Assessment of Fetal Lung Maturity by Estimation of Amniotic Fluid Palmitic Acid

The measurement of palmitic acid in amniotic fluid has been shown to be a rapid means of assessing the lecithin concentration. The level of palmitic acid increases quickly when the fetal lung matures at about 35 weeks'' gestation, and the level in amniotic fluid obtained 24 hours or less before delivery clearly distinguishes which infants are likely to develop respiratory distress syndrome and which are mature.     

Estimation of gestational age from study of amniotic fluid and clinical assessment.

Study of 108 samples of amniotic fluid obtained between 28 and 42 weeks'' gestation from 101 patients revealed that in normal pregnancies the creatinine concentration, lecithin/sphingomyelin (L/S) ratio and percentage of fat cells correlated better with the gestational age of the newborn--assessed by clinical criteria--than did the bilirubin and sodium concentrations. A creatinine concentration of 1.75 mg/dL or more, an L/S ratio of 4 or more and a fat cell percentage of 10 or more correlated significantly with a gestational age of 37 weeks or more. In abnormal pregnancies (those with obstetric or medical complications, or both) the mean creatinine concentration in the amniotic fluid was significantly less than expected for gestational age in fetal dysmaturity and greater than expected when the mother had diabetes. The mean L/S ratio in the amniotic fluid was elevated when the mother had hypertension or smoked and in cases of fetal dysmaturity or long interval between rupture of the membranes and delivery, whereas it was significantly lower than normal when the mother had diabetes. The mean bilirubin concentration in the amniotic fluid was significantly lower than normal when the mother had hypertension. When the mother had diabetes, maturity of the fetal lung, liver, skin and brain appeared to be delayed, according to the values for the amniotic fluid constituents.     

Origin and developmental patterns of lactase and other glycosidases in sheep amniotic and allantoic fluid.

Intestinal lactase activity (with its associated cellobiase, 4-methylumbelliferyl-beta-galactosidase and -beta-glucosidase activities) was used as a specific intestinal marker enzyme to study the release of protein and enzymes of intestinal origin in sheep amniotic fluid during gestation. In amniotic fluid, intestinal lactase activity peaked at 66--85 days of gestation and then decreased with gestation. This enzyme activity was very low or absent in allantoic fluid throughout gestation suggesting that there is no important transfer of amniotic fluid lactase towards the allantoic cavity. Maltase and 4-methylumbelliferyl-alpha-glucosidase showed no statistically significant variation with gestation in both amniotic and allantoic fluid whereas alpha-galactosidase and N-acetyl-beta-hexosaminidase which were first higher in allantoic than in amniotic fluid increased in amniotic fluid to reach allantoic fluid levels near term. Such patterns are consistent with the suggestion that the fetal urine is a source of alpha-galactosidase and N-acety-beta-hexosaminidase activities and that sheep urine is first accumulated in the allantoic sac via the urachus up to 86--90 days of gestation and thereafter passes more and more into the amniotic sac.     

Amniotic fluid embolism and leukotrienes--the role of amniotic fluid surfactant in leukotriene production.

Surfactant rich lipid (lipid) was extracted from cell free 10,000 x g pellets of amniotic fluid. White blood cells (WBC) were isolated from human donors. 36 x 10(7) WBC and 5 g rabbit lung were incubated with pretreated lipid or dipalmitoyl lecithin (lecithin). Leukotrienes (LTs) were identified by high performance liquid chromatography (HPLC) and bioassay, and quantified by radioimmunoassay. Peaks of LTC4 and LTD4 on HPLC and guinea-pig ileum contraction could be identified in lipid and lecithin groups, but not in the control group. LTC4 production by lipid and lecithin groups was significantly higher than that by the control group. An involvement of amniotic fluid surfactant in leukotriene production is suggested.     

Characteristics of ceruloplasmin synthesis in embryogenesis and in the early postnatal period of laboratory white rats

The dynamics of ceruloplasmin content was studied by immunochemical methods in the postimplantation rat embryos and postnatal animals. Ten to twenty two day old embryos contained ceruloplasmin (CP) in yolk sac, serum, and amniotic fluid. The highest CP levels were found in yolk sac. CP concentration profiles were almost identical in the serum and amniotic fluid being the highest on the 12th day (0.26 mg%) and the lowest (0.04) on the 16th day of gestation. CP concentration in the serum increased rapidly up to 3.5 mg% from the 17th day of gestation till the term (22nd day) while remaining at a constant and rather low level in the amniotic fluid. Within 16-18 days after birth, CP concentration in the serum remained at the level of 11 +/- 0.3 mg%. Later on it gradually increased and attained plateau (46-48 mg%) by the time of sex maturity. The maternal serum CP does not penetrate, in the embryo, as can be inferred from the experiments with 125I-CP injected into pregnant rats. Differences in the CP degradation rate and modes were found between the embryos and postnatal rats. It is suggested that CP is initially synthesized by the yolk sac endoderm during organogenesis (10-16 days of gestation) and predominantly by the liver during the foetal period (17-22 days).     

