Fetal lung maturity tests on the 10,000 X g pellet

Centrifugation has a profound effect on tests of fetal lung maturity performed on amniotic fluid. We have investigated the effect of a 700 X g centrifugal force for 10 min and a 10,000 X g force for 20 min on a battery of tests. While 91% of the OD650 was removed by the 10,000 X g centrifugal force, the supernatant fraction retained 34% and 38% of the L/S ratio and enzymatic lecithin respectively, when compared to the sample before centrifugation. Phosphatidylglycerol, when present in an amniotic fluid, was always absent from the 10,000 X g supernatant but present in the pellet formed by this centrifugal force. The pellet after 10,000 X g was unsuitable for OD650 and L/S ratio determinations but contained 63% of the enzymatic lecithin. When the pellet tests were subjected to a clinical trial, respiratory immaturity did not occur when phosphatidylglycerol was present or when the 10,000 X g pellet may be a useful means of detecting amniotic fluid surfactant and thus determining fetal lung maturity.     

Specificity and reproducibility of acetone precipitation in identifying surface-active phosphatidylcholine in amniotic fluid.

We show the thoretical and actual variability in the amount of surface-active lecithin precipitated in the acetone-precipitation step of determining lecithin/sphingomyelin (L/S) ratios in the clinical laboratory. Lecithins precipitated may range from 60 to 90% of the total surface-active lecithins present. The range is sufficient to account for the occasional "immature" L/S ratios (less than 2/1) found in amniotic fluid from women bearing mature fetuses.     

Interference by endogenous glycerol in an enzymatic assay of phosphatidylglycerol in amniotic fluid

We investigated the possibility of interference by endogenous glycerol with the enzymatic measurement of phosphatidylglycerol in amniotic fluid. Phosphatidylglycerol is an important indicator of fetal lung maturity. The concentrations of glycerol and phosphatidylglycerol in amniotic fluid were measured by using a coupled enzymatic assay with and without phospholipase D (EC 3.1.4.4). The precision of the assay was acceptable (within-run CV = 1.2%, between-run CV = 4.8%). Endogenous glycerol content was demonstrated to be approximately 10-20 times that of phosphatidylglycerol. This high proportion of endogenous glycerol in amniotic fluid would preclude the accurate enzymatic determination of amniotic fluid phosphatidylglycerol unless the glycerol is first removed. Nor can the actual phosphatidylglycerol concentration be determined by subtracting the endogenous glycerol concentration from the total glycerol, which includes that glycerol derived from phosphatidylglycerol. With a usual range of 9 +/- 7 mumol/L, the error for a given phosphatidylglycerol measurement of +/- 6.6 mumol/L (+/- 2 SD) clearly is too high for this assay to be clinically useful. There was no correlation between concentration of endogenous glycerol or apparent phosphatidylglycerol in amniotic fluid and the lecithin/sphingomyelin ratio of the sample.     

The assessment of foetal lung maturity by chemical analysis of amniotic fluid

Advances in the understanding of the neonatal respiratory distress syndrome have led to a proliferation of amniotic fluid tests. Measurement of pulmonary surfactant production is the most direct means of assessing pulmonary maturity. Assays of surfactant are subjected to certain pre-analysis sources of variation, such as variability in amniotic fluid volume, sample collection site, centrifugation speed and time, and contamination with blood and/or meconium. Amniotic fluid surfactant assays can be divided into biochemical and functional tests. When properly performed, both approaches yield results that correlate well with clinical findings. However, no single method has achieved the distinction of total reliability and universal applicability. In most tests the value for mature lungs is almost 99% accurate. On the other hand, immature values have very low accuracy. Therefore, it is advisable to perform an additional test or to repeat the determination. The determination of the lecithin/sphingomyelin ratio is characterized by sufficient accuracy for routine analyses. For scientific studies we recommend the use of a capillary gas-chromatographic method allowing an accurate assessment of dipalmitoylphosphatidylcholine, the most important surfactant constituent.     

Multicenter evaluation of TDx test for assessing fetal lung maturity

"TDx Fetal Lung Maturity," an automated assay that measures the relative concentrations of surfactant and albumin in amniotic fluid, was compared with the lecithin/sphingomyelin (L/S) ratio and phosphatidylglycerol determination at five clinical sites. A total of 695 amniotic fluid samples were analyzed, of which 312 were followed by delivery of the infant within three days of sample collection. Of these 312, 24 developed respiratory distress syndrome and seven developed transient tachypnea. With the cutoff for maturity set at a surfactant/albumin value of 50 mg/g, the assay showed a sensitivity of 0.96 and a specificity of 0.88 for all samples, compared with a sensitivity of 0.96 and a specificity of 0.83 for the L/S ratio. The combination of rapid assay (30 min), accurate results, and uniformity among testing centers makes the TDx assay a very promising method.     

