Annals of Clinical Biochemistry, 1979, 16, 184-190
Is the L/S ratio really a lecithin/sphingomyelin ratio?' CORAL G. DUCK-CHONGl AND LOUISE M. BROWW From the 1 Department of Histology and Embryology, University of Sydney, Sydney, NSW 2006 and 2Biochemistry Department, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia
Fetal lung maturity is commonly assessed by determining the ratio of lecithin/sphingomyelin in centrifuged amniotic fluid. In a variety of chromatographic systems currently used for the routine determination of the lecithin/sphingomyelin ratio, including the systems recommended in the original procedure, at least one and frequently two additional phospholipids, normally present in amniotic fluid, tend to chromatograph between or overlapping with lecithin and/or sphingomyelin. These phospholipids have been tentatively identified as phosphatidylserine and phosphatidylinositol. The extra phospholipids contribute significantly to the routine lecithin/sphingomyelin ratio with considerable variation between individual cases. Treatment of dried lipid extracts with cold acetone before chromatography, as suggested in the original lecithin/sphingomyelin ratio procedure, does not remove the interfering phospholipids. SUMMARY
The determination of the lecithin/sphingomyelin (L/S) ratio of amniotic fluid is now widely used for the assessment of fetal lung maturity in complicated pregnancies. The predictive value of the L/S ratio rests on the fact that, as the lung matures, lecithin accumulates in the amniotic fluid while the concentration of sphingomyelin parallels the increase in lecithin to about 32 weeks and then gradually declines (Gluck et al., 1971; Gluck and Kulovich, 1973). Since its introduction, the determination of the LIS ratio has been a controversial procedure; many technical problems have already been recognised (Biezenski, 1976). This paper draws attention to the presence of other phospholipids in amniotic fluid which may interfere with the L/S ratio determination, a long-standing problem that has not received the attention it deserves. The present investigation was undertaken when it was found that a clear separation of lecithin and sphingomyelin was not achieved in the routine procedure for determining the L/S ratio. Material and methods
Amniotic fluid collected by amniocentesis was centrifuged for 5 minutes at 3000 rpm (Clements Model GS 200 centrifuge, r a • = 13 cm) soon after collection and then chilled. None of the samples 1 These findings were presented to a meeting of the Australian Association of Clinical Biochemists, November 1978 (Duck-Chong and Brown. 1979)
contained blood or meconium. If the supernatant could not be used on the same-day it was stored frozen. The supernatant (2 ml) was mixed with 2 ml methanol, vortexed for 10 seconds, then mixed with 2 ml chloroform and vortexed for 1 minute before centrifugation for 10 minutes at 2700 rpm (Sorvall GLC-1 centrifuge, r a • = 13 em), The lower chloroform layer was carefully collected using a Pasteur pipette and evaporated to dryness under nitrogen with gentle warming at a temperature not exceeding 40 aC. Some samples were treated with cold acetone before chromatography; the procedure for 2 ml samples described by Gluck et al. (1974b) was followed strictly. The whole sample was dissolved in chloroform and applied to a plate for thin-layer chromatography. For two-dimensional chromatography, 4 ml amniotic fluid was extracted instead of 2 mI. The following solvents were used for thin-layer chromatography: Solvent A, chloroform/methanol/water (66 :25 :4) Solvent B, chloroform/methanol/acetic acid/butanol/ water (50:33:6'8:8'5:4'1) Solvent C, chloroform/methanol/water (65 :25:4) Solvent D, chloroform/methanol/water (72 :27 :4) Solvent E, chloroform/methanol/28 % aqueous NHa (65 :25 :4); all by volume. The following procedures were used for thin. layer chromatography: (1) The routine procedure used for the past four years in the Biochemistry Department of the Royal Prince Alfred Hospital: the sample was applied as
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Is the L/S ratio really a lecithin/sphingomyelin ratio?