Disappearance of prostaglandins F2α and 15-methyl F2α from amniotic fluid as measured by radioimmunoassay

A sensitive and relatively specific radioimmunoassay for 15 (S) 15 methyl prostaglandin F_2α was used to determine the levels of the drug in amniotic fluid after it had been injected intra-amniotically for termination of second trimester pregnancy. The disappearance of the free acid (tham salt) and methyl ester of the prostaglandin analogue were similar. The results of this preliminary study suggest that the drug rapidly equilibrates in the fluid and this is followed by a slow removal from the amniotic sac. A comparison with a similar study with PGF_2α, revealed that the analogue had a longer half-life in the amniotic fluid.     

Distribution of 3H in rat conceptus cultured in vitro following brief administration of [3H]thymidine

Rat embryos with intact visceral yolk sacs, explanted at 12 1/2 days of gestation, were cultured in vitro for up to 60 min in medium consisting of fetal calf serum, Eagle's MEM, and [3H]thymidine (1.2 kBq ml-1), using the roller bottle method. The total amount of 3H incorporated into the conceptus during the 60-min incubation was 79.2 Bq, and approximately 33, 23, and 44% of this activity was distributed to the embryo, the yolk sac, and the fluid in the exocoelom and amniotic cavity, respectively. The rate of 3H accumulation in conceptuses decreased with time in culture. It appeared that the decrease in the viability of the conceptus was not responsible for this phenomenon. The concentration of 3H in the yolk sac, i.e., 3H activity per gram wet weight, was 2.1 times that in the medium at the end of culture. In contrast, the 3H concentration in the embryo was significantly lower than that in the medium. These findings suggest that the visceral yolk sac of rat conceptuses may act as a barrier to the transport of tritiated thymidine between the medium and embryo.     

Disappearance of prostaglandins F2α and 15-methyl F2α from amniotic fluid as measured by radioimmunoassay

A sensitive and relatively specific radioimmunoassay for 15 (S) 15 methyl prostaglandin F_2α was used to determine the levels of the drug in amniotic fluid after it had been injected intra-amniotically for termination of second trimester pregnancy. The disappearance of the free acid (tham salt) and methyl ester of the prostaglandin analogue were similar. The results of this preliminary study suggest that the drug rapidly equilibrates in the fluid and this is followed by a slow removal from the amniotic sac. A comparison with a similar study with PGF_2α, revealed that the analogue had a longer half-life in the amniotic fluid.     

Fetal-maternal transfer of [H3]-6 beta-hydroxycortisol in the pregnant ewe

Distribution and fetomaternal transfer of 6 beta-hydroxycortisol (6 beta-OHF) was studied using serial sampling following injection of tritium labelled 6 beta-OHF into various fluid compartments in the chronically cannulated unaesthesized pregnant ewe. There was a rapid transfer of 6 beta-OHF from the fetal circulation into amniotic fluid and maternal blood. In contrast, the maternal----fetal transfer of this steroid metabolite was considerably less. The sequence of appearance of 6 beta-OHF in fetal blood and amniotic fluid following injection into maternal blood suggests that this steroid is first transferred across the placenta to fetal blood before gaining entry into the amniotic fluid space. The half-lives of 6 beta-OHF after initial equilibration in maternal plasma, fetal plasma and amniotic fluid were 2.0 h, 5.1 h and 8.9 h respectively. The amniotic sac appears to contain a relatively static pool of 6 beta-OHF and may act as a "trap" for 6 beta-OHF in the ovine conceptus.     

Relation of amniotic fluid lecithin/sphingomyelin ratio and fetal asphyxia to respiratory distress syndrome in premature infants.

