Phosphatidylcholine In Amniotic Fluid Research Articles

Analysis of dipalmitoyl phosphatidylcholine in amniotic fluid by high-performance liquid chromatography.

A novel method for the determination of dipalmitoyl phosphatidylcholine (DPPC) in amniotic fluid by high-performance liquid chromatography (HPLC) is described. Aliquots of 50 microliters of amniotic fluid were hydrolyzed with phospholipase C from Bacillus cereus and the resulting dipalmitoylglycerol analyzed by HPLC. Run-to-run and day-to-day precision for DPPC analysis were 4.2 and 6.1%, respectively, and analysis time was 10 min. Recoveries for DPPC ranged between 92 and 98%. In summarizing, this method provides a high precision and fast turnaround time means for the analysis of DPPC in amniotic fluid.

Fetal lung maturation in congenital diaphragmatic hernia

OBJECTIVE: Our purpose was to determine whether congenital diaphragmatic hernia is associated with abnormalities of fetal lung maturation. STUDY DESIGN: We measured surfactant protein A and saturated phosphatidylcholine in amniotic fluid from 19 pregnancies with a prenatal diagnosis of congenital diaphragmatic hernia (gestational age 16 to 40 weeks) and 48 control pregnancies (gestational age 16 to 39 weeks). Results were compared by analysis of covariance. RESULTS: Beyond 34 weeks of gestation there was a progressive rise in amniotic fluid surfactant protein A and saturated phosphatidylcholine in control pregnancies, whereas in most fetuses with prenatal diagnosis of congenital diaphragmatic hernia these values remained low ( p < 0.01). Amniotic fluid surfactant protein A was lower in fetuses with congenital diaphragmatic hernia who died or required extracorporeal membrane oxygenation than in survivors treated with conventional management (4.9 ± 2.9 vs 16.8 ± 5.7 μg/ml surfactant protein A, respectively, p < 0.05 by Mann-Whitney U test). CONCLUSIONS: There are decreased surfactant components in amniotic fluid in many pregnancies complicated by congenital diaphragmatic hernia, which may reflect fetal lung immaturity or hypoplasia.

Analysis of dipalmitoyl phosphatidylcholine in amniotic fluid by enzymatic hydrolysis and high-performance thin-layer chromatography reflectance spectrodensitometry.

A novel test for the determination of dipalmitoyl phosphatidylcholine (DPPC) in amniotic fluid (AF) as free dipalmitoylglycerol (DPG), is described. Aliquots of amniotic fluid were hydrolyzed with Bacilus cereus phospholipase C, and the resulting diglycerides analyzed by AgNO3-modified high-performance thin-layer chromatography (HPTLC)-reflectance spectrodensitometry. This HPTLC system provided resolution of DPG and palmitoylpalmitoleoylglycerol (POG) from other 1,2-diglycerides and cholesterol. The turn-around analysis time for triplicate aliquots of amniotic fluid was 40 min. Recoveries ranged between 90 and 98%. In summary, this method provides a quantitative, specific, highly reproducible, and fast turn-around means of analysis of DPPC in amniotic fluid.

Corticotrophin-Releasing Hormone Levels in Human Plasma and Amniotic Fluid During Gestation

Summary OBJECTIVE Corticotrophin releasing hormone, a hypothalamic neuropeptide also made in placenta, may regulate fetal maturation in a stress-responsive manner. The objectives of this study were: (1) to determine if levels of corticotrophin releasing hormone in the amniotic fluid correlate with fetal lung maturation; (2) to confirm that third trimester plasma levels of corticotrophin releasing hormone are increased in patients with pregnancy-induced hypertension compared to normotensives, and (3) to increase the recovery of extracted corticotrophin releasing hormone from plasma and amniotic fluid. DESIGN (1) Levels of corticotrophin releasing hormone in amniotic fluid during the third trimester were compared with those of saturated phosphatidyl choline. (2) Corticotrophin releasing hormone levels were measured in a group of normotensive pregnant women during the entire gestation period. Corticotrophin releasing hormone levels during the third trimester were compared in normotensives and patients with pregnancy-induced hypertension. PATIENTS Twenty-one non-pregnant normal volunteers and 63 pregnant women. MEASUREMENTS Blood pressure, corticotrophin releasing hormone in plasma and amniotic fluid, and saturated phosphatidyl choline in amniotic fluid. RESULTS Corticotrophin releasing hormone levels in amniotic fluid samples during the third trimester ranged from 12 to 98 pmol/l and positively correlated with the saturated phosphatidyl choline levels, but not with gestational age. A significant difference existed in plasma corticotrophin releasing hormone concentration between gestational age-matched third trimester normotensive and hypertensive gravids: corticotrophin releasing hormone levels were significantly lower in normotensives (223 ± 65 pmol/l) than in patients with pregnancy-induced hypertension (544 ± 106 pmol/l, P= 0.001). Plasma corticotrophin releasing hormone increased with gestational age from 51 pmol/l (range 8.4–85) at 25–32 weeks to 375 pmol/l (range 35–1386) at 33–40 weeks. During the third trimester the rise in plasma corticotrophin releasing hormone conformed to an exponential mathematical model of a positive feedback loop between placental corticotrophin releasing hormone and fetal adrenal Cortisol. CONCLUSIONS During the third trimester of pregnancy there is a positive correlation between the level of amniotic fluid corticotrophin releasing hormone and that of saturated phosphatidyl choline. The positive correlation between amniotic fluid corticotrophin releasing hormone and saturated phosphatidyl choline, but not between amniotic fluid corticotrophin releasing hormone and gestational age, suggests that a factor(s), such as stress, may affect both amniotic fluid corticotrophin releasing hormone and saturated phosphatidyl choline in parallel. Furthermore, our data are consistent with the hypothesis that the rise in placental corticotrophin releasing hormone is coupled to an increase in fetal glucocorticoid and lung maturation, and that stresses such as pregnancy-induced hypertension may accelerate this process.

