Profile of phospholipase A2 activity in subcellular fractions and lamellar bodies of developing, neonatal and adult rabbit lung. Correlation with intracellular levels of disaturated phosphatidylcholine.

Phospholipase A2 activity was determined in subcellular fractions and lamellar bodies of fetal, neonatal and adult rabbit lungs. Specific activity in most fractions decreased from the 24th to the 28th day of gestation. All fractions except the mitochondrial and the nuclear fractions exhibited a sharp increase in activity in the newborn lung. Specific activity in the adult lung generally declined in comparison to neonatal values. During gestation total enzyme activity per gram of lung was concentrated in the cytosolic fraction. With the exception of the lamellar body fraction, the total content of phospholipase A2 activity increased dramatically in all fractions from the neonatal lung. The lamellar body fractions displayed both low specific activity and low total enzyme activity during gestation. Specific activity increased dramatically in the neonatal and adult lung but still accounted for only a small fraction of the activity in comparison to the other subcellular fractions. The subcellular content of disaturated phosphatidylcholine (PC) appeared to correlate well with the activity of phospholipase A2 in the neonatal mitochondrial, microsomal and cytosolic fractions. Since decreasing prenatal enzyme levels are associated with increasing disaturated PC content, the alkaline and calcium-dependent phospholipase A2 may not be directly involved in disaturated PC synthesis in the fetus. However, postnatally, the correlation between the pattern of production of disaturated PC and the activity of the phospholipase A2 indicates a role for this enzyme in surfactant-related disaturated PC synthesis.

Convertase activity in alveolar surfactant and lamellar bodies in fetal, newborn, and adult rabbits.

Conversion of heavy-aggregate alveolar surfactant (H) to a light-aggregate, nonsurface active form (L) is believed to involve the activity of an enzyme, namely, convertase. This conversion can be reproduced in vitro by the surface-area cycling technique. The purpose of the present study was to use this technique to investigate the developmental aspects of convertase activity in fetal, newborn, and adult rabbits. H was isolated from alveolar lavage from term [31-day gestation (31d)] fetal rabbit pups, 1-, 4-, and 7-day-old newborns, and adults, and the percent conversion to L was determined. To assess lamellar bodies (LB) as a potential source of activity in this species, these structures were isolated from lung tissue of 27-day-gestation (27d) and 31d fetuses, 1-, 4-, and 7-day-old newborns, and adults and were cycled the same as for H. LB contained considerable activity at each developmental stage i.e., approximately 82% of a 27d LB preparation converted to L after 3 h of cycling. In the adult, this value was 78%. Very little conversion of H was obtained from fetal lung (i.e., <20% of the 31d fetal preparation converted to L), but, by postnatal day 4, this value was greatly increased (i.e., >80% conversion) and stayed elevated to adulthood. The activity for each H and LB fraction was temperature and concentration dependent and diminished with storage at 4 degreesC. These data suggest the LB as the source of convertase activity in the rabbit and demonstrate dramatic developmental changes in this activity after release of the LB contents to the alveoli.

Alveolar metabolism of natural vs. synthetic surfactants in preterm newborn rabbits.

We compared the recoveries of four surfactant preparations: two natural [term fetal rabbit surfactant (FRS) and adult rabbit surfactant (ARS)] and two commercially available preparations [apoprotein-based Survanta (S) and synthetic Exosurf (E)] from 27-day gestation rabbit pups treated at birth and ventilated up to 120 min. At 5, 60, and 120 min, we measured the recovery of the heavy-aggregate, metabolically active form (H) and the light-aggregate, nonsurface active metabolic breakdown form (L) of alveolar surfactant and determined the phospholipid content and composition of the intracellularly stored lamellar body (LB) pool. Pups treated with FRS had <15% loss of H by 2 h. ARS-treated pups had a >50% loss of H by 1 h, and E- and S-treated pups had approximately 50% loss by 5 min, with a slower rate of continuing loss of up to 80% by 2 h. The major losses of H phospholipid were not explained by the L-form recovery. LB phospholipid significantly increased only in the E-treated pups and only at 2 h. FRS provides a biologically active form (H) of surfactant that appeared to remain in the airway for a significantly longer time than the other surfactant preparations. The unique properties of FRS merit further study.