In a group of 81 prematurely delivered infants the amniotic fluid lecithin/sphingomyelin ratio was related to functional fetal lung maturity. Intrapartum fetal asphyxia occurred in 33% of the group. An association between fetal asphyxia and the development of respiratory distress syndrome (RDS) in the infant was observed. When pulmonary maturity was borderline according to the amniotic fluid assessment, intrapartum fetal asphyxia was associated with an increased risk for development of RDS in the infant.     

Total cortisol and L/S-ratio in amniotic fluid in late pregnancies complicated by diabetes mellitus

68 samples of amniotic fluid from 47 women with varying severity of diabetes mellitus and 48 samples from 43 normal women were obtained in the 31st to 40th week of pregnancy before onset of labour. The concentration of total cortisol and the L/S-ratio in amniotic flud were determined and related to gestational age. There was a continous rise of total cortisol with advancing gestational age in the diabetic pregnancies similar to that found in normal pregnancy. Diabetic pregnancies were associated with slightly lower amniotic fluid cortisol levels without significant difference to values found in normal pregnancy. The severity of the disease did not affect the cortisol levels in amniotic fluid. There was no correlation between total cortisol levels and L/S-ratios in amniotic fluid. Determination of total cortisol in amniotic fluid can thus not replace measurements of L/S-ratios to predict fetal lung maturation.     

Estimation of amniotic fluid phospholipids by high-performance liquid chromatography

We have developed a high-performance liquid chromatographic (HPLC) method for the analyses of surface-active amniotic fluid phospholipids, lecithin (L), sphingomyelin (S), phosphatidyl glycerol (PG), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), and phosphatidyl serine (PS), which are important in the prediction of fetal lung maturity. The method incorporates an internal standard in the amniotic fluid extract, and utilizes a 10-μl aliquot of a 2:1 chloroform—methanol extract of amniotic fluid injected onto a 5-μm DIOL or CN HPLC column, and a variable-wavelength detector set at 203 nm.Amniotic fluid phospholipid estimations were determined on 40 amniotic fluid samples by the HPLC method and by the routine thin-layer chromatographic (TLC) method. Good agreement was observed between the two methods for the L/S ratio, PG, and PI (r_PG 0.94, r_PI 0.95, r_L/S 0.97).The advantages of the HPLC procedure include: (i) Selective separation for PG, PI, PS, and PE, as well as L and S at the same time. (ii) The internal standard allows individual concentration of phospholipids to be estimated. (iii) The procedure is rapid: 16 min for a single assay compared with 50 min for the standard TLC procedure.     

THE CONCENTRATIONS OF FREE AMINO ACIDS AND OTHER ELECTROLYTES IN CEREBROSPINAL FLUID,IN VIVO DIALYSATE OF BRAIN,AND BLOOD PLASMA OF THE DOG*

The concentrations of free amino acids in plasma, CSF and in vivo dialysates of peripheral blood (neck sac fluid) and central nervous tissue (brain sac fluid) from each of five dogs (neck sac fluid from four of five dogs) were determined by ion-exchange chromatography. Dialysates were obtained by implanting small dialysis sacs filled with a dextran-saline solution into the subcutaneous tissue of the neck or the parenchyma of the brain at least 10 weeks before sample collection. The mean plasma concentration of most amino acids was within the range of values reported in the literature for human or dog plasma. The concentrations of most amino acids were higher in the neck sac fluid than in plasma; this discrepancy, however, was, for the most part, small and could most likely be accounted for by falling plasma free amino acid levels prior to sample taking. Previous conclusions that the CSF concentrations of most amino acids are lower than plasma concentrations are confirmed, although the present work indicates that there may be considerable individual variation in the CSF/plasma distribution ratio with respect to most amino acids. In the brain sac fluid the concentration of nearly every amino acid was consistently higher than that in CSF and lower than that in the neck sac fluid. The potassium concentration in the brain sac fluid was significantly higher than, and the total osmolality significantly lower than, those in the neck sac fluid. On the assumption that the brain sac fluid represents a dialysate of the brain extracellular fluid, these results contradict recent findings (Bito and Davson , 1965; 1966) indicating that the potassium concentration of the cortex extracellular fluid is lower than that of ventricular or cisterna magna CSF and certainly lower than that of plasma. Because of this and on the basis of consideration of the reaction of the brain to a foreign body, the possibility that the implanted brain sac lay on the‘blood side’of the bloodbrain barrier was suggested. Some implications of this possibility are discussed.     

Disappearance of prostaglandins F2alpha and 15-methyl F2alpha from amniotic fluid as measured by radiommunoassay.

