Marie V. Kulovich

Diagnosis of the respiratory distress syndrome by amniocentesis

Abstract Studies on 302 amniocenteses show that changes in phospholipids in amniotic fluid (PLAF) reflect those in the lung of the developing fetus. A sudden increase in lecithin concentration after 35 weeks heralds maturity of the pulmonary alveolar lining when respiratory distress syndrome will not occur should the fetus then be born. Clinical interpretation is made on a thin-layer chromatogram by inspection; a lecithin spot clearly larger than that of sphingomyelin marked pulmonary maturity in the fetus.

Biochemical Development of Surface Activity in Mammalian Lung: III. Structural Changes in Lung Lecithin during Development of the Rabbit Fetus and Newborn

Extract: This is a report of studies with gas-liquid chromatography of the fatty acids on the α- and β-carbons of surfae active and nonsurface active lecithins isolated from alveolar wash and from residual lung after wash in the developing rabbit fetus. The total fatty acids of lecithin show no clear tendency toward greater saturation with development. In alveolar wash, there was greater saturation of fatty acids (65%) compared with total lung (57%). Fatty acids separately determined on α- and β-carbons of lecithins showed significant developmental differences. Differnces in surface active acetone-precipitated lecithin and nonsurface active acetone-precipitated and acetone-soluble lecithin were in the β-carbon fatty acids. They were highly saturated (+70%) in surface active lecithin but only about 25% saturated in monsurface active lecithin. Concentration of acetone-precipitable lecithin in whole lung rises during fetal development to a peak (84%) with breathing. There was a marked increase in acetone-precipitable lecithin in alvcolar wash after 1 h of breathing (0.35 mg/g dry weight lung in nonbreathing full term to 9.8 mg/g), a 30-fold increase. During gestation, both α- and β-component palmitic acid (C16:0) rose abruptly after day 29 (α-45 to 67% and β-48 to 60%) when alveolar lecithin becomes surface active. The greatest increase in α- and β-palmetic acid followed the onset of breathing, and by day 2 of age, α-palmitic 85%, β-palmitic 62.5%. Thus, dipalmitoyl lecithin is the greatest single identifiable fraction of surface active lecithin isolated from rabbit alveolar wash. Acetone-soluble lecithins, even in the breathing animal, have only about 25% palmitic acid on the β-carbon. Early 29-day fetus, deliverd by cesarean section, synthesized de novo 100% of his surface active lecithin (CDP-choline +α –β-diglyceride pathway), predominantly α- and β-palmitic acid (69.7 and 61.2%, respectively). Phosphatidyl dimethylethanolamine (PDME), a surface active intermediate in the trimethylation of phosphatidyl ethanolamine (PE) to form lecithin, showed largely an α-palmitic/β-myristic acid (53/60%) distribution. Since the rate-limiting reaction is the first methyl group in PE, PDME represents the lecithin end product, another pulmonary surface active lecithin with α-palmitic/β-myristic acids.Speculation: Use of the β-carbon fatty acids as markers in studying the acetone-precipitated surface active fraction of lecithin isolated from alveolar wash will permit an assessment of the contributin of each of the two major pathways in the biosynthesis of surface active lecithin.

EXPERIMENTAL CARDIAC HYPERTROPHY: CONCENTRATIONS OF RNA IN THE VENTRICLES.

Banding of the aorta or pulmonary artery in puppies produces a selectively increased concentration of RNA in the ventricle with the increased hemodynamic load as compared to the opposite side or to normal hearts. The increase in concentration of RNA following distortion of the myocardial cell may represent a fundamental response of growth and the system described mayserve as a useful model for its study.

Fetal Lung Development: Current Concepts

Determination of the lecithin/sphingomyelin ratio in amniotic fluid and the newborn infant’s own tracheal fluid has proved of significant value in identifying the premature infant whose lungs have not yet matured to the point at which surface-active pulmonary lecithin will be synthesized rapidly enough after birth, and who are thus at risk for respiratory distress, with its high mortality and high percentage of associated neurologic and intellectual deficits.

RNA: A Marker in Embryonic Differentiation

A modified fluorochrome technique which identifies nucleic acids differentially in tissues permits following production of RNA in the embryo. Compact primordial chick embryo cells show a sequence to RNA production as differentiation begins: First in the nucleus, then in the perinuclear area, then in the cytoplasm. RNA increases in the cytoplasm with cell growth and continues as the cells merge to form an epithelium.

THE FETAL LUNG PROFILE: BEYOND THE L/S RATIO

Progressive expiratory atelectasis of RDS is attributed to deficient surfactant phospholipids. Fetal lung biosynthesis of the principal ones, lecithin (phosphatidyl choline, PC) the most abundant, and phosphatidyl inositol (PI) and phosphatidyl glycerol (PG), the acidic phospholipids, are determined developmentally during gestation. They appear in amniotic fluid and are used to monitor maturity of fetal lung and optimal time for delivery by means of a LUNG PROFILE, utilizing 2-dimensional thin layer chromatography of the extracted amniotic fluid lipids, comparing on a gestational scale L/S ratio, % disaturated PC, % PI and % PG with level of maturity. The leclthin/sphingomyelin (L/S) ratio relates concentrations of PC to sphingomyelin acting as an “internal standard”. With lung maturation PC increases and there is increasing disaturation of its fatty acid esters, measured as acetone precipitable fraction of PC. PI and PG are essential to the stability of PC; PI increases parallel to PC to 35-36 weeks, then declines. At 36-37 weeks, PG appears and increases; by term and in mature surfactant it is the 2d most abundant surfactant phospholipid. RDS is never seen once PG is present. On recovery from RDS increasing PI is seen; PG may not appear until near 36 gest wks. In infants with retained lung fluid, PG is not present in amniotic fluid but appears after birth in tracheal secretions as symptoms disappear. The lung profile greatly enhances both accuracy of prediction and understanding of lung development and eliminates non-diagnostic intermediate values.

THE TRACHEAL ASPIRATE LUNG PROFILE

The lecithin/sphingomyelin (L/S) ratio & percent phosphatidyl glycerol (PG) in amniotic fluid are used to evaluate fetal lung maturity, but their value in tracheal aspirates to predict respiratory distress syndrome (RDS) has not been defined. Phospholipid lung profiles on tracheal aspirates within 12 hrs of birth from 551 babies were analyzed by 2-dimensional thin layer chromatography & reflectance densitometry.Infants with respiration, asphyxia, pneumonia, syndromes, chorioamionitis, IUGR, or pulmonary hypoplasia were excluded. Radiologic & clinical criteria were used to classify the remaining patients as RDS (125), retained lung fluid (RLF) (50), or no respiratory disease (60). L/S best predicted RDS (78%) when values were ≤2.6 & identified normals (85%) above 2.6. The mean L/S for RLF (4.06) & normals (4.37) were higher than for RDS (2.3). Accuracy of PG in predicting RDS was best (96%) at trace (<1%) or less, identifying 83% of normals. Seventeen percent of normals & 56% of RLF had <1% PG, while 4% of RDS cases had ≥1% PG. Lung profile values accepted for amniotic fluid seem to overestimate pulmonary maturity when applied to tracheal aspirates. Tracheal aspirate L/S & PG together are highly useful in the biochemical diagnosis of RDS.