a 5 mm streak 1 ern from the bottom of a 0·5 mm thick Silica Gel 60, F254, precoated plate (Merck, Art. 5744). The 20 x 20 em plates were cut to a height of 10 ern and a width appropriate to the number of samples on the plate. A lecithin/sphingomyelin standard (61ll of a solution containing 5 mg of each phospholipid per ml) was applied to each plate. Chromatography was carried out in a sealed tank equilibrated with solvent A, until the solvent reached the top of the plate. The dry plate was sprayed with 50 % sulphuric acid and charred on a hot plate until the lecithin/sphingomyelin standard achieved a ratio of 1:1, assessed visually. After cooling, the plate was scanned in a Helena Quick Scan densitometer. For more precise quantitation, the area under the curve was determined using a Leitz ASM Semi-Automated Image Analyser. (2) As for procedure (1), using solvent B instead of solvent A. (3) The procedure of Borer et al. (1971), exactly as described by Gluck et al. (1974b), using solvent C. (4) The procedure of Gluck et al. (1974b), using heat-activated 0·25 mm plates prepared from Silica Gel H (Merck) containing 5 % ammonium sulphate, and solvent C. Similar plates prepared without ammonium sulphate were also used; these plates were visualised as described for procedure (1). (5) The sample was applied as a spot to a plate supplied by Helena Laboratories for the L/S ratio determination and developed in solvent D, the solvent recommended in the Helena Fetal Maturity Test Procedure (PRO 13-4-77). Visualisation was as described for procedure (1). The standard lecithin/sphingomyelin mixture was supplied by Applied Science Laboratories. Phosphatidylserine obtained from Sigma Chemical Company was purified by thin-layer chromatography, and phosphatidylinositol was isolated from fetal rat lung (two days pre-term). The identity of the latter two phospholipids was confirmed by co-chromatography with commercial standards from different sources using basic, acidic, and neutral solvent systems and by their response to ninhydrin staining (Johnston, 1971). All solvents were analytical reagent grade, Merck Uvasol or Mallinckrodt Nanograde. Chloroform was stored at room temperature and used within one to two weeks of the opening of the bottle. Results An extract of amniotic fluid was first chromatographed in the routine solvent, solvent A, then in the second dimension in solvent B. A large, irregularly shaped spot moved away from the regions
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C
Is the L/S ratio really a lecithin/sphingomyelin ratio?' CORAL G. DUCK-CHONGl AND LOUISE M. BROWW From the 1 Department of Histology and Embryology, University of Sydney, Sydney, NSW 2006 and 2Biochemistry Department, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia
Fetal lung maturity is commonly assessed by determining the ratio of lecithin/sphingomyelin in centrifuged amniotic fluid. In a variety of chromatographic systems currently used for the routine determination of the lecithin/sphingomyelin ratio, including the systems recommended in the original procedure, at least one and frequently two additional phospholipids, normally present in amniotic fluid, tend to chromatograph between or overlapping with lecithin and/or sphingomyelin. These phospholipids have been tentatively identified as phosphatidylserine and phosphatidylinositol. The extra phospholipids contribute significantly to the routine lecithin/sphingomyelin ratio with considerable variation between individual cases. Treatment of dried lipid extracts with cold acetone before chromatography, as suggested in the original lecithin/sphingomyelin ratio procedure, does not remove the interfering phospholipids. SUMMARY
The determination of the lecithin/sphingomyelin (L/S) ratio of amniotic fluid is now widely used for the assessment of fetal lung maturity in complicated pregnancies. The predictive value of the L/S ratio rests on the fact that, as the lung matures, lecithin accumulates in the amniotic fluid while the concentration of sphingomyelin parallels the increase in lecithin to about 32 weeks and then gradually declines (Gluck et al., 1971; Gluck and Kulovich, 1973). Since its introduction, the determination of the LIS ratio has been a controversial procedure; many technical problems have already been recognised (Biezenski, 1976). This paper draws attention to the presence of other phospholipids in amniotic fluid which may interfere with the L/S ratio determination, a long-standing problem that has not received the attention it deserves. The present investigation was undertaken when it was found that a clear separation of lecithin and sphingomyelin was not achieved in the routine procedure for determining the L/S ratio. Material and methods
