Results of a comparative study on the Salmonella pre-incubation and plate incorporation assays using test samples from the IPCS collaborative study.

Three complex mixtures (air particles, diesel particles and a coal tar fraction) and two pure compounds (benzo[a]pyrene and 1-nitropyrene) were tested in both the pre-incubation and the plate incorporation assay employing Salmonella typhimurium TA98 and TA100. Each experiment was conducted independently 2 or 4 times in duplicate in the presence and absence of metabolic activation. The mutagenic activities were calculated by least squares linear regression from the slope of the linear portion of each dose-response curve. Although slightly higher mutagenic activity was observed in the pre-incubation assay for the two pure compounds and with the plate incorporation assay for the diesel particulate sample, the overall data from both assays gave similar values and good correlations in TA100 and TA98. The results indicate that the pre-incubation assay could be used for these samples instead of the plate incorporation assay.     

Results of a comparative study on the Salmonella pre-incubation and plate incorporation assays using test samples from the IPCS collaborative study

Three complex mixtures (air particles, diesel particles and a coal tar fraction) and two pure compounds (benzo[a]pyrene and 1-nitropyrene) were tested in both the pre-incubation and the plate incorporation assay employing Salmonella typhimurium TA98 and TA100. Each experiment was conducted independently 2 or 4 times in duplicate in the presence and absence of metabolic activation. The mutagenic activities were calculated by least squares linear regression from the slope of the linear portion of each dose-response curve. Although slightly higher mutagenic activity was observed in the pre-incubation assay for the two pure compounds and with the plate incorporation assay for the diesel particulate sample, the overall data from both assays gave similar values and good correlations in TA100 and TA98. The results indicate that the pre-incubation assay could be used for these samples instead of the plate incorporation assay.     

Effects of drought on primary photosynthetic processes of cotton leaves

The localization of enzymes involved in the flow of carbon into and out of starch was determined in guard cells of Commelina communis. The guard cell chloroplasts were separated from the rest of the cellular components by a modification of published microfuge methods. The enzymes of interest were then assayed in the supernatant and chloroplast fractions. The chloroplast yield averaged 75% with 10% cytoplasmic contamination. The enzymes involved in starch biosynthesis, ADPglucose pyrophosphorylase, starch synthase, and branching enzyme, are located exclusively in the chloroplast fraction. The enzymes involved in starch degradation show a more complex distribution. Phosphorylase is located in both the supernatant and chloroplast fraction, 50% in each fraction. Most of the amylase and debranching enzyme activity is present in the supernatant (70%) fraction. The majority of the rest of the enzymes involved in the degradation of starch to malate and synthesis of starch from a hexose precursor were also investigated. All of the enzymes were present in the chloroplast except for hexokinase and phosphofructokinase. The inability to assay these enzymes could possibly have been due to the lack of or low activity of the enzymes or to nonoptimal assay conditions.     

Isolation and characterization of a small intestinal surfactant-like particle containing alkaline phosphatase and other digestive enzymes

Digestive brush-border enzymes in particulate form have been reported in the intestinal lumen in vivo and in medium from organ explants in vitro. It has been suggested that these particles derive from membrane shedding of the apical brush border. This study describes the isolation and characterization of particles derived from the 105,000 x g supernatant fraction of intestinal luminal washings and from light scrapings of the mucosa itself after fat feeding of rats. These fractions were separated in a continuous NaBr gradient, producing a visible band of 1.07-1.08 g/liter density and resulting in a 15-fold enrichment of intestinal alkaline phosphatase in the band fraction. Other brush-border hydrolases were represented in the banded fraction, but at specific activities only 1/5th to 1/36th that of the brush border. The major phospholipid in the fraction was phosphatidylcholine (58 +/- 15%), containing 75% saturated fatty acids. In contrast, the major brush-border phospholipid was phosphatidyl-ethanolamine. These characteristics showed that the particles derived from the lumen and mucosal surface were not identical to fragments of the brush border. Electron microscopy of the banded fraction revealed partially coiled membrane fragments. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots, some proteins (e.g. surfactant protein B, collagenous protein 4) were found in common between the intestinal particles and rat pulmonary surfactant. These data suggest the production of a particle secreted by rat intestine that differs from brush-border membranes and that shares some morphological and biochemical similarities with pulmonary surfactant.     

