels (index of alveolar endothelial/epithelial barrier disruption), arterial hypoxemia (index of impaired gas exchange) and surfactant phospholipid SA and LA fractions (index of the alveolar epithelium's ability to maintain a functional surfactant pool during sepsis).Results Ethanol ingestion decreased (p &lt; 0.05) lung lavage fluid glutathione levels, and this defect was prevented by procysteine. Although ethanol ingestion had no effect (p &lt; 0.05) on any of the indices of sepsis, it increased (p &lt; 0.05) lung lavage fluid protein levels, worsened hypoxemia, and decreased the functional (LA) surfactant phospholipid pool after sepsis, all of which was prevented by procysteine.Conclusions Ethanol ingestion, via glutathione depletion, increased sepsis‐mediated lung dysfunction, and these effects could contribute to the increased risk of ARDS seen in alcoholic patients.

Glutathione Replacement Preserves the Functional Surfactant Phospholipid Pool Size and Decreases Sepsis-Mediated Lung Dysfunction in Ethanol-Fed Rats

Background Alcohol abuse increases the incidence of acute respiratory distress syndrome (ARDS). Previous evidence from our laboratory links ethanol-mediated glutathione depletion to impaired surfactant production by alveolar epithelial cells in vitro and to endotoxin-mediated edematous injury in isolated lungs ex vivo. ARDS patients have an imbalance between the inactive small aggregate (SA) and the bioactive large aggregate (LA) forms of surfactant phospholipid (as reflected by increased SA/LA ratios). Therefore, we hypothesized that ethanol ingestion, via glutathione depletion, could alter surfactant phospholipid distribution between LA and SA forms and thereby exacerbate sepsis-mediated lung dysfunction in vivo. Methods Rats fed an isocaloric diet with or without ethanol (36% total calories) for 6 weeks were made septic via cecal ligation and perforation. Some ethanol-fed rats had their diets supplemented with the glutathione precursor procysteine (l-2-oxothiaxolidine-4-carboxylate). Sepsis physiology was assessed by determining respiratory rates, arterial blood pressures, and plasma lactate levels, and lung dysfunction was assessed by determining lung lavage fluid protein levels (index of alveolar endothelial/epithelial barrier disruption), arterial hypoxemia (index of impaired gas exchange) and surfactant phospholipid SA and LA fractions (index of the alveolar epithelium's ability to maintain a functional surfactant pool during sepsis). Results Ethanol ingestion decreased (p < 0.05) lung lavage fluid glutathione levels, and this defect was prevented by procysteine. Although ethanol ingestion had no effect (p < 0.05) on any of the indices of sepsis, it increased (p < 0.05) lung lavage fluid protein levels, worsened hypoxemia, and decreased the functional (LA) surfactant phospholipid pool after sepsis, all of which was prevented by procysteine. Conclusions Ethanol ingestion, via glutathione depletion, increased sepsis-mediated lung dysfunction, and these effects could contribute to the increased risk of ARDS seen in alcoholic patients.

Aspergillus fumigatus-induced allergic airway inflammation alters surfactant homeostasis and lung function in BALB/c mice.

The differential regulation of pulmonary surfactant proteins (SPs) is demonstrated in a murine model of Aspergillus fumigatus (Af )-induced allergic airway inflammation and hyperresponsiveness. BALB/c mice were sensitized intraperitoneally and challenged intranasally with Af extract. Enzyme-linked immunosorbent assay analysis of serum immunoglobulin (Ig) levels in these mice showed markedly increased total IgE and Af-specific IgE and IgG1. This was associated with peribronchial/perivascular tissue inflammation, airway eosinophilia, and secretion of interleukin (IL)-4 and IL-5 into the bronchoalveolar lavage fluid (BALF). Functional analysis revealed that in comparison with nonsensitized mice, allergic sensitization and challenge resulted in significant increases in acetylcholine responsiveness. To analyze levels of SPs, the cell-free supernate of the BALF was further fractionated by high-speed (20,000 x g) centrifugation. After sensitization and challenges, the pellet (large-aggregate fraction) showed a selective downregulation of hydrophobic SPs SP-B and SP-C by 50%. This reduction was reflected by commensurate decreases in SP-B and SP-C messenger RNA (mRNA) expression of the lung tissue of these animals. In contrast, there was a 9-fold increase in SP-D protein levels in the 20,000 x g supernate without changes in SP-D mRNA. The increased levels of SP-D showed a significant positive correlation with serum IgE (r = 0.85, P < 0.001). Tissue mRNA and protein levels of SP-A in either the large- or the small-aggregate fractions were unaffected. Our data indicate that allergic airway inflammation induces selective inhibition of hydrophobic SP synthesis accompanied by marked increases in the lung collectin SP-D protein content of BALF. These changes may contribute significantly to the pathophysiology of Af-induced allergic airway hyperresponsiveness.