Amniotic fluid collected by amniocentesis was centrifuged for 5 minutes at 3000 rpm (Clements Model GS 200 centrifuge, r a • = 13 cm) soon after collection and then chilled. None of the samples 1 These findings were presented to a meeting of the Australian Association of Clinical Biochemists, November 1978 (Duck-Chong and Brown. 1979)
contained blood or meconium. If the supernatant could not be used on the same-day it was stored frozen. The supernatant (2 ml) was mixed with 2 ml methanol, vortexed for 10 seconds, then mixed with 2 ml chloroform and vortexed for 1 minute before centrifugation for 10 minutes at 2700 rpm (Sorvall GLC-1 centrifuge, r a • = 13 em), The lower chloroform layer was carefully collected using a Pasteur pipette and evaporated to dryness under nitrogen with gentle warming at a temperature not exceeding 40 aC. Some samples were treated with cold acetone before chromatography; the procedure for 2 ml samples described by Gluck et al. (1974b) was followed strictly. The whole sample was dissolved in chloroform and applied to a plate for thin-layer chromatography. For two-dimensional chromatography, 4 ml amniotic fluid was extracted instead of 2 mI. The following solvents were used for thin-layer chromatography: Solvent A, chloroform/methanol/water (66 :25 :4) Solvent B, chloroform/methanol/acetic acid/butanol/ water (50:33:6'8:8'5:4'1) Solvent C, chloroform/methanol/water (65 :25:4) Solvent D, chloroform/methanol/water (72 :27 :4) Solvent E, chloroform/methanol/28 % aqueous NHa (65 :25 :4); all by volume. The following procedures were used for thin. layer chromatography: (1) The routine procedure used for the past four years in the Biochemistry Department of the Royal Prince Alfred Hospital: the sample was applied as
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Is the L/S ratio really a lecithin/sphingomyelin ratio?
a 5 mm streak 1 ern from the bottom of a 0·5 mm thick Silica Gel 60, F254, precoated plate (Merck, Art. 5744). The 20 x 20 em plates were cut to a height of 10 ern and a width appropriate to the number of samples on the plate. A lecithin/sphingomyelin standard (61ll of a solution containing 5 mg of each phospholipid per ml) was applied to each plate. Chromatography was carried out in a sealed tank equilibrated with solvent A, until the solvent reached the top of the plate. The dry plate was sprayed with 50 % sulphuric acid and charred on a hot plate until the lecithin/sphingomyelin standard achieved a ratio of 1:1, assessed visually. After cooling, the plate was scanned in a Helena Quick Scan densitometer. For more precise quantitation, the area under the curve was determined using a Leitz ASM Semi-Automated Image Analyser. (2) As for procedure (1), using solvent B instead of solvent A. (3) The procedure of Borer et al. (1971), exactly as described by Gluck et al. (1974b), using solvent C. (4) The procedure of Gluck et al. (1974b), using heat-activated 0·25 mm plates prepared from Silica Gel H (Merck) containing 5 % ammonium sulphate, and solvent C. Similar plates prepared without ammonium sulphate were also used; these plates were visualised as described for procedure (1). (5) The sample was applied as a spot to a plate supplied by Helena Laboratories for the L/S ratio determination and developed in solvent D, the solvent recommended in the Helena Fetal Maturity Test Procedure (PRO 13-4-77). Visualisation was as described for procedure (1). The standard lecithin/sphingomyelin mixture was supplied by Applied Science Laboratories. Phosphatidylserine obtained from Sigma Chemical Company was purified by thin-layer chromatography, and phosphatidylinositol was isolated from fetal rat lung (two days pre-term). The identity of the latter two phospholipids was confirmed by co-chromatography with commercial standards from different sources using basic, acidic, and neutral solvent systems and by their response to ninhydrin staining (Johnston, 1971). All solvents were analytical reagent grade, Merck Uvasol or Mallinckrodt Nanograde. Chloroform was stored at room temperature and used within one to two weeks of the opening of the bottle. Results An extract of amniotic fluid was first chromatographed in the routine solvent, solvent A, then in the second dimension in solvent B. A large, irregularly shaped spot moved away from the regions
185
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18 2B
C