A sensitive and relatively specific radioimmunoassay for 15 (S) 15 methyl prostaglandin F2alpha was used to determine the levels of the drug in amniotic fluid after it had been injected intra-amniotically for termination of second trimester pregnancy. The disappearance of the free acid (tham salt) and methyl ester of the prostaglandin analogue were similar. The results of this preliminary study suggest that the drug rapidly equilibrates in the fluid and this followed by a slow removal from the amniotic sac. A comparison with a similar study with PGF2Alpha revealed that the analogue had a longer half-life in the amniotic fluid.     

THE NATURE AND ORIGIN OF THE SOLUBLE PROTEIN IN HUMAN AMNIOTIC FLUID

1. Amniotic fluid surrounds the human fetus and is separated from the uterus by the amnion, chorion and placenta. The ability to obtain samples of amniotic fluid from women by a simple procedure has encouraged studies on the nature and origin of the fluid, and on its use for the diagnosis of a variety of clinical conditions. The fluid contains cells, which are of fetal origin, and can be grown in a tissue culture. Cyto-genetic and biochemical analyses can therefore be used to detect chromosomal aberrations and inborn errors of metabolism in the fetus. 2. The supernatant of amniotic fluid contains many of the solutes typical of extracellular fluid. In particular, it contains a wide range of proteins and those which are of fetal origin are likely to be of use in the prenatal diagnosis of fetal disease. This review examines the nature and origin of the soluble protein in amniotic fluid, and discusses the diagnostic uses of the proteins which are of fetal origin. 3. In other mammals, the arrangement of the fetal membranes is different from that in man, and these differences are reflected by changes in the nature of the amniotic fluid. Thus data from other animals have little applicability to man. 4. Electrophoresis and immunoelectrophoresis have established that the major proteins in amniotic fluid are also present in maternal and fetal sera. Their concentrations in the fluid are influenced by their molecular weight and proteins larger than about 2.5 times 10⁶ may be excluded. Towards term, phenotyping studies show that a number of serum proteins in amniotic fluid are of maternal origin. In the case of group-specific component (Gc) this has been shown to be so throughout pregnancy. Such proteins must enter the fluid by diffusing across either the chorion or the chorionic plate and then the amnion. 5. It has been previously claimed that various serum proteins in amniotic fluid are of fetal origin. For albumin and IgG there are data that strongly support a maternal origin. The evidence on the origin of insulin is inconclusive. The concentration of β₂-microglobulin in amniotic fluid exceeds that in maternal serum and is probably too high also for fetal serum to be its major source. It has a wide tissue distribution and probably enters the fluid from surrounding structures. 6. Alpha-fetoprotein in amniotic fluid is of fetal origin as it is present in maternal serum at far lower concentrations. It is found in fetal serum, urine and yolk sac, but it is not clear how it enters the amniotic fluid of normal fetuses. The concentrations of Gc and alpha-fetoprotein have been measured in amniotic fluid and in their sera of origin. The relative concentration of Gc in amniotic fluid was found to be much greater than that of alpha-fetoprotein and the concentration gradients of these marker proteins can be compared with data for other proteins. In this way further evidence has been obtained that the albumin, α₁,-antitrypsin and transferrin in amniotic fluid are mainly of maternal origin throughout pregnancy. 7. Immunological studies have shown that at least three proteins of non-serum origin are present in amniotic fluid and they have also been located in the amnion and uterine decidua. 8. The enzymes present in amniotic fluid are summarized. Many lysosomal enzymes are clearly of fetal origin since they show altered specific activities in the appropriate cases where the fetus is affected with an inborn error of metabolism. For other enzymes, analysis of specific activity gradients can help to decide the extent to which an enzyme is of serum origin, although this will not exclude the possibility of a maternal (uterine) contribution. The results of such analyses suggest that, relative to the serum protein in amniotic fluid, the greatest concentrations of the minor non-serum proteins in the fluid occurs between thirteen and eighteen weeks of pregnancy and also towards term. 9. Some inborn errors of metabolism may be diagnosed prenatally by measuring the specific activity of the respective enzyme in amniotic fluid. However, the presence of different enzymes with similar substrate specificities has prevented this in Pompe's disease. 10. In cases where the fetus is affected with anencephaly or spina bifida there is an increase in the concentration of alpha-fetoprotein in the amniotic fluid. This has provided a way of detecting these diseases early enough to allow termination of pregnancy. 11. The discovery of new proteins in fetal serum and in the tissues surrounding the amniotic cavity would seem to provide the best chance of extending the uses of amniotic fluid into the other areas of prenatal medicine.     