Analysis of saturated phosphatidylcholine in amniotic fluid by 31P NMR.

A technique using solubilization with sodium cholate to resolve 31P NMR resonances of phospholipid molecular species was applied to amniotic fluid samples from 16 subjects. Gestational ages from 25 to 40 weeks were represented, and two subjects were sampled sequentially. Fitting of the partially resolved 31P NMR signal of phosphatidylcholine (PC) generated an estimate of percent disaturated acyl PC (%dsPC) which correlated more highly with gestational age than did several other potential indices of fetal lung maturation, such as the ratio of PC to inorganic phosphate from the solubilized spectra, or PC to sphingomyelin from extract spectra. In a few cases, enzyme-catalyzed PC hydrolysis limited the precision, but did not appear to affect the accuracy, of the %dsPC estimates. Resolution of palmitoyl and oleoyl lyso-PC species was observed for both the 1- and 2-acyl isomers. Upfield shifts due to the presence of cis double bonds in the lone acyl chain of the lyso-PCs were analogous to those observed for the diacyl PCs.

Combined enzymatic assay of phosphatidylglycerol and phosphatidylcholine in amniotic fluid

We present here a combined, quantitative enzymatic procedure for determining amniotic fluid phosphatidylglycerol and phosphatidylcholine and relate these findings to the assessment of fetal lung maturity. Under the assay conditions described phospholipase C specifically hydrolyses phosphatidylglycerol (PG) and phosphatidylcholine (PC) but not sphingomyelin, precluding the need for removal of sphingomyelin prior to analysis. Solvent extraction of the phospholipids from the amniotic fluid is, however, employed to avoid spurious elevation of PG and PC results by endogenous glycerol and choline. Of 45 amniocentesis fluids examined, 28 yielded detectable PG concentrations (> 0.5 μmol/l) and all but three of these exhibited PC concentrations in excess of 10 μmol/l. One case of respiratory distress occurred in an infant of 29 wk gestation with severe intrauterine growth retardation. Of the remaining 17 fluids in which PG was undetected enzymatically (⩽ 0.5 μmol/l), 14 also contained PC concentrations ⩽ 10 μmol/l and all six cases of true respiratory distress syndrome came from within this sub-group. Strong correlations between the PC concentration and the lecithin : sphingomyelin ratio, r = 0.85 ( p < 0.001) and the PC and PG concentrations, r = 0.96 ( p < 0.001) were also found.

Assay of disaturated phosphatidylcholine in amniotic fluid as a test of fetal lung maturity: experience with 2000 analyses.

We determined concentrations of disaturated phosphatidylcholine (DSPC) in nearly 2000 amniotic fluid samples obtained either transabdominally or as vaginal pools. Here we report our comparison of these DSPC values with the lecithin/sphingomyelin (L/S) ratios for amniotic fluid samples obtained from diabetic and nondiabetic pregnancies and also between transabdominally or vaginally collected samples uncontaminated by blood or meconium. DSPC measurement is at least as good as the L/S ratio in predicting the absence of respiratory distress syndrome. DSPC concentrations were, however, lower in diabetic than in nondiabetic pregnancies, supporting the hypothesis that DSPC synthesis may be impaired in fetuses of diabetic mothers. Visually uncontaminated samples collected transabdominally or vaginally, when grouped according to length of gestation, have similar DSPC values but different L/S ratios. Thus, even in the absence of blood or meconium, DSPC may be a more useful test than the L/S ratio for vaginally pooled samples.

