The effect of intra-amniotic injection of triiodothyronine on pulmonary maturity in lambs at 130 days gestation.

Saline or triiodothyronine (T3) (50-1000 micrograms) was injected into the amniotic sac of 17 pregnant ewes under general anesthesia at 130 days gestation. Forty-eight h later, the lambs were delivered by hysterotomy. Cord plasma T3 and cortisol and amniotic fluid T3 were assayed, and the maturity of the fetal lung was assessed in terms of its pressure-volume response and its surfactant (lamellar body phospholipid) content. With the highest dose of T3, cord plasma T3 and cortisol were raised, and lung maturity was enhanced compared with saline-treated controls; the pressure-volume curve showed increased hysteresis on inflation and deflation, and the lung retained air on return to zero pressure. There was also an apparent, but not statistically significant, increase in the lamellar body phospholipid content of the lung. Irrespective of treatment, lungs which were more mature, in terms of their pressure-volume characteristics, tended to contain a higher proportion of lamellar body phospholipid relative to total phospholipid.

Methods for extracting phospholipids from human amniotic fluid compared.

Phospholipids were extracted from human amniotic fluid by various procedures, including the two most commonly applied to amniotic fluid for evaluation of fetal lung maturity. We find that the yield of phospholipid is greatly procedure dependent. This should be taken into account when one is considering the various reported methods of evaluating fetal lung maturity, because in some of them phospholipid data are expressed in terms of absolute concentration in the amniotic fluid. There were also significant differences in phospholipid composition in extracts prepared by the various procedures, but in general these were not large enough to influence evaluation of fetal lung maturity by methods in which phospholipid data are expressed in relative terms, as ratios or percentages--e.g., in the lecithin/sphingomyelin ratio and "lung profile" procedures. In the extraction method originally recommended for determination of the lecithin/sphingomyelin ratio, both the yield and composition of phospholipid depend on the extent of mixing.

Differential effect of detergents on the alkaline denaturation of haemoglobin in maternal and fetal blood, with particular reference to Triton X-100.

In this investigation, the alkaline denaturation of haemoglobin in the blood of pregnant women and in cord blood obtained from newborn infants was followed by measuring the increase in absorbance at 375 nm. As expected, in the absence of detergent, the haemoglobin of cord blood was much more resistant to alkaline denaturation than that of maternal blood. However, in the presence of Triton X-100, a non-ionic detergent, the sensitivity of fetal haemoglobin to alkali was comparable to that of adult haemoglobin. Similar results were obtained using the non-ionic detergents, Brij-35, Tween 80 and Nonidet P40, but the anionic detergent, sodium deoxycholate, was apparently without effect. These findings form the basis of a rapid and sensitive method for discriminating between maternal and fetal blood in biological specimens.

Rapid qualitative analysis of polar and nonpolar lipids in a single sample using "three-way" thin layer chromatography.

Plastic sheets, precoated with silica gel, are cut to give a square with a strip attached at the top right-hand corner. Lipid extract is applied to the square portion, just below the strip, and chromatographed in a solvent which moves nonpolar lipids on to the strip, leaving polar lipids at the origin. The strip is cut off and separation of the lipids on the strip and the remaining square portion of the plate is completed by conventional one- or two-dimensional chromatography, respectively. The application of this method and a multiple staining technique to the analysis of lipids in human bronchoalveolar lavage fluid is described.

Improved procedure for lecithin/sphingomyelin ratio in amniotic fluid reduces false predictions of lung immaturity.

Many laboratories have found that their procedure for determining lecithin/sphingomyelin (L/S)ratios gives an unacceptably high proportion of false predictions of fetal lung immaturity. We investigated each step in the procedure, in an attempt to improve the clinical performance of the test and to make the method more amenable to standardization between laboratories. L/S ratios were determined by the new procedure in amniotic fluid from 147 pregnancies, collected within two days of delivery of the infant. Four cases had an L/S ratio less than 2; all developed hyaline membrane disease. No other cases of hyaline membrane disease were encountered in this study. For 106 of these pregnancies, the L/S ratio was also determined by the procedure previously in use. This predicted lung immaturity for 16 infants, only four of whom developed hyaline membrane disease. All 12 cases incorrectly predicted as immature by the old procedure were correctly classified by the new procedure.

