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An explanation of the low lecithinsphingomyelin ratio of amniotic fluid in early pregnancy
Europ. J. Obstet. Gynec. reprod. Biol., 17 (1984) 383-386 Elsevier
383
E!Jo 00107
An explanation of the low lecithin/sphingomyelin ratio of amniotic fluid in early pregnancy D.O.E.
Gebhardt
‘, C.M. van Gent 2, H. van der Voort 2 and B.W. van der Meer 3
’ Department ofObstetrics and Gynecology, Unioersity Hospital, 2333 AA Leiden, ’ Gaubius Institute, Health Research Organization,
TNO, Leiden, and 3 Physiological Laboratory Netherlands
Medical Faculty, Leiden, The
Accepted for publication 2 March 1984
GEBHARDT, D.O.E., VAN GENT, CM., VAN DER VOORT, H. and VAN DER MEER, B.W. (1984): An explanation of the low lecithin/sphingomyelin ratio of amniotic fluid in early pregnancy. Europ. J. Obstet. Gynec. reprod. BioL, 17, 383-386. We have attempted to determine why the lecithin/sphingomyelin (L/S) ratio of amniotic fluid is lower than 2 during an early stage of pregnancy. We found that, at 16 wk of gestation, long before the fetal lung secretes lecithin into the amniotic fluid, the L/S ratio was about 1. High-density lipoprotein isolated from the amniotic fluid also had such a low L/S ratio. The L/S ratios of the high-density lipoprotein from umbilical cord blood and maternal blood, however, were much higher, viz. 3.7 ( f 0.25) and 6.4 ( f 0.33), respectively. The increase coincided with a decrease in their fluorescence polarization. We suggest that the low L/S ratio of 16 wk amniotic fluid is caused by lipolysis of its lecithin, which is derived from fetal or maternal high-density lipoproteins. amniotic fluid; lecithin/sphingomyelin ratio; hi&-density lipoprotein; fluorescence polarization; early pregnancy
Introduction
Although much has been written about the significance of an L/S ratio of amniotic fluid higher than 2 (Gluck et al., 1974), there is still no explanation for the well-documented observation that amniotic fluids from early pregnancies have L/S ratios lower than 2.0, e.g., Arvidson et al. (1972), Kling et al. (1973), Keniston et al. (1975) (these L/S ratios were all determined without the acetone precipitation step). It has been established that the increase in the ratio after about the 30th wk is due to an influx of lecithin from the fetal lung in the form of lamellar bodies (Duck-Chong et al., 1978; Oulton et al., 1982). Before that time the water-insoluble phospholipids are present in the amniotic fluid solely as high-density lipoprotein (HDL) or as a related apolipoprotein A-I-containing component (Gebhardt et al., 1979) (such lipid-protein complexes are soluble in an aqueous environment). One wouid there0028-2243/84/$03.00
0 1984 Elsevier Science Publishers B.V.