Androgen receptors influence the production of pulmonary surfactant in the testicular feminization mouse fetus.

A sexual dimorphism in fetal pulmonary maturation has been described in which the female fetal lung produces surfactant earlier in gestation than the male fetal lung. This is felt to be related to the increased incidence in male newborns of the Respiratory Distress Syndrome. Dihydrotestosterone will delay surfactant production in the female fetus, and a relationship between fetal sexual differentiation and fetal lung maturation has been proposed. We hypothesized that the dimorphism in fetal surfactant production is dependent on androgen receptor function. We measured phosphatidylcholine (PC), saturated phosphatidylcholine (SPC), and sphingomyelin (S) in the amniotic fluid of fetal mice of the mouse model of testicular feminization (Tfm mouse). In this model, male carriers of the X-linked Tfm gene have no functional androgen receptors. The mean amniotic fluid phosphatidylcholine to sphingomyelin ratio (PC/S ratio) was 28% higher in females than in normal males, and the amniotic fluid PC/S ratio of the Tfm male fetuses was the same as the females. The ratio of amniotic fluid saturated phosphatidylcholine to sphingomyelin (SPC/S ratio) was lowest in males, intermediate in females, and highest in Tfm males. A significant relationship between the fetal groups and the amniotic fluid SPC/S ratio was identified by analysis of variance. There were no differences in the whole lung phospholipid content between the three groups. To substantiate the effect of androgen receptors, dihydrotestosterone was injected into pregnant carriers of the Tfm mutation, 2.5 mg/d from day 10 of gestation through the day of sacrifice. The amniotic fluid PC/S ratio was decreased in the female fetuses (consisting of both homozygous normal and heterozygous carriers of the Tfm gene), but not in the Tfm male fetuses. The overall result was no significant difference between the male and female amniotic fluid PC/S ratio while the Tfm amniotic fluid PC/S ratio remained at the level of the untreated females. We conclude that androgens affect fetal lung development via a mechanism dependent on the presence of androgen receptors.

Amniotic fluid phosphatidylglycerol and prediction of fetal lung maturity in diabetic pregnancies

SummaryAmniocentesis was carried out in 35 pregnant diabetic patients within 72 hours before delivery. All patients had lecithin/sphingomyelin ratios of 22 or more. Four babies (114 per cent) developed respiratory distress syndrome. The remainder of the same 35 samples of amniotic fluid were used retrospectively to predict fetal lung maturity in two ways using phosphatidylglycerol. Measurement of amniotic fluid phosphatidylcholine (lecithin) and phosphatidylglycerol concentrations appears to predict fetal lung maturity more accurately than either the lecithin/ sphingomyelin ratio alone or when combined with simply recording the presence of phosphatidylglycerol.

Relationships among human amniotic fluid dipalmitoyl lecithin, postpartum respiratory compliance, and neonatal respiratory distress syndrome.

The major molecular species of amniotic fluid phosphatidylcholine were determined as diacylglycerol trimethylsilyl ether derivatives by gas-liquid chromatography with use of glass capillary columns. We studied amniotic fluid specimens from 48 pregnancies. As expected, the major disaturated species of amniotic fluid phosphatidylcholine, dipalmitoylphosphatidylcholine, and palmitoylmyristoylphosphatidylcholine increased with gestational age, whereas phosphatidylcholine species with 34, 36, and 38 carbon atoms in the acyl radicals decreased. Although the amniotic fluid samples were obtained shortly before parturition (4.2 +/- 3.7 h, mean +/- SD), the correlation between dipalmitoylphosphatidylcholine concentration in amniotic fluid and gestational age was better than between dipalmitoylphosphatidylcholine concentration and the compliance of the respiratory system of the newborns. In addition, the percentage of amniotic fluid phosphatidylcholine species present as dipalmitoylphosphatidylcholine seemed to be a more reliable predictor of lung maturity than was the absolute dipalmitoylphosphatidylcholine concentration in amniotic fluid.

Improved thin-layer chromatography of disaturated phosphatidylcholine in amniotic fluid.

We describe an indirect test of fetal lung maturity: the quantitation of disaturated phosphatidylcholine in amniotic fluid. The lipids in samples of amniotic fluid from 172 patients were reacted with osmium tetroxide, and disaturated phosphatidylcholine was then isolated by thin-layer chromatography. Interfering substances were retained by a pre-adsorbent layer. The charred disaturated phosphatidylcholine, quantitated by densitometry, was compared to standard dipalmitoyl phosphatidylcholine. Both within-run and between-run coefficients of variation were about 10%. Blood and meconium do not interfere. Six infants developed respiratory distress when disaturated phosphatidylcholine concentrations of amniotic fluid drawn within 72 h of delivery were less than 5.5 mg/L. A concurrently determined lecithin/sphingomyelin ratio falsely predicted lung maturity in one of these. In seven other samples for which lecithin/sphingomyelin ratios suggested lung immaturity but disaturated phosphatidyl-choline predicted maturity, none of the infants developed respiratory distress. In normal pregnancies, measurement of disaturated phosphatidylcholine in amniotic fluid appears to be a better predictor of fetal lung maturity than is measurement of the lecithin/sphingomyelin ratio. Further studies are needed to determine if this analysis is a better predictor in diabetic pregnancies.

