Quantitation of disaturated phosphatidylcholine and phosphatidylglycerol in amniotic fluid by fluorescence diminution: Methodology and clinical results

Abstract

The authors report the determination of disaturated phosphatidylcholine and phosphatidylglycerol extracted from amniotic fluid specimens. The method involves isolating the two compounds on thin-layer chromatographic plates, immersing the plates for 3 to 5 s in a cold acetone dip containing 2,7-dichlorofluorescein, and quantitating the phospholipid spots by the diminution in fluorescence emission at 504 nm when the excitation wavelength is set at 312 nm on a scanning spectrodensitometer. The method in sensitive to as little as 15 ng of dipalmitoyl phosphatidylcholine; standard curves are nearly linear to 1 μg; and the coefficients of variation are routinely less than 6% for quantities of phosphatidylglycerol and dipalmitoyl phosphatidylcholine between 100 and 1000 ng. We used this technique to evaluate a scheme for extracting the two compounds from amniotic fluid. Analytical recoveries were ≥92% for dipalmitoyl phosphatidylcholine concentrations ranging from 15 to 80 mg/L in amniotic fluid, ≥74% for phosphatidylglycerol concentrations ranging from 2.0 to 24.2 mg/L. We assayed 99 amniotic fluid specimens obtained during the final trimester of normal and complicated pregnancies. For infants delivered with normal respiratory status, mean concentrations of phosphatidylglycerol, disaturated phosphatidylcholine, and the ratio of the two were, respectively: at 33-35 weeks, 1.8 mg/L, 20 mg/L, 0.11; at 36-37 weeks, 3.4 mg/L, 33 mg/L, 0.11; at 38-39 weeks, 4.2 mg/L, 43 mg/L, 0.10; and at 40-45 weeks, 6.4 mg/L, 66 mg/L, 0.14. In cases of intrauterine growth retardation, and nontoxemic maternal hypertension, concentrations of phosphatidylglycerol and disaturated phosphatidylcholine consistently exceeded mean values for the population. Values obtained in cases of maternal diabetes were appropriate for gestational age. In six cases, amniotic fluid was obtained within 72 h of delivery of infants who developed the respiratory distress syndrome. In five of the six cases, phosphatidylglycerol concentrations were ≤ 2 mg/L, disaturated phosphatidylcholine concentrations ≤ 3 mg/L. The sixth case was a macrosomic infant of a diabetic mother. In our population, we found that the amniotic fluid concentration of disaturated phosphatidylcholine better indicated fetal respiratory status than did phosphatidylglycerol concentration.