羊水における肺サーファクタソト・リン脂質マーカーの酵素的定量に関する研究

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  • Studies on Enzymatic Assays of Phospholipid Markers for Pulmonary Surfactant in Amniotic Fluid

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The lecithin/sphingomyelin （L/S） ratio, disaturated phosphatidylcholine （DSPC）, phosphatidylglycerol （PG） and surfactant protein A （SP-A） in amniotic fluid have been demonstrat- ed to be specific surfactant markers. It is important for their clinical use that the determination of these markers be simple, rapid and sensitive. In the present study, we improved the enzymatic as- says of DSPC, L/S ratio and PG in order to make the determination of these phospholipid markers more sensitive. An enzymatic assay method using phosphatidylinositol （PI） was developed for the first time in this study. Using these assay methods, the phospholipid markers of amniotic fluid at various gestational ages were determined. In the enzymatic assays of DSPC, PC, sphingomyelin and PG, 2-hydroxy-3,5-dichlorobenzene sulfonate （HDCBS） was used as a coupling agent with 4-aminoantipyrine and with hydroperoxide produced from either choline or glycerophosphate and their oxidases. The use of HDCBS was much more sensitive than phenol. For more specific and accurate determination of DSPC, we used phos- pholipase C from Bacillus cereus instead of bacterial or cabbage phospholipase D. PI was treated by phospholipase D to release myo-inositol, which was then determined enzymatically. The L/S and PG/PI ratios in amniotic fluid determined by the enzymatic methods correlated well with those obtained by the thin-layer chromatographic method. The DSPC concentration in amniotic fluid increased gradually from 30 weeks gestation, and the development profiles of DSPC and the L/S ratio were well correlated. The increase of PG concentration in amniotic fluid appeared from 35 weeks gestation, but PI concentration increased until 35 weeks gestation, and then decreased. The PG/PI ratio, therefore, increased dramatically from 35 weeks gestation. The development profile of SP-A in amniotic fluid correlated with that of DSPC, L/S ratio, PG and PG/PI ratio. In conclusion, the enzymatic assay methods improved or developed here were useful for the microdetermination of surfactant phospholipid markers in amniotic fluid.

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