384
fore expect the L/S ratio of amniotic fluid from early pregnancy to resembie the L/S ratio of HDL from maternal serum or possibly from umbilical cord serum. A study of the literature made it clear that the L/S ratio of adult serum HDL was in any case higher than 4 (Schmitz et al., 1983; Gebhardt, 1984). No information, however, was available on the L/S ratio of umbilical cord HDL. We have, therefore, now determined this value and have compared it to the L/S ratio of the adult HDL and HDL derived from a pool of 16 wk amniotic fluids. The L/S ratio of each of 10 amniotic fluids from early pregnancies was also measured. Furthermore we have determined the fluorescence polarization (FP) of all samples, since we wished to evaluate the work of Shinitzky et al. (1976). According to this group of workers there is an inverse correlation between the L/S ratio of a lipid complex and its FP as long as other factors, such as the cholesterol content, remain unchanged. Material and methods The quantitative determination of the L/S ratio of HDL from maternal and umbilical cord serum and from amniotic fluid proceeded as follows. 1 ml samples of HDL or 5 ml of amniotic fluid were extracted with 5 ml methanol and 10 ml chloroform (the amniotic fluid had first been centrifuged for 5 min at 600 x g to remove cells). After separation of the layers by low-speed centrifugation, the chloroform layer was concentrated to 50 ~1. This concentrated extract was transferred to TLC plates and the phospholipids were separated according to Touchstone et al. (1980). The lecithin and sphingomyelin bands were visualized by spraying with a solution of rhodamine 6G. The bands were scraped into test tubes and the phosphorus content was determined according to Bhagwanani et al. (1972). For estimation of the total cholesterol content we used the method of Abel et al. (1952) in combination with the technique of van Gent (1968) for measurement of the neutral lipids. For the isolation of the HDL from 16 wk amniotic fluid, two pools of 1 liter were made of about 200 samples received from the Department of Human Genetics, University of Amsterdam, and the Department of Obstetrics, University of Groningen. The pools were concentrated to 70 ml (at 4’ C) in an Amicon cell 402 (Amicon, Danvers, MA, U.S.A.), using an Amicon UM2 filter. The 70 ml were then subjected to sequential ultracentrifugation according to Hatch and Lees, 1968. Due to scarcity of material only two pools of HDL could be prepared from 16 wk amniotic fluid. Statistical analysis was therefore not feasible in this case. The HD.L (density 1.063-1.21 g/ml) from the maternal and umbilical cord sera at 40 wk of pregnancy was also obtained by the sequential flotation procedure. For this purpose we used pools of 70 ml serum derived from about 20 individuals. The fluorescence polarization measurements were done as described by Shinitzky et al. (1976) using the Elscint MV-1 microviscosimeter (Elscint, Haifa, Israel). Results and discussion The results of our investigation are shown in Table I. It can be seen that the L/S ratio and the FP of the 16 wk amniotic fluids are similar to the L/S ratio and FP of the HDL isolated from a pool of such samples. This makes it qlikely that the L/S
385 TABLE I Number of samples
L/S
FP
Total cholesterol (weight %)
10
1.0 f 0.2 (S.D.)
0.360 f 0.01 (SD.)
33
Amniotic fluids from 16 wk pregnancy HDL from amniotic fluids of 16 wk pregnancy HDL from umbilical cord serum
2 pools
1.3, 0.9
0.350, 0.365
34
4 pools a
3.4, 3.7, 3.8, 4.0
0.302, 0.291
31
HDL from maternal serum at term
4 pools
6.0, 6.3, 6.4, 6.8
0.274, 0.270
29
a Significantly different from L/S ratios of HDL from maternal serum when tested at 0.05 level using the Mann-Whitney test.
ratio and the FP of amniotic fluid from early pregnancy are determined by the composition of its HDL. The L/S ratio of HDL from umbilical cord serum was more than 3-times as high and that from maternal HDL more than 6-times as high as the L/S ratio of amniotic fluid HDL. It is therefore not possible to determine the origin of the HDL in amniotic fluid by a comparison of its L/S ratio with that of maternal and fetal HDL. The relatively low L/S ratios of early amniotic fluid and its HDL can be explained with the aid of work published by various authors. Olofsson et al. (1975) for instance, incubated HDL at 37OC and found some degradation of its lecithin. Furthermore, Merger et al. (1980) described a lipase in amniotic fluid which may specifically hydrolyse lecithin (Bamberger et al., 1983). A phospholipase has also been described in the fetal membranes (Oka.zaki et al., 1981; di Renzo et al., 1981) which may reduce the amount of lecithin of the HDL before this passes from the blood into the amniotic fluid. From Table I it also follows that an increase in L/S ratio is associated with a decrease in FP, a result in agreement with the work of Shinitzky et al. (1976). The lecithin and sphingomyelin, which are situated on the surface of the HDL (Shen et al., 1977), seem to determine its FP (Borochov et al., 1977) as long as the cholesterol content remains unchanged (Jonas et al., 1978). It is of interest that the L/S ratio and FP of umbilical cord HDL differ greatly from the respective values found for maternal HDL. This is probably caused by the difference in nutrition of the mother and her fetus. In fact, Berlin and Young (1983) have shown that the fat level and type of fat has a profound influence on the fluidity and physical state of the plasma lipoproteins. Acknowledgements We would like to thank Drs. van Bhtterswijk, van Hoeven, Leschot and Mr. de Widt of Amsterdam, Dr. de Bruyn of Groningen and Mrs. Beintema, Mrs. Deierkauf and Professor Miiller of Leiden for their help with this work.
