Changes in Surfactant Phospholipids in Fetal Rat Lungs from Normal and Diabetic Pregnancies

Abstract

ABSTRACT. The purposes of this study were to adapt and evaluate further a pulmonary surfactant isolation method applicable to unperfused fetal rat lung, to quantitate key phospholipids phosphatidylcholine (disaturated phosphatidylcholine, and phosphatidylglycerol) of the isolated material during the last 3 days of gestation, and to determine if abnormalities in surfactant phospholipids were present in fetuses of diabetic pregnancies. A simplified scheme of sucrose gradient centrifugation proved useful for small scale preparations of material enriched in the phospholipids most characteristic of pulmonary surfactant. It was shown that fetal blood phospholipids did not contaminate the surfactant fraction and therefore would not produce artifacts in assessment of lung maturational changes. Analyses of subcellular fractions isolated at 19.5, 20.5, and 21.5 days revealed that the percentages of disaturated phosphatidylcholine relative to total phospholipids were 23–44% in the surfactant preparations and 14–21% in the residual (nonsurfactant) fractions, while the disaturated phosphatidylcholine/phosphatidylcholine ratios were 0.62 ± 0.06 and 0.41 ± 0.03, respectively. Summation of the amounts of individual phospholipids in the two fractions yielded data that were nearly identical to the concentrations of these compounds in whole fetal lung samples analyzed independently, implying that losses during the surfactant isolation technique were negligible. The concentrations of phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, and total phospholipids increase markedly (more than 10-fold) and progressively in surfactant fractions prepared from normal fetal rat lung at 19.5, 20.5, and 21.5 days of gestation. In contrast, the residual fractions showed no changes from 19.5 to 20.5 days and then relatively modest increases from 20.5 to 21.5 days, except for phosphatidylglycerol, which increased markedly. The appearance of phosphatidylglycerol was first noted in the surfactant fraction at 20.5 days, but the level of this phospholipid did not show a marked increase until 21.5 days. These data are in agreement with previous morphologic and physiologic observations on fetal rat lung during late gestation and are also in keeping with clinical results from amniotic fluid analyses. Assessment of diabetic pregnancies revealed that at 20.5 days all phospholipids measured were significantly decreased in the surfactant fraction, but not in the residual material. Neither surfactant nor residual phospholipids were decreased in diabetic pregnancies at 19.5 or 21.5 days of gestation. The transient nature of abnormal fetal lung surfactant phospholipids in diabetic pregnancies suggests impaired timing of the pulmonary maturation processes.