Microanatomical changes in alveolar type II cells in juvenile mice intratracheally exposed to Stachybotrys chartarum spores and toxin.

Stachybotrys chartarum is an important environmental fungus. We have shown recently that alveolar type II cells are sensitive to exposure to Stachybotrys chartarum spores and to the trichothecene, isosatratoxin-F, both in vitro and in vivo, in a juvenile mouse model. This sensitivity is manifest as significant changes in the composition and normal metabolic processing of pulmonary surfactant. This study evaluated the effects of a single intratracheal exposure of S. chartarum spores and toxin on ultrastructure and dimensions of alveolar type II cells from juvenile mice. This was to determine whether there are concurrent morphological and dimensional changes in the alveolar type II cell that reflect the metabolic alterations in pulmonary surfactant that we observed in the treated mice. Marked ultrastructural changes were associated with alveolar type II cells in both S. chartarum and isosatratoxin-F treated animals compared to untreated, saline, and Cladosporium cladosporioides spore treated animals. These ultrastructural changes included condensed mitochondria with separated cristae, scattered chromatin and poorly defined nucleolus, cytoplasmic rarefaction, and distended lamellar bodies with irregularly arranged lamellae. Point count stereological analysis revealed a significant increase (p < 0.05) in lamellar body volume density in S. chartarum and isosatratoxin-treated animals after 48 h exposure. Mitochondria volume density was significantly lower in the isosatratoxin-F (48 h exposure) and S. chartarum treated (24 and 48 h exposure) animals compared to those in the other treatment groups. These results reveal that exposure to S. chartarum spores and toxin elicit cellular responses in vivo differently from those associated with exposure to spores of a nontoxigenic mold species. They also indicate that accumulation of newly secreted pulmonary surfactant in the alveolar space of S. chartarum and isosatratoxin-F treated animals might be a consequence of cellular trauma resulting in lamellar body volume density changes leading to increased release of pulmonary surfactant into the alveolar space.

Methylmercury-induced alterations in lung and pulmonary surfactant properties of adult mice.

Exposure to methylmercuric chloride (MMC) has been shown to significantly affect development of the lung and pulmonary surfactant system of the fetus. Preliminary results suggest it may also affect adult lung and associated bronchoalveolar lavage (BAL), which represents the extracellular surfactant pool. To determine if mercury exposure has the potential to alter surfactant function, adult mice were treated with MMC, 15 mg/kg by intragastric intubation on 4 successive days. BAL was collected by repeated intratracheal lavage 24 h after the last treatment. Nucleated cell numbers in lavage were determined. Tissue was prepared for scanning electron microscopy (SEM). Lavage fluid was extracted into chloroform:methanol and phospholipid concentration determined. A sample of the extract was used at a constant phospholipid concentration to measure surface activity on a bubble surfactometer. Lung weight to body weight ratio increased whereas total numbers of nucleated cells in BAL were not altered by MMC. SEM of samples from lungs of animals exposed to MMC showed normal architecture. Surface tension measurements suggest that the mean time to minimum surface tension and the minimum surface tension were greater in BAL from mice exposed to MMC for 4 days. In addition samples of BAL were prepared for Fourier-transform infrared spectrophotometry (FT-IR). Spectra showed changes in both lipid and protein components of BAL. Morphometric analyses of micrographs showed that mean alveolar diameter was reduced and wall thickness increased after mercury exposure. These results suggest that methylmercury exposure may significantly affect surface tension characteristics and composition of BAL, possibly through leakage of edematous interstitial tissue.

Fourier-transform infrared spectroscopic analysis of rabbit lung surfactant: subfraction-associated phospholipid and protein profiles.