Characterization of organic extracts from standard reference materials 1649, 'urban dust/organics,' and 1650, 'diesel particulate matter', using a microsuspension assay. A WHO/IPCS/CSCM study.

Mutagenicity associated with replicate organic extracts from standard reference materials 1649 'urban dust/organics' (air particles), and 1650, 'diesel particulate matter' (diesel particles), was determined using a Salmonella microsuspension assay. The results indicate that the mutagenicity of samples such as these can readily be determined using the microsuspension assay with only 5% of the mass required for the standard plate incorporation assay. In general, 80% of the variation in mutagenic activity was due to the bioassay procedure and 20% to the extraction process. Extracts from both samples had primarily direct-acting mutagenicity as there were no significant differences in responses with and without metabolic activation (S9). The TA98-S9 mean air particles mutagenic activities (C.V., %) based on mass of extractable organics or particles were 4.4 (4.7%) and 0.29 (3.6%) revertants/micrograms, respectively, and for the diesel particles were 66 (44%) and 12 (29%) revertants/microgram, respectively. More of the observed direct-acting mutagenicity in the diesel particles extracts was due to nitro-substituted compounds because there were significant reductions in activity with TA98NR (45% of TA98 -S9) and TA98-1,8-DNP6 (21% of TA98 -S9). In the air particles extracts, the TA98NR activities were not significantly different from TA98 -S9 but the TA98-1,8-DNP6 levels were.     

Characterization of organic extracts from standard reference materials 1649, ‘urban dust/organics,’ and 1650, ‘diesel particulate matter’, using a microsuspension assay. A WHO/IPCS/CSCM study

Mutagenicity associated with replicate organic extracts from standard reference materials 1649 ‘urban dust/organics’ (air particles), and 1650, ‘diesel particulate matter’ (diesel particles), was determined using a Salmonella microsuspension assay. The results indicate that the mutagenicity of samples such as these can readily be determined using the microsuspension assay with only 5% of the mass required for the standard plate incorporation asssay.In general, 80% of the variation in mutagenic activity was due to the bioassay procedure and 20% to the extraction process. Extracts from both samples had primarily direct-acting mutagenicity as there were no significant differences in responses with and without metabolic activation (S9). The TA98 - S9 mean air particles mutagenic activities (C.V., %) based on mass of extractable organics or particles were 4.4 (4.7%) and 0.29 (3.6%) revertants/μg, respectively, and for the diesel particles were 66 (44%) and 12 (29%) revertants/μg, respectively. More of the observed direct-acting mutagenicity in the diesel particles extracts was due to nitro-substituted compounds because there were significant reductions in activity with TA98NR (45% of TA98 -S9) and TA98-1,8-DNP6 (21% of TA98 -S9). In the air particles extracts, the TA98NR activities were not significantly different from TA98 - S9 but the TA98-1,8-DNP6 levels were.     

Pulmonary phosphatidic acid phosphatase: evidence for a membrane-bound phosphatidic acid-dependent activity associated with the high speed supernatant of rat lung.

The 104,000 × g supernatant fraction from rat lung contains a greater proportion of the phosphatidic acid phosphatase activity toward membrane-bound phosphatidic acid than the microsomal fraction. The microsomal fraction is more effective in hydrolyzing aqueously dispersed phosphatidic acid. The effects of various ions and chelators, particularly Mg²⁺ and EDTA, suggest that these two activities are distinct. These results indicate that the supernatant fraction of rat lung contains a phosphatidic acid phosphatase activity which may play an important role in pulmonary glycerolipid synthesis.     