386
References Abel, L.L., Levy, B.B., Brodie, B.B. and Kendall, F.E. (1952): A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity . J. Biol. Chem., 195, 357-366. Arvidson, A., Ekelund, H. and Astedt, B. (1972): Phospholipid composition of human amniotic fluid during gestation and at term. Acta obstet. gyncc. stand., 51, 71-75. Bamberger, M., Glick, J.M. and Rotblat, G.H. (1983): Hepatic lipase stimulates the uptake of HDL cholesterol by hepatoma cells. J. Lipid Res., 24, 869-876. Bhagwanani, S.G., Fahmy, D. and Turnbull, A.C. (1972): Quick determination of amniotic fluid lecithin concentration for the prediction of neonatal respiratory distress. Lancet, ii, 66-67. Berlin, E.‘and Young, C. (1983): Effect of fat level, feeding period and source of fat on lipid fluidity and physical state of rabbit plasma lipoproteins. Artherosclerosis, 48, 15-27. Borochov, H., Zahler, P., Wilbrandt, W. and Shinitrky, M. (1977): The effect of phosphatidylcholine to sphingomyelin mole ratio on the dynamic properties of sheep erythrocyte membrane. B&him. Biophys. Acta, 470, 382-288. Di Renzo, G.C., Johnston, J.M., Okazaki, T., Okita, J.R., Macdonald, P.C. and Bleasdale, J.E. (1981) Phosphatidylinositol-specific phospholipase C in fetal membranes and uterine deeidua. J. Chn. Invest., 67, 847-856. Duck-Chong, C.G. (1978): The isolation of lamellar bodies and their membraneous content from rat lung, lamb tracheal fluid and human amniotic fluid. Life Sci., 22, 2025-2030. Gebhardt, D.O.E., Beintema, A., Reman, F.C. and Van Gent, C.M. (1979): The lipoprotein composition of amniotic fluid. Clin. Chim. Acta, 94, 93-100. Gebhardt, D.O.E. (1984): What is the ‘correct’ lecithin/sphingomyelin ratio of HDL. Clin. Chem. 30, 164. Gluck, L., Kulovitch, M.V. and Borer, R.C. (1974): The interpretation and significance of the lecithin/ sphingomyehn ratio in amniotic fluid. Amer. J. Obstet. Gynec., 120, 142-147. Hatch, F: and Lees, R. (1968): Practical methods for plasma lipoprotein analysis. Adv. Lipid Res., 6, l-68. Jonas, A., Hesterberg, L.K. and Drengler, SM. (1978): Incorporation of excess cholesterol by high-density serum lipoproteins. B&him. Biophys. Acta, 528, 47-57. Keniston, R.C., Pemoll, M.L., Buist, N.R.M., Lyon M. and Swanson, J.R. (1975): A prospective evaluation of the lecithin/sphingomyelin ratio and the rapid surfactant test in relation to fetal pulmonary maturity. Amer. J. Obstet. Gynec., 121, 324-332. Khng, O.R., Crosby, W.M. and Merill, J.A. (1973): Amniotic fluid correlates of fetal maturity and perinatal outcome. Gynec. Invest., 4, 38-49 Merger, C., Valette, A., Ruf, H. and Boyer, J. (1980): Characterization of triacylglycerol lipase activity in human amniotic fluid. Amer. J. Obstet. Gynec., 138, 68-72. Olofsson, S.O., Gustafson, A. and Svanberg, U. (1975): Studies on human high-density lipoproteins (HDL). IV. Isolation of lipoprotein families after incubation of HDL. Stand. J. Lab. Invest., 35, 363-371. Okazaki, T., Sagawa, N., Bleasdale, J.E., Okita, J.R., Macdonald, P.C. and Johnston, J.M. (1981): Initiation of human parturition. Phospholipase C, phospholipase A, and diacylglycerol lipase activities in fetal membranes and decidue vera tissues from early and late gestation. Biol. Reprod., 25, 103-109. Oulton, M., Bent, A.E., Gray, J.H., Luther, E.R. and Peddle, L.J. (1982): Assessment of fetal pulmonary maturity by phospholipid analysis of amniotic