Surfactant obtained from bronchoalveolar lavage (BAL) can be separated into subfractions based on sedimentation characteristics. It has been suggested that the 10,000 x g, 60,000 x g and 100,000 x g subfractions isolated by this approach represent stages of surfactant extracellular processing. These three subfractions have been reported to differ in their morphology, composition and ability to lower surface tension. We wished to determine if infrared spectroscopy, which may be applied as a non-invasive technique could potentially prove useful for characterization and quantification of bronchoalveolar lavage (BAL) protein and phospholipid, and if this approach could detect differences in intermediate surfactant processing stages. Subfractions were collected from adult rabbit lungs by BAL and differential centrifugation and analyzed by Fourier transform infrared (FT-IR) spectroscopy. Biochemical assay of phospholipid and protein showed differences between subfractions that correlated well with the phospholipid/protein ratios obtained from FT-IR spectra (r = 0.939; r2 = 0.882). The subfraction sedimenting at 100,000 x g (P100) exhibited spectral shifts in the Amide I band, suggesting that the protein secondary structure was different compared to other fractions. Spectra obtained after separation of lipids and protein components showed an apparent disordering of protein secondary structure but little or no effect on the structure or mobility of phospholipids. These results support the idea that subfractions represent various processing stages of surfactant. In addition, they show that results from FT-IR analyses correlate significantly with traditional biochemical assay methods which may prove of clinical use.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-PS-dependent protein kinase C and surfactant protein A in isolated fetal rabbit type II alveolar cells and surfactant-related material.

The fetal lung secretes significant quantities of surfactant during late gestation to prepare for initiation of respiration at birth. However, the mechanism by which this occurs has not been determined. Since Ca2+-phosphatidylserine (PS)-dependent protein kinase C has been implicated in surfactant secretion in adult lung, the present study was done to determine whether this enzyme is also involved in the initiation of surfactant release from fetal type II cells. Type II cells isolated from gestational day-24 fetal rabbits were used. Cells were prelabelled with [32P] and [3H]choline and exposed to 4beta phorbol ester (10(-5) M) for 2 h. Secretion product and subcellular fractions were isolated by removing the culture medium, mixing with homogenate from adult rabbit lung, and subfractionating by centrifugation on a sucrose gradient. Samples of secretion product were also prepared for electron microscopy. Ca2+-PS-dependent protein kinase C was also assayed in some samples, and an add-back technique was used to determine whether enzyme activity in the intracellularly stored surfactant fraction was due to contamination. The results showed that material released by fetal type II cells after exposure to phorbol ester coprecipitated with adult rabbit lung lamellar bodies and microsomes. Morphologically, a range of forms, including lamellar-body-like structures, was detected. The released material originated largely from the lamellar body compartment of the fetal type II cells and displayed immunoreactivity with antibody to surfactant protein A (SP-A) at 35 and 70 kDa apparent molecular mass. Assay of protein kinase C in fetal type II cells showed that exposure to conditioned medium, which induces differentiation, increased activity. Incubation with phorbol ester induced translocation of activity to the microsomal fraction. Add-back assays suggested that protein kinase C activation by treatment with phorbol ester induced translocation of enzyme activity to the lamellar body fraction; none was detected prior to treatment. These results support a role for Ca2+-PS-dependent protein kinase C in initiation of surfactant release by interaction with the developing lamellar body compartment in fetal type II cells.

Analysis of pulmonary surfactant by Fourier-transform infrared spectroscopy following exposure to Stachybotrys chartarum (atra) spores.