Studies on guanine deaminase and its inhibitors in rat tissue

1. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, as in brain and liver, the enzymic activity recovered in the supernatant was higher than that in the whole homogenate. The particulate fractions of kidney, especially the heavy mitochondria, brought about powerful inhibition of the supernatant guanine-deaminase activity. 2. In spleen, as in kidney, guanine-deaminase activity was localized in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, the particulate fractions did not inhibit the activity of the supernatant. 3. Guanine-deaminase activity in rat brain was absent from the cerebellum and present only in the cerebral hemispheres. The inhibitor of guanine deaminase was located exclusively in the cerebellum, where it was associated with the particles sedimenting at 5000g from sucrose homogenates. 4. Homogenates of cerebral hemispheres, the separated cortex or the remaining portion of the hemispheres had significantly higher guanine-deaminase activity than homogenates of whole brain. The enzymic activity of the subcellular particulate fractions was nearly the same. 5. Guanine deaminase was purified from the 15000g supernatant of sucrose homogenates of whole brain. The enzyme separated as two distinct fractions, A and B, on DEAE-cellulose columns. 6. The guanine-deaminase activity of the light-mitochondrial fraction of whole brain was fully exposed and solubilized by treatment with Triton X-100, and partially purified. 7. Tested in the form of crude preparations, the inhibitor from kidney did not act on the brain and liver supernatant enzymes and the inhibitor from cerebellum did not act on kidney enzyme, but the inhibitor from liver acted on both brain and kidney enzyme. 8. The inhibitor of guanine deaminase was purified from the heavy mitochondria of whole brain and liver and the 5000g residue of cerebellum, isolated from iso-osmotic homogenates. The inhibitor appeared to be protein in nature and was heat-labile. The inhibition of the enzyme was non-competitive. 9. Kinetic, immunochemical and electrophoretic studies with the preparations purified from brain revealed that the enzyme from light mitochondria was distinct from enzyme B from the supernatant. A distinction between the two forms of supernatant enzyme was less certain. 10. Guanine deaminase isolated from light mitochondria of brain did not react with 8-azaguanine or with the inhibitor isolated from heavy mitochondria.     

Translation and identification of the viral mRNA species isolated from subcellular fractions of vesicular stomatitis virus-infected cells.

The cytoplasm of vesicular stomatitis virus (VSV)-infected BHK cells has been separated into a fraction containing the membrane-bound polysomes and the remaining supernatant fraction. Total poly(A)-containing RNA was isolated from each fraction and purified. A 17S class of VSV mRNA was found associated almost exclusively with the membrane-bound polysomes, whereas 14,5S and 12S RNAs were found mostly in the postmembrane cytoplasmic supernatant. Poly(A)-containing VSV RNA synthesized in vitro by purified virus was resolved into the same size classes. The individual RNA fractions isolated from VSV-infected cells or synthesized in vitro were translated in cell-free extracts of wheat germ, and their polypeptide products were compared by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The corresponding in vivo and in vitro RNA fractions qualitatively direct the synthesis of the same viral polypeptides and therefore appear to contain the same mRNA species. By tryptic peptide analysis of their translation products, the in vivo VSV mRNA species have been identified. The 17S RNA, which is compartmentalized on membrane-bound polysomes, codes for a protein of molecular weight 63,000 (P-63) which is most probably a nonglycosylated form of the viral glycoprotein, G. Of the viral RNA species present in the remaining cytoplasmic supernatant, the 14.5S RNA codes almost exclusively for the N protein, whereas the 12S RNA codes predominantly for both the NS and M proteins of the virion.     

Stereospecificity of hepatic L-tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase [L-tryptophan--oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.11] has been reported to act solely on the L-isomer of tryptophan. However, by using a sensitive assay method with D- and L-[ring-2-14C]tryptophan and improved assay conditions, we were able to demonstrate that both the D- and L-stereoisomers of tryptophan were cleaved by the supernatant fraction (30000 g, 30 min) of liver homogenates of several species of mammals, including rat, mouse, rabbit and human. The ratio of activities toward D- and L-tryptophan was species variable, the highest (0.67) in ox liver and the lowest (0.07) in rat liver, the latter being hitherto exclusively used for the study of hepatic tryptophan 2,3-dioxygenase. In the supernatant fraction from mouse liver, the ratio was 0.23 but the specific activity with D-tryptophan was by far the highest of all the species tested. To identify the D-tryptophan cleaving enzyme activity, the enzyme was purified from mouse liver to apparent homogeneity. The specific activities toward D- and L-tryptophan showed a parallel rise with each purification step. The electrophoretically homogeneous protein had specific activities of 0.55 and 2.13 mumol/min per mg of protein at 25 degrees C toward D- and L-tryptophan, respectively. Additional evidence from heat treatment, inhibition and kinetic studies indicated that the same active site of a single enzyme was responsible for both activities. The molecular weight (150000), subunit structure (alpha 2 beta 2) and haem content (1.95 mol/mol) of the purified enzyme from mouse liver were similar to those of rat liver tryptophan 2,3-dioxygenase. The assay conditions employed in the previous studies on the stereospecificity of hepatic tryptophan 2,3-dioxygenase were apparently inadequate for determination of the D-tryptophan cleaving activity. Under the assay conditions in the present study, the purified enzyme from rat liver also acted on D-tryptophan, whereas the pseudomonad enzyme was strictly specific for the L-isomer.     