fluid lamellar bodies. Amer. J. Obstet. Gynec., 142, 684-691. Schmitz, G., Jabs, H.U. and Assmann, G. (1983): Densitometry of phosphatidylcholine and sphingomyelin in high-density lipoproteins. Clin. Chem. 29, 1435-1437 Shen, B.W., Scanu, A.M. and Kezdy, FKJ. (1977): Structure of human serum lipoprotein inferred from compositional analysis. Proc. Natl. AcadSci. USA, 74, 837-841. Shinitsky, M., Goldfisher, A., Bruck, A., Goldman, B. and Stem, E. (1976): A new method for assessment of fetal lung maturity. Brit. J. Obstet. Gynaec., 83, 838-844. Touchstone, J.C., Chen, J.C. and Beaver, K.M. (1980): Improved separation of phospholipids in thin-layer chromatography. Lipids, 15, 61-62. Van Gent, C.M. (1968): Separation and micro determination of lipids by thin-layer chromatography followed by densitometry. Z. analyt. Chernie, 236 344-350.
383
E!Jo 00107
An explanation of the low lecithin/sphingomyelin ratio of amniotic fluid in early pregnancy D.O.E.
Gebhardt
‘, C.M. van Gent 2, H. van der Voort 2 and B.W. van der Meer 3
’ Department ofObstetrics and Gynecology, Unioersity Hospital, 2333 AA Leiden, ’ Gaubius Institute, Health Research Organization,
TNO, Leiden, and 3 Physiological Laboratory Netherlands
Medical Faculty, Leiden, The
Accepted for publication 2 March 1984
GEBHARDT, D.O.E., VAN GENT, CM., VAN DER VOORT, H. and VAN DER MEER, B.W. (1984): An explanation of the low lecithin/sphingomyelin ratio of amniotic fluid in early pregnancy. Europ. J. Obstet. Gynec. reprod. BioL, 17, 383-386. We have attempted to determine why the lecithin/sphingomyelin (L/S) ratio of amniotic fluid is lower than 2 during an early stage of pregnancy. We found that, at 16 wk of gestation, long before the fetal lung secretes lecithin into the amniotic fluid, the L/S ratio was about 1. High-density lipoprotein isolated from the amniotic fluid also had such a low L/S ratio. The L/S ratios of the high-density lipoprotein from umbilical cord blood and maternal blood, however, were much higher, viz. 3.7 ( f 0.25) and 6.4 ( f 0.33), respectively. The increase coincided with a decrease in their fluorescence polarization. We suggest that the low L/S ratio of 16 wk amniotic fluid is caused by lipolysis of its lecithin, which is derived from fetal or maternal high-density lipoproteins. amniotic fluid; lecithin/sphingomyelin ratio; hi&-density lipoprotein; fluorescence polarization; early pregnancy
Introduction
Although much has been written about the significance of an L/S ratio of amniotic fluid higher than 2 (Gluck et al., 1974), there is still no explanation for the well-documented observation that amniotic fluids from early pregnancies have L/S ratios lower than 2.0, e.g., Arvidson et al. (1972), Kling et al. (1973), Keniston et al. (1975) (these L/S ratios were all determined without the acetone precipitation step). It has been established that the increase in the ratio after about the 30th wk is due to an influx of lecithin from the fetal lung in the form of lamellar bodies (Duck-Chong et al., 1978; Oulton et al., 1982). Before that time the water-insoluble phospholipids are present in the amniotic fluid solely as high-density lipoprotein (HDL) or as a related apolipoprotein A-I-containing component (Gebhardt et al., 1979) (such lipid-protein complexes are soluble in an aqueous environment). One wouid there0028-2243/84/$03.00
0 1984 Elsevier Science Publishers B.V.