Influence of phospholipid saturation on classical thin-layer chromatographic detection methods and its effect on amniotic fluid lecithin/sphingomyelin ratio determinations

We compared seven techniques, commonly used for detection of amniotic fluid phospholipids in thin-layer chromatography, with respect to their sensitivity to saturation of the fatty acid carbon-chain of lecithin. The techniques fell into two classes: sensitive and insensitive; those classed as saturation sensitive were less than or equal to 10% as sensitive to fully saturated lecithin as to lecithin with singly unsaturated acid moieties. Color development increased with the number of carbon-carbon double bonds per molecule but required only a single unsaturated acid ester in either the alpha or the beta position. Mixtures of lecithins with defined saturation, when detected by saturation-sensitive methods, mimicked the uneven coloration of amniotic fluid lecithin bands, supporting the hypothesis that this uneven coloration results from natural saturation heterogeneity. Detection techniques representative of these classes are shown to give widely differing values for the lecithin/sphingomyelin ratio for actual specimens of amniotic fluid.     

Amniotic fluid palmitic acid concentrations and prediction of fetal lung maturity.

A method for rapid determination of total esterified palmitic acid concentration (TEPAC) in amniotic fluid is described. The correlation coefficient between the TEPAC and the lecithin concentration was 0.93 in 123 samples of amniotic fluid obtained during the last trimester. The respiratory distress syndrome (RDS) occurred in 73% of the cases studied with TEPAC less than mmol/l in predelivery samples. The RDS was not observed with higher concentrations except in cases of maternal diabetes mellitus. The predictive value of total esterified fatty acid studies was confirmed to the concentration of palmitic acid recorded, and no specific fatty acid distributions or ratios were reliable in identifying RDS or estimating gestational age.     

Studies on serum apolipoproteins and lipids in amniotic fluid and neonatal urine

Amniotic fluid and neonatal urine were examined for the presence of lipids and serum apolipoproteins. Human apolipoproteins A-I, A-II, and ApoD found principally in serum high density lipoproteins were identified in both neonatal urine and amniotic fluid. A lecithin/sphingomyelin ratio of greater than 5 associated with fetal lung maturity was accompanied by the disappearance of A-II from amniotic fluid. Dissimilarities of total fatty acid composition of amniotic fluid when compared to cord serum or neonatal urine indicate other tissue sources for fatty acids found in amniotic fluid. In addition, the presence of serum apolipoproteins and lipids in both amniotic fluid and neonatal urine suggests that a least a portion of these constituents could be derived from fetal urine.     

Measurement of the ratio of lecithin to sphingomyelin in amniotic fluid by fast atom bombardment mass spectrometry

We describe a fast atom bombardment mass spectrometric method to determine the ratio of lecithin to sphingomyelin in amniotic fluid. Between m/z 400 and 1000, amniotic fluid extracts showed clear peaks for only sphingomyelin and lecithin. The molecular species of fatty acid in the phospholipids were analyzed quantitatively and quickly in a small volume of the extracts. The dipalmitoyllecithin/palmitoylsphingomelin ratio, determined by the proposed method, showed a reasonable correlation to the lecithin/sphingomyelin ratio measured by thin-layer chromatography. This method allows rapid and sensitive measurement of these phospholipids.     

Improved fluorescence polarization assay for use in evaluating fetal lung maturity. II. Analytical evaluation and comparison with the lecithin/sphingomyelin ratio

We assessed the analytical performance of an improved fluorescence polarization assay for use in evaluating fetal lung maturity and compared results with the lecithin/sphingomyelin ratio. During a three-month period 150 patients' samples were assayed by clinical laboratory personnel with both techniques. Values for the lecithin/sphingomyelin ratio correlate closely with net fluorescence polarization values (r = -0.85), less closely with net fluorescence intensity (r = 0.65). Background fluorescence intensity and polarization varied widely, indicating a need to correct measurements for endogenous fluorescence. Net fluorescence polarization values have a CV of 0.32% within-run, 1.07% between-day. A comparison of two amniotic fluid centrifugation procedures showed no significant difference in such values. For both methods, however, such values are slightly but significantly higher than those obtained for amniotic fluids without prior centrifugation. Short-term storage (less than 30 days) of uncentrifuged amniotic fluid samples at -20 degrees C does not significantly affect results.     