A particle concentration fluorescence immunoassay for prostaglandin D synthase in the rat central nervous system

A solid phase, particle concentration fluorescence immunoassay (PCFIA) was developed for the measurement of prostaglandin (PG) D synthase in the 100,000g supernatant of various regions of the rat central nervous system. In this assay, the enzyme (in the range of 1-25 micrograms protein of brain supernatant or 1-100 ng of the purified enzyme) is attached to submicrometer carboxypolystyrene beads coated with polyclonal anti-rat brain PGD synthase IgG. The total particle-bound enzyme is assayed with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-PGD synthase IgG after incubation for 1 h. The optimum assay condition was obtained when carboxyl particles coated with ca. 500 micrograms/ml of polyclonal IgG at pH 5.0 and 5 micrograms/ml of FITC-IgG were used. No significant fluorescence was observed when FITC conjugates or carboxyl particles were prepared using IgG from nonimmunized rabbits. Heat treatment of the brain supernatant decreased the specific binding of the enzyme in parallel with the loss of enzyme activity, indicating that the denatured enzyme is not recognized by this assay method. The PGD synthase immunoreactivity was widely distributed in the brain regions and was highest in the paraflocculus. Although slight discrepancy was observed between the concentration by PCFIA and the enzyme activity measured by using [14C]PGH2 in some brain regions, there is a considerable correlation (0.727) between the values by both methods in the same brain regions. The PCFIA now developed showed higher sensitivity (around 10 times), greater reliability, and larger number of samples measurable at once than the radio-TLC assay using [14C]PGH2. This method could provide valuable information concerning the regulatory mechanisms of PGD synthase.     

Heterogeneity of immunohistochemical staining with pulmonary surfactant protein A among fractionated alveolar macrophages which involves metabolism of pulmonary surfactant.

Alveolar macrophages (AM) which are separated into four fractionated subpopulations (I, II, III and IV), represented differential immunohistochemical staining with antibody against pulmonary surfactant protein A (SP-A). In light microscopy, the least dense AM (fraction I) were intensely stained with antibody to SP-A in numerous granules of the cytoplasm, whereas the most dense cells (fraction IV) showed immuno-reactivity with the antibody in several granules distributed in the spreading and elongating cytosol. By Western blot analysis, antibody to SP-A recognized a triplet of nature molecules of SP-A in AM lysate. However, the antigen of the AM lysate almost disappeared when the cells were cultured for more than two days, which indicate that AM do not synthesize SP-A and have digested intracellular SP-A during the cultivation. Immunoelectron microscopically, AM of fraction IV sometimes had very large vacuoles including lamellar body-like structures, probably pulmonary surfactant immediately after taken up from the alveolar lumen by them, which were heavily deposited with gold particles indicating antigenic site of SP-A. Whereas cells of fraction I contained numerous cytoplasmic vacuoles that were frequently labelled with the immuno-gold particles and were not associated with lamellar body-like structures, which may indicate that the materials in the vacuoles are digesting. The results of this experiments suggest that pulmonary surfactant, layered on the alveolar epithelium, is in part taken up by higher dense AM and is digested during a process of their maturation in the direction of lower dense cells, which undergo an important role of metabolism of pulmonary surfactant by AM subpopulations.     

A comparative study of bile acid CoA:amino acid:N-acyltransferase (BAT) from four mammalian species.

1. Bile acid CoA:amino acid:N-acyltransferase (BAT) was partially purified from dog, human, pig and rat livers. The interspecies variation in substrate specificity and kinetics were determined for glycine and taurine. 2. BAT activity from dog liver formed bile acid conjugates with taurine exclusively, whereas BAT activity from each of the other species formed conjugates with both taurine and glycine. 3. Biliary composition of glycine and taurine bile acid conjugates could partly be accounted for by substrate affinity (Km) and turnover number (Vmax) of BAT activity. 4. A monospecific anti-human BAT polyclonal antibody reacted on Western blot analysis with a 40 kDa band in a 100,000 g supernatant fraction from rat liver. 5. Immunoabsorption chromatography using an anti-human BAT antibody-Sepharose affinity column showed that both the immunoreactive protein band and BAT activity were removed from the 100,000 g supernatant fraction from human and rat livers.     