This month in J Lab Clin Med: Issue highlights for June 2001

This month in J Lab Clin Med: Issue highlights for June 2001

Pneumocystis carinii pneumonia alters expression and distribution of lung collectins SP-A and SP-D

Surfactant proteins SP-A and SP-D, members of the collectin family, have been shown to play a significant role in lung host defense. Both proteins selectively bind Pneumocystis carinii (PC) organisms and modulate the interaction of this pathogen with alveolar macrophages. We hypothesized that the expression and distribution of lung collectins SP-A and SP-D is altered by PC lung infection. PC organisms (2 × 105) were inoculated intratracheally into C.B–17 scid/scid mice that do not require steroids for immunosuppression. Four weeks after inoculation, bronchoalveolar lavage (BAL) fluid was fractionated into three fractions—cell pellet, large aggregate (LA), and small aggregate (SA) surfactant—and each fraction was analyzed for the expression of surfactant components. In uninfected mice, the majority of SP-A (62% ± 10%) was found in association with lipids in the LA fraction, while 55% ± 14% of SP-D was distributed in the SA fraction. In contrast, both hydrophobic proteins SP-B and SP-C were associated exclusively with LA. PC infection resulted in major changes in the expression of all surfactant components. Total protein content of LA was unchanged by PC infection (115% ± 18% of control), whereas SA protein content markedly increased (240% ± 18% of control level, P < .001). In contrast, the phospholipid content of LA was significantly decreased (53% ± 5% of control level, P < .001), whereas the SA phospholipid content of infected mice was increased (172% ± 16% of control level, P < .001). By Western blotting, PC pneumonia (PCP) induced a 3-fold increase in the total alveolar SP-D protein that was reflected mainly in increases in SA SP-D (454% ± 135% of control, P < .05). The total alveolar SP-A protein content was also increased in PCP because of a large increase in SP-A in SA (720% ± 115% of control, P < .05); SP-A levels in LA were unchanged. The increases in lung collectin expression were selective, because PCP resulted in the down-regulation of both SP-B and SP-C in LA (5% ± 2% and 13% ± 2% of control, respectively, P < .001). We conclude that PCP induces marked elevations in alveolar collectin levels because of increased expression and accumulation of SP-A and SP-D protein in SA surfactant. (J Lab Clin Med 2001;137:429-39)

Study of Interaction of Poly(ethylene imine) with Sodium Dodecyl Sulfate in Aqueous Solution by Light Scattering, Conductometry, NMR, and Microcalorimetry

Light scattering studies show that in aqueous solution poly(ethylene imine) (PEI) exists largely in the form of individual macromolecules plus a small fraction of aggregates. The aggregates make a large contribution to the scattering signal but only a very small contribution to the solution viscosity. Addition of sodium dodecyl sulfate (SDS) to the solution has a number of interesting consequences. Microcalorimetry experiments show that well below the critical micelle concentration of SDS, individual SDS molecules add to the PEI through an exothermic process. At higher SDS concentrations, there is a noncooperative adsorption, which is endothermic in nature, of SDS micelles onto the polymer chains. The surfactant−polymer complex likely contains several polymer molecules. These solutions are characterized by a higher specific conductivity than can be explained by the sum of the conductivities of all the individual ions in solution, even if the Na+ and DS- ions were free in solution and not bound to the polymer. Pulsed-gradient NMR measurements were carried out to examine the Na+ and DS- ion mobility in the solutions. These measurements showed that surfactant binding to the polymer released sodium ions from the SDS micelles. The increase in pH showed that this binding also releases a small amount of OH- into the solution. These two effects by themselves are not large enough to account for the measured conductivity of the solutions. We speculate that there is high ionic mobility inside the polymer−surfactant complex that adds to the overall conductivity of the solution.