384
fore expect the L/S ratio of amniotic fluid from early pregnancy to resembie the L/S ratio of HDL from maternal serum or possibly from umbilical cord serum. A study of the literature made it clear that the L/S ratio of adult serum HDL was in any case higher than 4 (Schmitz et al., 1983; Gebhardt, 1984). No information, however, was available on the L/S ratio of umbilical cord HDL. We have, therefore, now determined this value and have compared it to the L/S ratio of the adult HDL and HDL derived from a pool of 16 wk amniotic fluids. The L/S ratio of each of 10 amniotic fluids from early pregnancies was also measured. Furthermore we have determined the fluorescence polarization (FP) of all samples, since we wished to evaluate the work of Shinitzky et al. (1976). According to this group of workers there is an inverse correlation between the L/S ratio of a lipid complex and its FP as long as other factors, such as the cholesterol content, remain unchanged. Material and methods The quantitative determination of the L/S ratio of HDL from maternal and umbilical cord serum and from amniotic fluid proceeded as follows. 1 ml samples of HDL or 5 ml of amniotic fluid were extracted with 5 ml methanol and 10 ml chloroform (the amniotic fluid had first been centrifuged for 5 min at 600 x g to remove cells). After separation of the layers by low-speed centrifugation, the chloroform layer was concentrated to 50 ~1. This concentrated extract was transferred to TLC plates and the phospholipids were separated according to Touchstone et al. (1980). The lecithin and sphingomyelin bands were visualized by spraying with a solution of rhodamine 6G. The bands were scraped into test tubes and the phosphorus content was determined according to Bhagwanani et al. (1972). For estimation of the total cholesterol content we used the method of Abel et al. (1952) in combination with the technique of van Gent (1968) for measurement of the neutral lipids. For the isolation of the HDL from 16 wk amniotic fluid, two pools of 1 liter were made of about 200 samples received from the Department of Human Genetics, University of Amsterdam, and the Department of Obstetrics, University of Groningen. The pools were concentrated to 70 ml (at 4’ C) in an Amicon cell 402 (Amicon, Danvers, MA, U.S.A.), using an Amicon UM2 filter. The 70 ml were then subjected to sequential ultracentrifugation according to Hatch and Lees, 1968. Due to scarcity of material only two pools of HDL could be prepared from 16 wk amniotic fluid. Statistical analysis was therefore not feasible in this case. The HD.L (density 1.063-1.21 g/ml) from the maternal and umbilical cord sera at 40 wk of pregnancy was also obtained by the sequential flotation procedure. For this purpose we used pools of 70 ml serum derived from about 20 individuals. The fluorescence polarization measurements were done as described by Shinitzky et al. (1976) using the Elscint MV-1 microviscosimeter (Elscint, Haifa, Israel). Results and discussion The results of our investigation are shown in Table I. It can be seen that the L/S ratio and the FP of the 16 wk amniotic fluids are similar to the L/S ratio and FP of the HDL isolated from a pool of such samples. This makes it qlikely that the L/S
385 TABLE I Number of samples
L/S
FP
Total cholesterol (weight %)
10
1.0 f 0.2 (S.D.)