New method for determining the extent of proline hydroxylation by measuring changes in the ratio of [4-3H]:[14C]proline in collagenase digests

A sensitive colorimetric assay for proteases and certain polysaccharidases is based on the digestion of proteoglycan from bovine nasal cartilage. This high-molecular-weight substrate is trapped in the interstices of a polyacrylamide gel. The gel is then dispersed as small particles. Enzymes can diffuse into these particles and digestion products can diffuse out. Following digestion, the particles are centrifuged off and the digestion products in the supernatant are quantitated by reaction with the metachromatic dye 1,9-dimethylmethylene blue or by assay of their uronic acid content with carbazole reagent. Proteases from each of the four major classes can be quantitated at levels of 1–200 ng. The method is particularly suitable for the study of cartilage proteases that degrade matrix proteoglycans.     

Intra- and extracellular compartmentalization of the surface-active fraction in dog lung

Adult mongrel dogs were killed at various times after injection of (3)H-labeled palmitate. The lungs were removed and subjected to an extensive saline lavage. The surface-active fraction was isolated from the lavage and from homogenized residual lung by a procedure based upon differential centrifugation in sucrose solutions. The material isolated from the lavage was designated extracellular surfactant; material from the residual lung was designated intracellular surfactant. Both had similar chemical composition and surface activity. The results of the isotopic labeling studies demonstrate that the two fractions have distinctly different specific activity curves. Label was incorporated into the intracellular surfactant rapidly and reached a peak at 1 hr. No radioactivity was found in the extracellular surfactant for the first 15 min, and the specific activity increased much more slowly than in the intracellular surfactant. These results demonstrate at least two anatomically distinct metabolic "pools" of pulmonary surfactant in the lung. While our data are not conclusive, one possible interpretation is that the biosynthesis of pulmonary surfactant takes place intracellularly with a subsequent secretion onto the alveolar surface.     

Monoclonal antibodies against human pulmonary surfactant apoproteins: specificity and application in immunoassay

Monoclonal antibodies were prepared against pulmonary surfactant apoproteins which were isolated from lung lavages of patients with alveolar proteinosis with the following steps: solubilization of the surface-active fraction by Triton X-100, delipidation with butanol-ethanol extraction followed by column chromatographies on Blue-Sepharose and DEAE-Toyopearl in the presence of dithiothreitol. The fraction including 62 and 36 kDa proteins, i.e., pulmonary surfactant apoproteins, was used for the immunization. Monoclonal antibodies against the pulmonary surfactant apoproteins were prepared using hybridoma technology. The monoclonal antibodies prepared, PC6 and PE10, recognized the same proteins, i.e., 62 and 36 kDa proteins, in the patients' lavages. They also recognized 37 and 34 kDa proteins in human lung lavage and amniotic fluid. Quantitation of the apoproteins by enzyme-immunoassay using the monoclonal antibodies has been developed. A combination of PC6 and PE10 was found to be useful for a two-site sandwich enzyme-linked immunosorbent assay (ELISA), where it gave a good dose response and was capable of measuring 10-1280 ng of the apoprotein/ml. The specificity of the monoclonal antibodies in animal species was tested by this sandwich ELISA. The results indicated that the monoclonal antibodies obtained in this study are specific for the human lung.     