Lung cells are among the first tissues of the body to be exposed to air-borne environmental contaminants. Consequently the function of these cells may be altered before other cells are affected. As gas exchange takes place in the lungs, changes in cellular function may have serious implications for the processes of oxygen uptake and carbon dioxide elimination. In order for these processes to occur, the lung must maintain a high degree of expandability. This latter function is accomplished in part by the pulmonary surfactant which is synthesized and released by alveolar type II cells. Earlier studies have shown that exposure to gas phase materials such as smoke or organic solvents can alter the composition and function of the surfactant. The present study examines the ability of highly toxigenic mold spores to alter surfactant composition. Stachybotrys chartarum spores suspended in saline were instilled into mouse trachea as described earlier. After 24 h, the lungs were lavaged and the different processing stages of surfactant isolated by repeated centrifugation. Intracellular surfactant was isolated from the homogenized lung tissue by centrifugation on a discontinuous sucrose gradient. Samples were extracted into chloroform-methanol, dried and analyzed by Fourier-Transform infrared spectroscopy (FTIR). Exposure to S. chartarum induced an overall reduction of phospholipid among the three surfactant subfractions. The intermediate and spent surfactant fractions in particular were reduced to about half of the values observed in the saline-treated group. The relative distribution of phospholipid was also altered by spore exposure. Within the intracellular surfactant pool, higher levels of phospholipid were detected after spore exposure. In addition, changes were observed in the nature of the phospholipids. In particular strong intramolecular hydrogen bonding, together with other changes, suggested that spore exposure was associated with absence of an acyl chain esterified on the glycerol backbone, resulting in elevated levels of lysophospholipid in the samples. This study shows that mold spores and their products induce changes in regulation of both secretion and synthesis of surfactant, as well as alterations in the pattern of phospholipid targeting to the pulmonary surfactant pools.

Subcellular distribution of disaturated phosphatidylcholine in developing rabbit lung.

To determine the subcellular distribution of disaturated phosphatidylcholine (DSPC) in lung tissue during perinatal development, fetal rabbits at 24, 26, 28 and 31 (term) days gestation and newborns were studied. Following alveolar lavage, fractions enriched in nuclei-cellular debris, mitochondria, microsomes, surfactant (lamellar bodies) and cytosol were prepared from the residual tissue homogenate, and their DSPC content was determined. The DSPC content of the unfractionated residual lung tissue homogenate progressively and significantly increased during fetal development, rising from 9.09 +/- 0.91 to 17.45 +/- 2.88 mg/g dry lung between 24 days gestation, and term. Between 24 and 26 days gestation the overall increase in tissue DSPC was due to a two-fold increase in the mitochondrial, microsomal and cytosolic pools. Lamellar bodies were first isolable at 26 days gestation. The DSPC content of this fraction increased six-fold (from 0.10 +/- 0.02 to 0.67 +/- 0.15 mg/g dry lung) between 26 and 28 days gestation and a further seven-fold (to 4.63 +/- 1.06 mg/g dry lung) by term, accounting for the overall increase in the tissue homogenate value during this time period. By the first postnatal day, microsomal and cytosolic DSPC increased another two-fold, but no significant change occurred in the other subcellular fractions. Alveolar lavage DSPC progressively increased over the time period studied. While there was no change in the lamellar body DSPC/total PC ratio during fetal development, each of the mitochondrial, microsomal and cytosolic ratios decreased between days 26 and 28 of gestation and then increased at term.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse alveolar surfactant: characterization of subtypes prepared by differential centrifugation.

To characterize the properties of alveolar surfactant subfractions obtained from mouse lung by differential centrifugation, lavage fluid, following a preliminary centrifugation at 140 x g for 5 min to yield a cellular pellet (Pc), was sequentially centrifuged at 10,000 x g for 30 min, 60,000 x g for 60 min and 100,000 x g for 15 h; and the resultant pellets, respectively referred to as P10, P60 and P100, were harvested for electron microscopy, phospholipid analysis and surface tension measurements. Ultrastructural differences were observed, in that P10 contained large multilamellated structures which were typical of newly secreted surfactant, P100 contained small unilamellar vesicular structures, typical of catabolic end products of alveolar surfactant and P60 appeared to contain a mixture of structures present in P10 and P100 in addition to numerous, large unilamellar vesicles which were not present in either P10 or P100. Slight but significant differences were found in the phospholipid compositions of the three subfractions but not in the fatty acid composition of their phosphatidylcholine (PC) component. There were no significant differences in their disaturated PC/total PC ratios, but significant differences in their phospholipid/protein ratios. P60 had the highest proportion of phospholipid to protein. P10 and P60 demonstrated surface activity but P100 did not. Total alveolar surfactant phospholipid was evenly distributed among the three fractions. This pattern of distribution was significantly different from that observed in rabbit subfractions prepared by the same procedure. These data indicate that mouse alveolar surfactant consists of three distinct subfractions or subtypes which can be separately and quantitatively isolated by differential centrifugation.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of absorbance at 650 nm with the presence of phosphatidylglycerol in amniotic fluid.