0.360 f 0.01 (SD.)
33
Amniotic fluids from 16 wk pregnancy HDL from amniotic fluids of 16 wk pregnancy HDL from umbilical cord serum
2 pools
1.3, 0.9
0.350, 0.365
34
4 pools a
3.4, 3.7, 3.8, 4.0
0.302, 0.291
31
HDL from maternal serum at term
4 pools
6.0, 6.3, 6.4, 6.8
0.274, 0.270
29
a Significantly different from L/S ratios of HDL from maternal serum when tested at 0.05 level using the Mann-Whitney test.
ratio and the FP of amniotic fluid from early pregnancy are determined by the composition of its HDL. The L/S ratio of HDL from umbilical cord serum was more than 3-times as high and that from maternal HDL more than 6-times as high as the L/S ratio of amniotic fluid HDL. It is therefore not possible to determine the origin of the HDL in amniotic fluid by a comparison of its L/S ratio with that of maternal and fetal HDL. The relatively low L/S ratios of early amniotic fluid and its HDL can be explained with the aid of work published by various authors. Olofsson et al. (1975) for instance, incubated HDL at 37OC and found some degradation of its lecithin. Furthermore, Merger et al. (1980) described a lipase in amniotic fluid which may specifically hydrolyse lecithin (Bamberger et al., 1983). A phospholipase has also been described in the fetal membranes (Oka.zaki et al., 1981; di Renzo et al., 1981) which may reduce the amount of lecithin of the HDL before this passes from the blood into the amniotic fluid. From Table I it also follows that an increase in L/S ratio is associated with a decrease in FP, a result in agreement with the work of Shinitzky et al. (1976). The lecithin and sphingomyelin, which are situated on the surface of the HDL (Shen et al., 1977), seem to determine its FP (Borochov et al., 1977) as long as the cholesterol content remains unchanged (Jonas et al., 1978). It is of interest that the L/S ratio and FP of umbilical cord HDL differ greatly from the respective values found for maternal HDL. This is probably caused by the difference in nutrition of the mother and her fetus. In fact, Berlin and Young (1983) have shown that the fat level and type of fat has a profound influence on the fluidity and physical state of the plasma lipoproteins. Acknowledgements We would like to thank Drs. van Bhtterswijk, van Hoeven, Leschot and Mr. de Widt of Amsterdam, Dr. de Bruyn of Groningen and Mrs. Beintema, Mrs. Deierkauf and Professor Miiller of Leiden for their help with this work.
386
References Abel, L.L., Levy, B.B., Brodie, B.B. and Kendall, F.E. (1952): A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity . J. Biol. Chem., 195, 357-366. Arvidson, A., Ekelund, H. and Astedt, B. (1972): Phospholipid composition of human amniotic fluid during gestation and at term. Acta obstet. gyncc. stand., 51, 71-75. Bamberger, M., Glick, J.M. and Rotblat, G.H. (1983): Hepatic lipase stimulates the uptake of HDL cholesterol by hepatoma cells. J. Lipid Res., 24, 869-876. Bhagwanani, S.G., Fahmy, D. and Turnbull, A.C. (1972): Quick determination of amniotic fluid lecithin concentration for the prediction of neonatal respiratory distress. Lancet, ii, 66-67. Berlin, E.