The assessment of fetal lung maturity by analysis of amniotic fluid

This thesis begins with an introduction which describes the role of pulmonary surfactant and the evidence that a pulmonary surfactant deficiency in the neonate can lead to Respiratory Distress Syndrome. Respiratory Distress Syndrome is a serious problem producing a high incidence of mortality and morbidity in the premature neonate. Lung maturation and the production and properties of surfactant are outlined. The synthesis of two of the more important pulamonary surfactant phospholipids, lecithin and phosphatidylglycerol, are briefly explained. Attention is drawn to the fact that fetal lung maturity cannot be inferred from gestational age, emphasising the necessity of biochemical teats to assess fetal lung status. The introduction concludes with a brief description of some of the teats of fetal lung maturity. The principal aims of the work described in this thesis were: (a) to assess the lecithin/sphingomyelin (L/S) ratio, lecithin concentration, the palmitic acid concentration, percentage palmitic acid and palmitic acid/stearic acid ratio, using the acetone precipitable lipids extracted from amniotic fluid samples, and also to evaluate the more rapid surfactant tests, the bubble stability test and the spectrophotometric analysis of amniotic fluid, and to compare their reliability for predicting fetal lung maturity. (b) to assess the value of a test which determines the fetal lung phospholipid profile, a technique which is used in North American Centres, but as fax as is known, not yet used in Britain. (c) to determine the effect of gestational age on the results obtained from the various tests. (d) to separate the various phospholipids by h.p.l.c. This is a technique which has as yet only been used to separate lecithin and sphingomyelin and not the other phospholipids. The use of this technique as a clinical tool for determining fetal lung maturity was to be assessed. (e) to investigate the effect of acetone precipitation on amniotic fluid phospholipids, in terms of selective precipitation of phospholipids containing saturated fatty acids and a comparison of the reliability of the L/S ratio determined on the total lipid extract with the L/S ratio determined on the acetone precipitable extract. The assessment of the L/S ratio, lecithin concentration, spectrophotometric analysis and bubble stability test confirmed that the problem is not in indicating fetal lung maturity, but rather in identifying fetal lung immaturity which was much more difficult to predict reliably. The fatty acid analysis of amniotic fluid lipids allowed determination of the percentage palmitic acid, palmitic acid concentration, and the palmitic acid/stearic acid ratio, which although numbers were small, gave promising results. The fetal lung phospholipid profile (the main features of which were the L/S ratio and the presence or absence of phosphatidylglycerol) was determined by a standard 2D t.l.c. technique and it was found to be most accurate in identifying fetal lung status. Therefore, for laboratories with ready access to t.l.e. techniques its use would be highly recommended for determining fetal lung maturity. The effect of gestational age on the L/S ratio, lecithin concentration and spectrophotometric analysis showed an exponential relationship with an obvious increase in surfactant around 34 weeks of gestation. No relationship was found between the palmitic acid content of the amniotic fluid lipids and gestational age. However, in all tests there was a considerable individual variation at each gestational age. With suitable eluting columns and solvents the h.p.l.c. was shown to be effective in separating the amniotic fluid phospholipids and the results compared well with those obtained by t.l.c. and phosphate analysis. However, the h.p.l.c. would be too unreliable for use as a clinical tool, but may have a role as a useful research technique. Acetone precipitation was found to precipitate the acidic phospholipids, and sphingomyelin. Only 50% of the lecithin was precipitated. Not all the unsaturated fatty acids were removed by acetone precipitation, nor were the saturated fatty acids completely precipitated however, the lipid extract was enriched with saturated fatty acids after this step. It was concluded that the L/S ratio was a more reliable predictor of fetal lung maturity after the amniotic fluid lipid extract had been acetone precipitated