Amniotic fluid absorbance at 650 nm was correlated with the presence of phosphatidylglycerol (PG) in the isolated surfactant fraction (10,000-g pellet). Shake test results were included. Two hundred ninety-seven samples were analyzed. PG was present in 222 of 226 samples in which the absorbance was greater than or equal to 0.250 and absent from 48 of 71 with an absorbance less than 0.250. PG was present in all 166 samples with a positive shake test and absent in 52 of 131 samples with a negative one. In 65 samples in which the shake test was negative and the absorbance greater than or equal to 0.250, PG was present in all but 4. The false-positive rate for the prediction of respiratory distress syndrome was 0.8% for the Shake test and 0.6% for the absorbance measurement. The results support the usefulness of the absorbance measurement as a simple and reliable procedure for assessing fetal lung maturity.

The role of centrifugation in the measurement of surfactant in amniotic fluid.

Analysis of amniotic fluid fractions obtained by differential centrifugation has revealed the presence of intact globular-like structures of approximately 1 mu dimensions. These particles, which appear to be representative of fetal lung surfactant, begin precipitating at very low g forces and the extent of their removal by centrifugation was found to significantly alter L/S ratio measurements. This effect is particularly significant when moderate levels of these structures are present, which is usually between 34 and 37 weeks' gestation. False-negative L/S ratios could be attributed to this effect. Centrifugation can be employed to harvest these structures in reasonably pure form and measurement of their concentration, by phospholipid analysis, showed good correlation with fetal lung function in a trial study with 91 patients. These findings suggest that measurement of these surfactant particles harvested from amniotic fluid may provide a simple yet specific index of fetal pulmonary maturity.

Comparative effects of chronic exposure to glucose or sodium butyrate on surfactant development in fetal rabbits.

BACKGROUND: Infants of diabetic mothers (IDM) often have delayed lung development and are thus at an increased risk of Respiratory Distress Syndrome (RDS). Both hyperglycemia and/or hyperinsulinemia have been implicated in this delay but the precise mechanism has not been clarified. Another metabolite, sodium butyrate, which is increased in IDM has been shown to decrease surfactant production in vitro but its effects on the development of the fetal lung surfactant system in vivo have not been studied. AIM: To investigate the in vivo effects of high glucose and sodium butyrate treatment on maternal and fetal glucose and insulin levels and on fetal lung surfactant maturation using timed-pregnant New Zealand White rabbits. METHODS: On the 24th day of gestation the doe was implanted s.c. with time release pellets containing either glucose (300 mg), sodium butyrate (200 mg) or matching placebo. On the 27th or 30th day maternal (ear vein) and fetal (cardiac puncture) blood samples were drawn for glucose and insulin determinations. Fetal surfactant pools (both intra- and extracellular) were quantitatively harvested using differential and density gradient centrifugation and their phospholipid profiles determined. Data were statistically compared with ANOVA and Duncan's Multiple Range Test. RESULTS: Neither glucose nor sodium butyrate affected maternal plasma glucose or insulin. Both metabolites significantly increased fetal plasma insulin, decreased fetal plasma glucose but did not delay any of the parameters of surfactant maturation examined. CONCLUSIONS: Fetal hyperinsulinemia, whether attained by prolonged exposure to elevated glucose or sodium butyrate in vivo does not appear to be the causative agent for delayed lung maturity which frequently occurs in infants of diabetic mothers.

Characterization of myelin of chick sciatic nerve during development.