‘and Young, C. (1983): Effect of fat level, feeding period and source of fat on lipid fluidity and physical state of rabbit plasma lipoproteins. Artherosclerosis, 48, 15-27. Borochov, H., Zahler, P., Wilbrandt, W. and Shinitrky, M. (1977): The effect of phosphatidylcholine to sphingomyelin mole ratio on the dynamic properties of sheep erythrocyte membrane. B&him. Biophys. Acta, 470, 382-288. Di Renzo, G.C., Johnston, J.M., Okazaki, T., Okita, J.R., Macdonald, P.C. and Bleasdale, J.E. (1981) Phosphatidylinositol-specific phospholipase C in fetal membranes and uterine deeidua. J. Chn. Invest., 67, 847-856. Duck-Chong, C.G. (1978): The isolation of lamellar bodies and their membraneous content from rat lung, lamb tracheal fluid and human amniotic fluid. Life Sci., 22, 2025-2030. Gebhardt, D.O.E., Beintema, A., Reman, F.C. and Van Gent, C.M. (1979): The lipoprotein composition of amniotic fluid. Clin. Chim. Acta, 94, 93-100. Gebhardt, D.O.E. (1984): What is the ‘correct’ lecithin/sphingomyelin ratio of HDL. Clin. Chem. 30, 164. Gluck, L., Kulovitch, M.V. and Borer, R.C. (1974): The interpretation and significance of the lecithin/ sphingomyehn ratio in amniotic fluid. Amer. J. Obstet. Gynec., 120, 142-147. Hatch, F: and Lees, R. (1968): Practical methods for plasma lipoprotein analysis. Adv. Lipid Res., 6, l-68. Jonas, A., Hesterberg, L.K. and Drengler, SM. (1978): Incorporation of excess cholesterol by high-density serum lipoproteins. B&him. Biophys. Acta, 528, 47-57. Keniston, R.C., Pemoll, M.L., Buist, N.R.M., Lyon M. and Swanson, J.R. (1975): A prospective evaluation of the lecithin/sphingomyelin ratio and the rapid surfactant test in relation to fetal pulmonary maturity. Amer. J. Obstet. Gynec., 121, 324-332. Khng, O.R., Crosby, W.M. and Merill, J.A. (1973): Amniotic fluid correlates of fetal maturity and perinatal outcome. Gynec. Invest., 4, 38-49 Merger, C., Valette, A., Ruf, H. and Boyer, J. (1980): Characterization of triacylglycerol lipase activity in human amniotic fluid. Amer. J. Obstet. Gynec., 138, 68-72. Olofsson, S.O., Gustafson, A. and Svanberg, U. (1975): Studies on human high-density lipoproteins (HDL). IV. Isolation of lipoprotein families after incubation of HDL. Stand. J. Lab. Invest., 35, 363-371. Okazaki, T., Sagawa, N., Bleasdale, J.E., Okita, J.R., Macdonald, P.C. and Johnston, J.M. (1981): Initiation of human parturition. Phospholipase C, phospholipase A, and diacylglycerol lipase activities in fetal membranes and decidue vera tissues from early and late gestation. Biol. Reprod., 25, 103-109. Oulton, M., Bent, A.E., Gray, J.H., Luther, E.R. and Peddle, L.J. (1982): Assessment of fetal pulmonary maturity by phospholipid analysis of amniotic fluid lamellar bodies. Amer. J. Obstet. Gynec., 142, 684-691. Schmitz, G., Jabs, H.U. and Assmann, G. (1983): Densitometry of phosphatidylcholine and sphingomyelin in high-density lipoproteins. Clin. Chem. 29, 1435-1437 Shen, B.W., Scanu, A.M. and Kezdy, FKJ. (1977): Structure of human serum lipoprotein inferred from compositional analysis. Proc. Natl. AcadSci. USA, 74, 837-841. Shinitsky, M., Goldfisher, A., Bruck, A., Goldman, B. and Stem, E. (1976): A new method for assessment of fetal lung maturity. Brit. J. Obstet. Gynaec., 83, 838-844. Touchstone, J.C., Chen, J.C. and Beaver, K.M. (1980): Improved separation of phospholipids in thin-layer chromatography. Lipids, 15, 61-62. Van Gent, C.M. (1968): Separation and micro determination of lipids by thin-layer chromatography followed by densitometry. Z. analyt. Chernie, 236 344-350.