Comparisons of the concentrations of palmitate and cortisol in amniotic fluid for the prediction of pulmonary maturity

The concentrations of palmitic acid, derived from lecithin, and of cortisol in samples of amniotic fluid collected during the last trimester of human pregnancy, ranged from 28 to 420 mumol/l and from 16 to 104 nmol/l, respectively. There was a weak (r = 0.43) but significant (p less than 0.01) correlation between the concentrations of cortisol and palmitate in the samples. Measurement of the cortisol concentration in amniotic fluid provided an unreliable index of pulmonary immaturity, whether this was assessed with reference to the concentration of palmitate or from subsequent development of the respiratory distress syndrome. Measurement of cortisol in amniotic fluid cannot be recommended for this purpose.     

Critical assessment of phospholipid measurement in amniotic fluid.

We assessed several methods of inorganic phosphate assay for their suitability in estimating phospholipids in digested extracts of amniotic fluids. The extraction and digestion procedures used for phospholipids from amniotic fluid were also examined critically. The effect of contamination by blood or obstetric cream has been examined. Accordingly, we suggest a method for measuring total phospholipids in amniotic fluids, and results of it are compared with the lecithin/sphingomyelin ratio measurement in some clinical situations.     

Human serum Zn-alpha2-glycoprotein in amniotic fluid

Human serum Zn-alpha2-glycoprotein (Zn-alpha2-GP) was found to be present in the amniotic fluid in the mean concentration of 0.98 +/- 0.40 mg/100 ml, which represents about one-tenth of its concentration in the maternal serum (9.65 +/- 1.18 mg/100 ml). Its concentration in the amniotic fluid was proportional to the amniotic fluid total protein and very approximately to the maternal serum Zn-alpha2-GP. The relationship between the maternal serum Zn-alpha2GP and the maternal serum total protein as well as between the amniotic fluid total protein and the maternal serum total protein was found to be not significant. The amniotic fluid Zn-alpha2-GP as well as the amniotic fluid total protein showed some increase during gestation to reach the highest values at the end of the second trimester. At present both the origin and significance of the amniotic fluid Zn-alpha2-GP are not known.     

Is the L/S ratio really a lecithin/sphingomyelin ratio?

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.     

A rapid electrochemical assay of lecithin in amniotic fluid using a fluoride ion-sensitive electrode

An electrochemical method is described for the determination of lecithin in rat and human amniotic fluid. Choline is released from lecithin enzymatically by phospholipase D and the hydrogen peroxide released by the action of choline oxidase is quantitatively determined by peroxidase-catalyzed rupture of the covalent C-F bond of 4-fluorophenol. The concentration of F- ions in solutions is determined by a fluoride sensitive electrode from the resulting cell potential difference recorded before and 10 min after addition of a solution containing phospholipase D, choline oxidase and horseradish peroxidase. Lecithin levels in rat amniotic fluid increased from about 10 mumol/l on the 20th day of gestation to 80 mumol/l on day 21, which corresponds to the time of spontaneous delivery. In human amniotic fluid the lecithin concentrations determined with this new method parallel those already reported. They were approximately 10 to 50 mumol/l between the 15th and 18th weeks of gestation and increased from 5- to 7-fold between the 37th and 41st weeks of pregnancy. This method was only slightly influenced by the presence of blood or meconium contamination in the amniotic fluid.     

Assessment of fetal lung maturity by colorimetric phospholipid determination without digestion.

A previously described method for the determination of phospholipid concentration in amniotic fluid without digestion has been modified to make it more suitable for use in a routine laboratory. Results compare well with those by the original procedure. Results from nearly 70 amniotic fluids, collected at delivery, were compared with lecithin/sphingomyelin (L/S) ratios determined on the same fluids. Statistical analysis of these data showed that for the prediction of lung immaturity, determination of total phospholipid concentration was at least as good as the L/S ratio.     