Myelin was isolated from the sciatic nerves of chicks of ages 18-day embryonic, 1-day, 4-day, 7-day post-hatch, and adult to study developmental changes in lipid composition of this structure. The yield of myelin increased throughout the early stages of development and the preparations were of high purity. Although the lipid content of the myelin did not change, significant changes took place in lipid composition during development. The most significant changes were a relative increase in cerebrosides, phosphatidalethanolamine and long-chain fatty acids of cerebrosides, and a relative decrease in the content of phosphatidylserine and phosphatidylethanolamine. A second fraction ("lower band") was obtained during the isolation procedure. This "lower band" was present at all developmental stages and layered consistently at the interface of 1.2 and 0.8 M sucrose on a discontinuous gradient. The quantity of this fraction did not change during development and it differed from myelin in electron microscopic appearance. Its lipid composition, which did not change, resembled that of 18-day embryonic myelin in its high phospholipid:cholesterol ratio and low galactolipid content. The enzyme, 2':3'-cyclic-nucleosidemonophosphate phosphodiesterase was found to be present in both the myelin and "lower band" fractions; however there was no enrichment of this enzyme in purified myelin.

Beta-adrenergic-induced surfactant synthesis, secretion, and reutilization in fetal rabbit lung and isolated differentiating type II alveolar cells.

In vivo and in vitro approaches were used to examine the role of beta-adrenergic agonists in the regulation of surfactant synthesis and secretion in the lung. Rabbit fetuses of either 28 or 30 gestational days were treated with isoxsuprine. Fetuses from half of the does in each group were removed and allowed to breathe for 30 minutes. The others were left in utero. Intracellular and extracellular surfactant pools were isolated. Breathing increased secreted surfactant. On the twenty-eighth day without breathing, isoxsuprine treatment increased secretion of surfactant. The reverse effect was noted in the group that received the drug and also breathed. In contrast, on the thirtieth day, the drug inhibited surfactant release in those fetuses that did not breathe. In in vitro studies, undifferentiated type II alveolar cells were isolated and stimulated to differentiate. Subsequent exposure to isoxsuprine (5 or 10 mumol/L) stimulated both the synthesis and secretion of radiolabeled disaturated phosphatidylcholine. Concurrent incubation of those cells exposed to 10 mumol/L isoxsuprine with either unsaturated or disaturated phosphatidylcholine that was carbon 14 labeled showed a strong preference for incorporation of the latter phospholipid into total cellular phosphatidylcholine. These results suggest that beta-adrenergics may inhibit as well as stimulate secretion of surfactant by type II alveolar cells and that these cells may reincorporate secreted disaturated phospholipid.

Characterization and comparison of the role of beta-agonists on in vivo and in vitro surfactant-related phospholipid synthesis and secretion by fetal rabbit lung and isolated type II alveolar cells.

The role of beta-adrenergic stimulation in surfactant synthesis and secretion was investigated in the fetal lung. Fetuses were treated with isoxsuprine or saline on gestational day 24 by ip injection. Three days later the fetal lungs were lavaged and intracellular surfactant was isolated on a sucrose gradient. Concurrently undifferentiated type II alveolar cells were isolated from 24-day fetal rabbit lung and grown in vitro. In the in vivo portion of the study, examination of surfactant pool sizes revealed that only saline treatment produced a significant elevation in tissue-stored or secreted surfactant compared to untreated controls. Isoxsuprine appeared to inhibit the saline-induced increase. In the case of the intracellular surfactant, the phosphatidylcholine content per gram of lung was significantly increased after saline treatment. In vitro response of isolated type II alveolar cells to isoxsuprine was dependent on prior incubation of the cells for 24 h with conditioned medium. Isoxsuprine stimulated a dose-dependent decrease in the intracellular stores of radioactively labeled DSPC after 24 h of exposure to the drug. A corresponding increase in labeled DSPC in the culture medium was observed. Forth-eight hours after exposure to the drug, those cells that had secreted the highest level of DSPC displayed the highest levels of renewed synthesis of DSPC. This study indicates that the immature fetal lung can be induced to synthesize surfactant-related phospholipid by the stress of laparotomy and/or drug administration. Short-term exposure to beta-agonists is insufficient to stimulate secretion of surfactant stores. In contrast, isolated type II alveolar cells exposed to isoxsuprine respond by secreting DSPC.