Diesel Exhaust Particle Exposure In Vitro Alters Monocyte Differentiation and Function

Air pollution by diesel exhaust particles is associated with elevated mortality and increased hospital admissions in individuals with respiratory diseases such as asthma and chronic obstructive pulmonary disease. During active inflammation monocytes are recruited to the airways and can replace resident alveolar macrophages. We therefore investigated whether chronic fourteen day exposure to low concentrations of diesel exhaust particles can alter the phenotype and function of monocytes from healthy individuals and those with chronic obstructive pulmonary disease. Monocytes were purified from the blood of healthy individuals and people with a diagnosis of chronic obstructive pulmonary disease. Monocyte-derived macrophages were generated in the presence or absence of diesel exhaust particles and their phenotypes studied through investigation of their lifespan, cytokine generation in response to Toll like receptor agonists and heat killed bacteria, and expression of surface markers. Chronic fourteen day exposure of monocyte-derived macrophages to concentrations of diesel exhaust particles >10 µg/ml caused mitochondrial and lysosomal dysfunction, and a gradual loss of cells over time both in healthy and chronic obstructive pulmonary disease individuals. Chronic exposure to lower concentrations of diesel exhaust particles impaired CXCL8 cytokine responses to lipopolysaccharide and heat killed E. coli, and this phenotype was associated with a reduction in CD14 and CD11b expression. Chronic diesel exhaust particle exposure may therefore alter both numbers and function of lung macrophages differentiating from locally recruited monocytes in the lungs of healthy people and patients with chronic obstructive pulmonary disease.     

The proteins of human lung surfactant

Human pulmonary surfactant was purified from bronchoalveolar lavage of patients. The proteins present in surfactant were analyzed by SDS-polyacrylamide gel electrophoresis into serum and non-serum components. One non-serum surfactant protein (Mr = 43 000) was then identified in the 100 000 X g supernatant of a lung homogenate on the basis of phospholipid binding. This lung protein was purified and partially characterized. The presence of 3-methyl histidine and reaction in Western blot analysis with antibody against chicken muscle actin both strongly suggested that the 43 000 Da protein of human surfactant is indeed cytoplasmic actin. It is proposed that this surfactant protein is involved in the secretion and not necessarily in the function of surfactant.     

Pulmonary antioxidants exert differential protective effects against urban and industrial particulate matter

This investigation focuses on the application of an in vitro assay in elucidating the role of lung lining fluid antioxidants in the protection against inhaled particles, and to compare the toxicities of different airborne particulate matter (PM), PM10, collections from South Wales, UK. PM collections from both urban and industrial sites caused 50% oxidative degradation of DNA in vitro at concentrations as low as 12.9 +/- 2.1 microg ml(-1) and 4.9 0.9 mg ml-1 respectively. The primary source of this bioreactivity was found to be the soluble fraction of both particle collections. The coarser PM(10-2.5) fraction also showed greater oxidative bioreactivity than the PM(2.5-0.1) in both cases. When repeated in the presence of a low molecular weight fraction of fresh pulmonary lavage fluid, as well as in artificial lung lining fluid (200 microM urate, glutathione and ascorbate), the DNA damage was significantly reduced in all cases (P < 0.05). The antioxidants exerted a greater effect on the industrial samples than on the urban samples, and on the PM(10-2.5) fractions than on the PM(2.5-0.1) fractions, supporting the previous findings that respirable PM and urban samples contain fewer free radical sources than inhalable PM and industrial samples.     

Quantification of surfactant pool sizes in rabbit lung during perinatal development

Methods are presented for the quantitative isolation of surfactants from fetal and newborn rabbit alveolar lavage returns and post-lavaged lung tissue homogenates. The phospholipid content of both fractions progressively increased between 27 days gestation and term (31 days). The tissue-stored fraction increased approximately 16-fold (from 0.48 +/- 0.13 to 7.83 +/- 0.86 mg/g dry lung) and the alveolar fraction more than 30-fold (from 0.08 +/- 0.02 to 2.69 +/- 0.52 mg/g dry lung). Developmental changes in phospholipid composition were also observed. Tissue-stored surfactant was prepared using differential and density gradient centrifugation. Alveolar surfactant was isolated during fetal development as a high-speed pellet following a one-step differential centrifugation. There was little change in the phospholipid content of fetal alveolar lavage supernatant (range 0.12 +/- 0.04 to 0.28 +/- 0.09 mg/g dry lung). By the first postnatal day the phospholipid content of both lavage fractions significantly increased (pellet, 7.51 +/- 1.79; supernatant, 4.01 +/- 1.36 mg/g dry lung) and both were identified as surfactant. This increase in alveolar surfactant was accompanied by an approximately twofold decrease (to 3.81 +/- 1.1 mg/g dry lung) in the tissue-stored fraction. These data provide a quantitative profile of surfactant accumulation and secretion in developing rabbit lung.