Methods of evaluating fetal lung maturity.

Respiratory distress syndrome occurs in infants born with immature lungs. The immature fetal lung lacks an adequate supply of surfactant, a phospholipid-rich substance which is produced in the type II cells of the alveolar epithelium. In the fetus, surfactant is secreted into the potential air spaces of the lung and passes into the amniotic fluid as gestation proceeds. It is now clear that most methods currently in use for assessing fetal lung maturity depend on the detection of a sudden release of surfactant into the amniotic fluid as the lung reaches a critical stage of maturity. These methods, which include the lecithin/sphingomyelin ratio, the lung profile, total phospholipid or lecithin concentration, fluorescence depolarization, lamellar body phospholipid concentration, and the "shake" test, are reviewed in the light of recent understanding of the nature of surfactant. In assessing each method, we have examined possible sources of error in performing the test in the laboratory, factors which could theoretically limit its ability to reflect the state of fetal lung maturity and current information regarding its reliability, in terms of clinical performance. Guidelines for future research in this area are also suggested.

Measurement of "lamellar body phospholipid" in amniotic fluid as a method for assessing fetal lung maturity.

A simple, rapid micro-method, suitable for use in a routine clinical laboratory, is described for isolating a surfactant fraction from 0.1 mL of human amniotic fluid and measuring its phospholipid content. We determined the phospholipid content of this fraction, referred to as "lamellar body phospholipid," in 451 samples of amniotic fluid collected within two days of delivery and related the data to the respiratory performance of the newborn in every case; 112 of the infants were delivered at 28-37 weeks gestation. The incidence of hyaline membrane disease was inversely related to the concentration of lamellar body phospholipid in the amniotic fluid. Eleven of 12 infants with lamellar body phospholipid values less than 25 mg/L and four of 44 infants with lamellar phospholipid values between 25 and 50 mg/L developed hyaline membrane disease or other serious respiratory problems possibly related to lung immaturity, whereas all of 395 infants with lamellar body phospholipid values of 50 mg/L or more were free from respiratory problems of this nature. The incidence of transient tachypnea was greatest when the lamellar body phospholipid value was between 25 and 50 mg/L, suggesting that this condition may be related to a degree of lung maturity.

Fetal lung maturation assessment after administration of glucocorticoid.

The concentration of lamellar body (or surfactant) phospholipid in amniotic fluid generally increases as gestation proceeds and the fetal lungs mature. However, when glucocorticoid is administered prior to 35 weeks' gestation to accelerate fetal lung maturation, the ensuing increase in lamellar body phospholipid, to levels indicative of lung maturity, may be followed by a decrease to levels which suggest a reversion to lung immaturity. Estimations of fetal lung maturity should be interpreted with caution following the administration of glucocorticoid.

Sedimentation of lung-derived phospholipid during low-speed centrifugation of amniotic fluid.

In most methods proposed for the assessment of fetal lung maturity, amniotic fluid is subjected to a preliminary low-speed centrifugation in an attempt to separate whole cells and cell debris from lung-derived surfactant phospholipid (lamellar body phospholipid). However, because lamellar body phospholipid is present in amniotic fluid in a membranous or particulate form, it is also partly sedimented by this procedure. The sedimentation of total phospholipid and lamellar body phospholipid by low-speed centrifugation has been quantitated for 49 samples of amniotic fluid from pregnancies of 30--41 weeks gestation. Isopycnic density-gradient centrifugation in a small air-driven ultracentrifuge was used to isolate lamellar body fractions from whole and centrifuged amniotic fluid. Centrifugation for 5 min at 1000 x g removed 0--70% of total phospholipid or lamellar body phospholipid, the mean values being 34 or 29%, respectively. There was an appreciable increase in lamellar body phospholipid relative to total phospholipid as a result of centrifugation in only 51% of the samples. In general, the effects of centrifugation were not related to gestational age of the fetus or the state of maturity of its lungs.