High pressure liquid chromatography of standard and amniotic fluid phospholipids

AbstractA method for the HPLC separation of phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylcholine (PC), and sphingomyelin (SPH) was achieved using five in‐series columns packed with LiChrosorb, Partisil, and μ‐Porasil adsorbents, a solvent mixture of chloroform/methanol/ammonium hydroxide (50 : 36 : 6.7, by volume), and a Pye LCM2 Moving Wire (FID) detector. The same phospholipid mixture was also separated using four μ‐Porasil columns with the same eluent and detector. The latter conditions were found to be suitable for the analysis of phospholipids obtained after centrifuging, extraction, and precipitation of surface‐active lipid components of patient amniotic fluid collected at amniocentesis section. The lecithin/sphingomyelin (L/S) ratios, determined by the HPLC method, correlated well with those determined by the TLC technique in four normal pregnancies, whereas results of shake tests did not correlate too well with L/S ratios determined by the above two chromatographic methods. Besides the lecithin/sphingomyelin ratio, the present method was able to supply additional information: the concentrations of phosphatidylglycerol and phosphatidylinositol, for prediction of fetal lung maturity, and the palmitic acid content of amniotic fluid phosphatidylcholine.

Saturated Phosphatidylcholine in Amniotic Fluid and Prediction of the Respiratory Distress Syndrome

Abstract The lecithin/sphingomyelin (L/S) ratio in amniotic fluid is widely used to predict the risk of respiratory-distress syndrome. However, the results are unreliable if the specimen is contaminated or obtained during a complicated pregnancy. We therefore compared the predictive value of the L/S ratio with that of the concentration of saturated phosphatidylcholine (SPC) in 322 amniotic-fluid samples, 75 per cent of which were contaminated or obtained during complicated pregnancies or both. A positive result is one that predicted the development of respiratory-distress syndrome, taken as an L/S ratio equal to or less than 2/1 or an SPC below 500 μg per deciliter. The respiratory-distress syndrome was correctly predicted in 25 of 45 cases (55.5 per cent) with L/S ratios equal to or less than 2/1, and in 35 of 42 cases (82 per cent) with SPC's less than 500 μg per deciliter. When L/S ratios were greater than 2/1, there were 13 of 277 (4.7 per cent) false negatives, and when SPC's were above 500 μg per de...

Determination of phosphatidylcholine in amniotic fluid

A relatively rapid method for measuring phosphatidylcholine (lecithin) quantitatively in amniotic fluid has been described that requires 1 ml or less of sample for fluids having a total phospholipid concentration greater than 25 mg/liter. Following extraction with chloroform-methanol, the solvent is passed through a calcium hydroxyphosphate column which removes the acidic phospholipids and allows passage of the phosphatidylcholine and sphingomyelin. Hydrolysis with periodate-sulfuric acid selectively releases inorganic phosphate from the phosphatidylcholine that is measured by reduction of the formed phosphomolybdate complex to the usual blue color. Various mixtures of phospholipids were carried through the entire procedure with excellent recoveries. Phosphatidylcholine added to an amniotic fluid pool was also quantitatively recovered, so that the method appeared completely suitable for routine clinical laboratory use.

Amniotic fluid phospholipids measured by continuous-development thin-layer chromatography.

We describe a one-dimensional thin-layer chromatographic system for separation of amniotic fluid phosphatidylcholine, sphingomyelin, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine in amniotic fluid. We utilize short-bed continuous development and "high performance" thin-layer chromatography. Phospholipids are detected with an antimony molybdate staining reagent and quantitated by transmittance densitometry. This system is more sensitive to changes in lecithin/sphingomyelin ratios than are planimetric evaluations.

Amniotic fluid phospholipids measured by continuous-development thin-layer chromatography.

Abstract We describe a one-dimensional thin-layer chromatographic system for separation of amniotic fluid phosphatidylcholine, sphingomyelin, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine in amniotic fluid. We utilize short-bed continuous development and "high performance" thin-layer chromatography. Phospholipids are detected with an antimony molybdate staining reagent and quantitated by transmittance densitometry. This system is more sensitive to changes in lecithin/sphingomyelin ratios than are planimetric evaluations.

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.