羊水样本卵磷脂和鞘磷脂检测超高效液相色谱串联质谱方法的建立及应用

目的建立检测羊水中卵磷脂和鞘磷脂(L/S)的超高效液相色谱串联质谱(UPLC-MS/MS)方法,以便准确、高效地预测胎儿肺成熟度。方法收集孕32~39周孕妇分娩时的羊水样本23份。依据新生儿Apgar评分标准,有3例胎儿胎肺未成熟、20例胎儿胎肺成熟。另收集孕18周孕妇羊水样本7份作为基线对照,建立检测羊水中卵磷脂和鞘磷脂水平的UPLC-MS/MS方法,计算卵磷脂/鞘磷脂(L/S)比值,同时采用板层小体计数(LBC)法检测板层小体(LB),评价2种方法在预测胎肺成熟度中的价值。结果建立的检测羊水中卵磷脂和鞘磷脂的UPLC-MS/MS方法精密度良好,离子峰强度和保留时间均在可检测范围内,主成分分析(PCA)显示6个质控样本聚类良好。以L/S比值=10作为判断胎肺成熟度与不成熟的临界值,UPLC-MS/MS的敏感性和特异性均为100%。以LB=50×10^9/L作为判断胎肺成熟度与不成熟的临界值,LBC法的敏感性和特异性分别为80%和95%。结论建立了检测羊水中卵磷脂和鞘磷脂水平的UPLC-MS/MS方法,其结果可靠,可以准确、高效地预测胎肺成熟度。     

Phosphatidylglycerol in 261 samples of amniotic fluid from normal and diabetic pregnancies, as measured by one-dimensional thin-layer chromatography.

We describe a one-dimensional thin-layer chromatographic method for separating lecithin, sphingomyelin, phosphatidylglycerol, and other phospholipids. The occurrence of phosphatidylglycerol in relation to the lecithin/sphingomyelin ratio is reported for 261 amniotic fluid samples. This compound does not consistently appear until the ratio exceeds about 3.1, and occurs less often in samples from diabetic patients than in those from normal patients. The respiratory distress syndrome did not occur when phosphatidylglycerol was present in amniotic fluid although the reverse was not necessarily true. Thus the presence of phosphatidylglycerol offers additional assurance of pulmonary maturity.     

Effects of tomatoes on the lipid profile

PURPOSE: The Mediterranean diet has been reported to reduce cardiovascular mortality and morbidity considerably. Tomatoes and lycopene are considered potent antioxidants. Our purpose was to study the effects of a tomatoe-rich diet on the lipid profile following 300g daily of tomatoes for one month. METHODS: Plasma concentrations of triglyceride, total cholesterol, HDL-cholesterol, LDL-cholesterol and VLDL-cholesterol were determined in 98 apparently healthy volunteers (mean age 45.5+/-14.1 yr) before and after one month of follow-up. Fifty volunteers (34 women and 16 men) ate tomatoes 300g daily and 48 volunteers (32 women and 16 men) continued their regular diet without eating tomatoes for a month. RESULTS: In the regular diet group, there were no changes in the lipid profile: triglyceride level (169.6+/-156.8 vs. 147.6+/-93.4mg/dl; P=0.33), total cholesterol level (198.3+/-41.2mg/dl vs. 204.2+/-70.9mg/dl; P=0.23), HDL-cholesterol level (50.6+/-12.2mg/dl vs. 47.6+/-10.8mg/dl; P=0.79), and LDL-cholesterol level (122.7+/-39.4mg/dl vs. 120.2+/-32.2mg/dl; P=0.24) before and after the 1 month offollow-up. In the tomato-rich diet group: triglyceride level 170.8+/-85.4mg/dl to 167.4+/-99.4mg/ dl (P=0.98), total cholesterol level 207.5+/-44.3mg/ dl to 204.1+/-45.1mg/dl (P=0.68), HDL-cholesterol level 46.1+/-10.6mg/dl to 53.4+/-13.3mg/dl (P=0.03), and LDL-cholesterol level 127.7+/-41.8mg/dl to 119.1+/-41.7mg/dl (P=0.57). CONCLUSION: We found that tomatoes'-rich diet (300g daily for one month) increased HDL-cholesterol level significantly by 15.2%.     

Determination precise du rapport lecithine/ sphingomyeline dans le liquide amniotique: Proposition d'une methode de referenceExact determination of the lecithin Isphingomy elm ratio in amniotic fluid. Suggestion of a reference method

Pulmonary maturity of the fetus can be evaluated by the lecithin/sphingo-myelin (L/S) ratio in amniotic fluid. To existing methods of lipid extraction, precipitation with acetone and chromatography, we add a simple and accurate estimation of sphingomyelins (S) and precipitated lecithins (Lp) without acid digestion. The method is reproducible (C.V. < 9%) for the measurement of Lp/S ratio and gives with accuracy the concentrations of Lp, avoiding possible errors in interpretation of Lp/S. Our results show that at 35 weeks of normal gestation, Lp/S ratio is about 2 and Lp concentration, 10 mg/l.