Health effects of sulfur-related environmental air pollution: the pulmonary surfactant system is not disturbed by exposure to acidic sulfate and neutral sulfite aerosols.

The goal of this study was to assess the impact of long-term exposure to environmental sulfur-related aerosols on the biochemical and biophysical properties of lung surfactant. Eight Beagle dogs were housed under clean air conditions for 450 days, followed by an exposure period of 400 days to 0.36 mg/m3 of sulfite (16.5 h/d) and to 5.66 mg/m3 of sulfate (6 h/d) equivalent to a pulmonary hydrogen burden of 15 mumol/m3. Other dogs kept in clean air for the whole study period were additional controls. Serial bronchoalveolar lavages (BALs) were analyzed for total phospholipid concentration, content and ratio of a surfactant-rich large aggregate (LA) fraction and a small aggregate (SA) fraction, in vitro surface area cycling of LAs into SAs as a measure of alveolar extracellular pulmonary surfactant aggregate metabolism, and surface activity of native and lipid-extracted LA. No significant changes over time and no differences between the clean air period and the exposure period were observed. Thus, long-term environmental exposure of dogs to the sulfur-related air pollution tested does not lead to alterations in the amount, extracellular metabolism, or surface-active properties of pulmonary surfactant.

Analytical Formulas for Interfacial Transition Zone Properties

Two analytical results are presented that are of use to concrete material technologists. Using a model of concrete in which the aggregates are spherical, but with an arbitrary size distribution, a result from statistical geometry can be used to accurately give the total interfacial transition zone (ITZ) volume for any width ITZ and any volume fraction of aggregates. In reality, the ITZ contains a gradient of porosity and therefore a gradient of properties. When only a small volume fraction of aggregates is present (called the dilute limit), it is possible to analytically solve for the effect of the ITZ on the overall concrete properties. This calculation can be carried out for the effective linear elastic moduli, linear electrical conductivity/ionic diffusivity, and linear thermal/moisture shrinkage/expansion. The details of the calculation are summarized and applications described.

Analytical formulas for interfacial transition zone properties

Abstract Two analytical results are presented that are of use to concrete material technologists. Using a model of concrete in which the aggregates are spherical, but with an arbitrary size distribution, a result from statistical geometry can be used to accurately give the total interfacial transition zone (ITZ) volume for any width ITZ and any volume fraction of aggregates. In reality, the ITZ contains a gradient of porosity and therefore a gradient of properties. When only a small volume fraction of aggregates is present (called the dilute limit), it is possible to analytically solve for the effect of the ITZ on the overall concrete properties. This calculation can be carried out for the effective linear elastic moduli, linear electrical conductivity/ionic diffusivity, and linear thermal/moisture shrinkage/expansion. The details of the calculation are summarized and applications described.

Preferential uptake of small-aggregate fraction of pulmonary surfactant in vitro.

Homeostasis of pulmonary surfactant requires metabolic clearance of surfactant forms with decreased surface activity. Rabbit pulmonary surfactant was labeled in vivo with rhodamine-labeled dipalmitoylphosphatidylethanolamine (R-DPPE), isolated, and fractionated into large- and small-aggregate subfractions by differential centrifugation. Endocytosis of large (LA)- and small (SA)-aggregate surfactant by a mouse lung epithelial cell line (MLE-12) was evaluated in vitro by epifluorescence microscopy. More SA than LA surfactant was taken up by MLE-12 cells. Endocytosis of SA and LA surfactant was inhibited by preincubation of the subfractions with surfactant protein A and 3.3 mM Ca2+. The difference in uptake between SA and LA surfactant was lost for reconstituted organic extracts of the subfractions. Much of the difference in uptake of SA and LA surfactant may be attributed to the greater concentration of surfactant protein A in LA surfactant.

Investigation of physicochemical changes to L-asparaginase during freeze-thaw cycling.