Gestation-dependent effects of the combined treatment of glucocorticoids and thyrotropin-releasing hormone on surfactant production by fetal rabbit lung.

The effects of cortisol (0.1 mg per dose, administered intraperitoneally to fetal rabbits at 24 to 27 days' gestation), thyrotropin-releasing hormone (40 micrograms/kg per dose administered intravenously to the doe at 24 to 26 days' gestation), or a combination of the two on surfactant pool size (both intracellular and extracellular) at 27 or 28 days' gestation was investigated. Cortisol increased both surfactant pools only when administered on the twenty-fourth or twenty-fifth gestational day. Thyrotropin-releasing hormone, whether administered in single or multiple doses, had no effect on the extracellular pool but increased the intracellular pool; the magnitude of the response (approximately twofold) was similar to that observed with the cortisol response. All combinations of cortisol and thyrotropin-releasing hormone resulted in an increased response over either drug given alone. The greatest response (almost tenfold) resulted from cortisol administration at 24 days' gestation plus thyrotropin-releasing hormone administration at 24+ 25+ 26 days. These data demonstrate differential effects of glucocorticoids and thyrotropin-releasing hormone on developing lung and furthermore show that the timing of their combined treatment may be crucial to achieving maximal response.

Developmental study of a lamellar body fraction isolated from human amniotic fluid.

Several properties of a pellet fraction obtained on centrifuging amniotic fluid at 10,000 X g for 20 min were investigated. From these analyses, we defined a developmental profile which appears to describe the maturational process of the fetal lung surfactant system. At 14 to 18 wk gestation, the pellet fraction consisted of membrane-bound vesicles without internal lamellae. The phospholipid composition did not resemble that of surfactant, the major phospholipid being sphingomyelin. This stage, designated as presurfactant, persisted until 30 to 32 wk gestation. After this time, the phospholipid concentration of the pellet fraction increased continuously throughout development, and gradual but continuous changes in phospholipid composition were observed. Lecithin and phosphatidylinositol increased between 30 and 35 wk gestation. Interruption of pregnancies at this stage, termed onset of surfactant synthesis, resulted in 100% incidence of respiratory distress syndrome. From 36 wk gestation to postterm, the pellet fraction contained structures with the characteristic morphology of the lamellar inclusion bodies. The presence of single membrane components in these preparations did not contribute to the phospholipid composition. Early formed lamellar bodies lacked phosphatidylglycerol and had a high content of phosphatidylinositol. Once phosphatidylglycerol appeared in the lamellar body fraction, it continued to increase, accompanied by a decrease in phosphatidylinositol with little change in lecithin. A phosphatidylglycerol value of greater than 1% of the total phospholipids appeared to represent the stage of maturity at which there was no risk of respiratory distress syndrome.

Assessment of fetal lung maturity: relationship of gestational age and pregnancy complications to phosphatidylglycerol levels.

Phosphatidylglycerol (PG) was measured in the pellet fraction of 863 amniotic fluid samples, and charts were reviewed for maternal disease, duration of gestation at collection, and outcome of pregnancy. PG was present at 32 to 34 weeks' gestation in 24.1% of samples; at 35 to 36 weeks, in 52.3%; and at 37 weeks, in 85.4%. Pre-eclamptic toxemia/hypertension, diabetes, premature rupture of membranes, and preterm labor all had earlier appearance of PG than a comparison group. There was no delay in lung maturity in gestational diabetics or Rh isoimmunization. Infants of patients with overt diabetes with PG greater than or equal to 0.5% did not develop respiratory distress syndrome. This value appeared in 30% of diabetic patients by 35 to 36 weeks and in 76.9% by 37 weeks' gestation.