Lamellar body phospholipid content of amniotic fluid and L/S ratio compared in assessing fetal lung maturity.

A micro-method has been devised for isolating a lung-derived membranous fraction from human amniotic fluid. The phospholipid content of this fraction, known as lamellar body phospholipid, provides an indication of fetal lung maturity (Ann. Clin. Biochem 16: 191, 1979). This method has now been applied to 479 samples of amniotic fluid from 330 pregnancies. The lecithin/sphingomyelin ratio has also been determined for each of the samples by the routine method currently in use in the hospitals providing the samples. Hyaline membrane disease was associated with a low concentration of lamellar body phospholipid (less than 35 mg/L) in all eight cases encountered in this study. In contrast, in 182 of the 185 cases where the lamellar body content of the amniotic fluid, collected within two days of delivery, exceeded 35 mg/L, the infants were free from serious respiratory problems. Data are presented which suggests that the lecithin/sphingomyelin ratio falsely indicated lung immaturity in many cases, amounting to 44% or more of all values indicating immaturity that were reported.

Lamellar body phospholipid content of amniotic fluid: a possible index of fetal lung maturity.

Lamellar bodies, produced by secretory cells in the alveolar epithelium, are the major source of surfactant phospholipid. As the fetal lung matures, the membranous content of the lamellar bodies is secreted into the alveolar spaces and passes into the amniotic fluid, from which it can be isolated in a morphologically recognisable form. A method is described for the rapid isolation of a lamellar body fraction from amniotic fluid using a small air-driven clinical ultracentrifuge. The lamellar body phospholipid content of amniotic fluid increases towards the end of gestation, but the time of onset and the rate of this increase show wide individual variation. Preliminary results suggest that the lamellar body phospholipid content of amniotic fluid may be a useful index of fetal lung maturity.

Is the L/S ratio really a lecithin/sphingomyelin ratio?

Fetal lung maturity is commonly assessed by determining the ratio of lecithin/sphingomyelin in centrifuged amniotic fluid. In a variety of chromatographic systems currently used for the routine determination of the lecithin/sphingomyelin ratio, including the systems recommended in the original procedure, at least one and frequently two additional phospholipids, normally present in amniotic fluid, tend to chromatograph between or overlapping with lecithin and/or sphingomyelin. These phospholipids have been tentatively identified as phosphatidylserine and phosphatidylinositol. The extra phospholipids contribute significantly to the routine lecithin/sphingomyelin ratio with considerable variation between individual cases. Treatment of dried lipid extracts with cold acetone before chromatography, as suggested in the original lecithin/sphingomyelin ratio procedure, does not remove the interfering phospholipids.

Fine structure of the secretion granules in the mandibular gland of the echidna, Tachyglossus aculeatus (Monotremata).

The cells of the secretory tubules in the mandibular gland of the echidna are packed with fairly large birefringent granules, which show a lamellated structure consisting of alternating thin and thick layers or shells of protein. This presumably rigid substructure collapses during exocytosis and the shells unravel as sheets that form a tangled mass in the lumen of the secretory tubule. Relatively pure fractions were obtained of the relevant granules and protein sheets, which should allow a further study to be made on the secretory proteins in this gland.

A reassessment of the phospholipid dependence of membrane-bound enzymes, with special reference to glucose-6-phosphatase and Na+, K+-dependent adenosine triphosphatase.

It is suggested that the specific involvement of phospholipid in either the expression or constraint of glucose-6-phosphatase activity is not conclusively established by the existing experimental evidence. The physiological significance of an apparent requirement for phosphatidylserine for Na+, K+-dependent adenosine triphosphatase activity is also questioned. The need for a critical reassessment of past conclusions in the light of present knowledge is emphasized.