L-Asparaginase derived from Erwinia chrysanthemi which is being investigated as an alternative to E. coli for the treatment of lymphoblastic leukaemia has been found in our laboratory to lose activity upon exposure to consecutive freeze-thaw cycles. An investigation was undertaken using several techniques to characterize fully the physicochemical changes L-Asparaginase is undergoing during freeze-thaw cycling leading to the loss of its activity. A total protein assay suggested that the loss of some enzyme activity was a result of protein precipitation. Circular dichroism (CD) studies showed a decrease of alpha-helical structure with a concomitant increase in beta sheet and random coil content, suggesting alterations in the secondary structure leading to unfolding, the first step of denaturation processes. The elution profiles obtained from size-exclusion chromatography (SEC) studies indicated the formation of several species during the process of freezing and thawing. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) studies showed bands corresponding to 1-3 kDa and 32 kDa, suggesting that some of the species are fragments and shortened monomers resulting from cleavage of monomers. The molecular weight distribution obtained using SEC-linked light scattering indicated a substantial fraction of polydispersed fragments ranging from 900 Da to 3 kDa and a small fraction of aggregates corresponding to 300 kDa. A scheme was proposed to explain the cascade of events leading to the loss of soluble protein and accompanying loss of enzyme activity. Tetramers of the enzyme dissociate into monomers some of which are cleaved into small fragments. The shortened monomers then aggregate and precipitate.

Alveolar surfactant aggregate conversion in ventilated normal and injured rabbits.

Alveolar surfactant can be separated into two subtypes; large aggregates and small aggregates. Large aggregates represent the surface active form of surfactant and are the metabolic precursors of small aggregates. Previous studies examined the mechanism by which large aggregates are converted into small aggregates in vitro. We used intratracheal injection of radiolabeled large aggregates in rabbits to probe the aggregate conversion in vivo. After this injection, animals were mechanically ventilated for 60 min. After the animals were killed, the lungs were lavaged, and the percentage of radiolabel present in the small aggregate fraction was determined. Our results showed that ventilation resulted in aggregate conversion and that increases in tidal volume, but not in respiratory rate, correlated with increased conversion. Aggregate conversion in rabbits with acute lung injury correlated significantly with severity of injury. We conclude that a change in surface area (i.e., respiration) is necessary for aggregate conversion in vivo and that the ventilation strategy can affect this conversion. Furthermore, increased aggregate conversion in injured lungs might contribute to increased small-to-large aggregate ratios in these lungs compared with normal lungs.

Earthworm populations and stability of soil structure in a silt loam soil of a recently reclaimed polder in the Netherlands

Earthworms are believed to be beneficial to the quality of the soil structure. An important aspect of quality of soil structure is the stability of aggregates. The influence of earthworms on soil structure stability was investigated in a silt loam soil under “conventional” (high-input) and “integrated” (reduced-input) management. Aggregates from the top 5 cm of soil were sampled, air dried and analysed for their size distribution and water stability. Dry-sieved aggregate size distribution did not differ appreciably among fields and between years, so aggregates of a certain size represented a similar fraction of the soil for all fields. The wet-sieving procedure, used to measure aggregate stability, yielded highly reproducible results. Within-field variability of the stability was lower than between-field variability, so in further sampling only one mixed sample per field was analysed for soil structure stability. Analysing only a large and a small aggregate fraction was enough to evaluate aggregate stability in this study, as there was a gradual increase in stability from large to smaller aggregates. A 2 year time series of samples was analysed for earthworm population density and aggregate stability. Aggregate stability showed a seasonal pattern and was correlated significantly with earthworm density. Other factors influencing aggregate stability were management history in the conventional and integrated fields and, for a wider range of fields, organic matter content of the soil. The lower plastic limit, another stability parameter, was influenced mainly by organic matter and clay content, and not by any of the seasonally changing factors.

Cardiopulmonary bypass significantly reduces surfactant activity in children

Lung injury remains an important problem after cardiopulmonary bypass. The contribution of altered surfactant concentration or activity to pulmonary dysfunction after cardiopulmonary bypass is unclear. Recent evidence indicates that alveolar surfactant exists in specific aggregate forms that differ with respect to density, phospholipid composition, and function. A transition from surface active, higher density, large aggregates of surfactant to lower density, small aggregates that possess reduced surface activity has been demonstrated after experimental lung injury. The purpose of the present study was to examine surfactant aggregate fractions before and after bypass in children. Twelve acyanotic patients, aged 2 to 12 years, underwent intraoperative pulmonary function testing followed by bronchoalveolar lavage before incision and approximately 1 hour after termination of cardiopulmonary bypass. Saturated phosphatidylcholine pool sizes and total protein content of the small- and large-aggregate fractions of bronchoalveolar lavage fluid were determined. One hour after termination of cardiopulmonary bypass, the ratio of saturated phosphatidylcholine in small-aggregate as compared with that in large-aggregate fractions increased (mean +/- standard error) from 0.19 +/- 0.03 to 0.37 +/- 0.07 (p < 0.02), as did the ratio of saturated phosphatidylcholine to protein in the small-aggregate fraction (from 0.04 +/- 0.01 to 0.08 +/- 0.02, p < 0.05). Reductions in forced vital capacity (-19% +/- 5%), inspiratory capacity (-15% +/- 3%), and small airway flow rates (-32% +/- 6%) were also observed after bypass. These changes were accompanied by a fivefold increase in alveolar polymorphonuclear leukocyte content. The present study suggests that cardiopulmonary bypass of moderate duration in relatively healthy children is associated with surfactant changes that are similar in type and magnitude to those observed in experimental lung injury.

Changes in Exogenous Surfactant in Ventilated Preterm Lamb Lungs

Preterm lambs were treated with either a surfactant from bovine lung (Survanta) or three synthetic surfactants (Exosurf), a 69:22:9 mixture of dipalmitoylphosphatidylcholine, phosphatidylglycerol, and palmitic acid prepared by heat annealing (Lipid Mixture 1) or with glass beads (Lipid Mixture 2). After 5 h of ventilation, large and small aggregate surfactant fractions were isolated from alveolar washes by centrifugation. SP-A was used as an indicator for the association of endogenous surfactant components with the treatment surfactants. The large aggregate fraction from Survanta-treated lambs contained more SP-A than did the fractions from the lambs treated with the other surfactants (p < 0.05). The surfactants used to treat the sheep and the large aggregate surfactants from alveolar washes increased compliances when tested in surfactant-deficient, immature rabbits, relative to that in control animals. The large aggregate fractions in alveolar washes from lambs treated with Survanta, Lipid Mixture 1, and Lipid Mixture 2 improved compliances in the preterm rabbits to a greater extent than did the surfactants used to treat the lambs. The small aggregate fractions were inactive as surfactants. The function of exogenous surfactant can be improved after exposure to the preterm lung. The improvement may result from the association of exogenous surfactant with components of endogenous surfactant.

Degradation of surfactant-associated protein B (SP-B) during in vitro conversion of large to small surfactant aggregates

Pulmonary surfactant obtained from lung lavages can be separated by differential centrifugation into two distinct subfractions known as large surfactant aggregates and small surfactant aggregates. The large-aggregate fraction is the precursor of the small-aggregate fraction. The ratio of the small non-surface-active to large surface-active surfactant aggregates increases after birth and in several types of lung injury. We have utilized an in vitro system, surface area cycling, to study the conversion of large into small aggregates. Small aggregates generated by surface area cycling were separated from large aggregates by centrifugation at 40,000 g for 15 min rather than by the normal sucrose gradient centrifugation. This new separation method was validated by morphological studies. Surface-tension-reducing activity of total surfactant extracts, as measured with a pulsating-bubble surfactometer, was impaired after surface area cycling. This impairment was related to the generation of small aggregates. Immunoblot analysis of large and small aggregates separated by sucrose gradient centrifugation revealed the presence of detectable amounts of surfactant-associated protein B (SP-B) in large aggregates but not in small aggregates. SP-A was detectable in both large and small aggregates. PAGE of cycled and non-cycled surfactant showed a reduction in SP-B after surface area cycling. We conclude that SP-B is degraded during the formation of small aggregates in vitro and that a change in surface area appears to be necessary for exposing SP-B to protease activity.