Evaluation of intra-amniotic surfactant administration for lung maturation in preterm sheep

We aimed to evaluate the effects of intra-amniotic surfactant administration on alveolar lecithin/sphingomyelin ratio, density of type II pneumocytes, and fetal lung function in preterm merino sheep. Pregnant ewes at 119 days gestation either received 200 mg intra-amniotic surfactant (n=4) or saline solution (n=4). After 24 h, the lambs were delivered by hysterotomy and mechanically ventilated. Lecithin/sphingomyelin ratios in alveolar fluid, inflating pressure–volume relationships, and type II pneumocyte counts in histological specimens were compared among the groups. All of the lambs completed the protocol. Mean lecithin/sphingomyelin ratio increased significantly in amniotic (p=0.03) and alveolar fluid (p=0.03) samples of surfactant-treated animals. Lung function in terms of pressure–volume curves did not differ between two groups. Type II pneumocyte density tended to be higher (p=0.057) after intra-amniotic surfactant administration. Single-dose treatment with intra-amniotic surfactant seems to improve amniotic and alveolar lecithin/sphingomyelin ratio questionably by increasing alveolar type II cells. Pressure–volume relationships from inflation of the lungs might be unaltered with intra-amniotic surfactant treatment.     

Bubble Stability Test Compared with Lecithin Assay in Prediction of Respiratory Distress Syndrome

A simple test for surfactant, utilizing bubble stability in ethanol, was performed in 106 samples of amniotic fluid obtained from 94 patients. Of these patients 80 delivered within 48 hours of the collection of the sample. The results were compared with the lecithin concentration in the same amniotic fluid samples and with the quality of respiration in the neonate. The test was “positive,” indicating fetal pulmonary maturity, in 37 cases and none of these infants developed respiratory distress syndrome (R.D.S.). In only one of these cases, however, was gestation less than 37 weeks. The test was “intermediate” or “negative” in 43 cases but in 35 of these infants respiration at birth was perfectly normal.Performed by the method described by its originators, this simple test gives too many false negative results to be of value in routine clinical practice, although a positive result is helpful. The concept of the test is ingenious, however, and further developments may be expected.     

High-performance liquid chromatographic determination of the lecithin/sphingomyelin ratio in amniotic fluid

A high-performance liquid chromatographic (HPLC) procedure has been developed for the separation of phospholipids commonly found in amniotic fluid. The chromatographic separation was achieved on a 25-cm column packed with LiChrosorb DIOL (10 μm). A 3-cm column packed with silica was fitted between the injector and the DIOL column to provide complete separation of lecithin (L) and sphingomyelin (S) from the remaining amniotic fluid phospholipids. The eluted phospholipids were quantitated employing an ultraviolet absorption detector set at 203 nm. The new HPLC separation described herein has improved the resolution and peak sharpness of L and S. Furthermore, phosphatidyl glycerol and phosphatidyl inositol were completely separated and quantitated. Amniotic fluid L/S ratios determined by this technique have been compared to those of an established thin-layer chromatographic procedure.     

Kinetic and metabolic studies of 15-methyl-prostaglandin F2α administered intra-amniotically for induction of abortion

The metabolism of tritium labeled 15-methyl-PGF_2α, administered intra-amniotically, was studied in seven cases of mid-trimester abortion. The disappearance of the compound from the amniotic sac was a very slow process. A metabolite, dinor-15-methyl-PGF_2α, was found in small amounts in the amniotic fluid and also in extracts of placenta and fetal liver, lung, and kidney. Plasma levels of 15-methyl-PGF_2α in the maternal circulation were determined.     

The experimental infection of the amniotic sac of sheep

The amniotic sac of 10 ewes was catheterised at 90–105 days of gestation. In eight ewes a suspension of a β-haemolytic strain of Staphylococcus aureus containing between 3.1 × 10⁸ and 4.4 × 10⁹ colony-forming units was infused 7–16 days after surgery. Two of the ewes were not infused with the test inoculum because fungi were identified in samples of amniotic fluid 13 days after catheterisation.Where possible, daily samples of fluid were withdrawn before abortion occurred (five ewes), hysterotomy was performed (two ewes), and death of the ewe (one ewe). In six of the ewes which were infused with the test organism a partial and temporary inhibition of bacterial multiplication occurred, ranging from 3 to 13 days after inoculation; this was followed by a phase of rapid multiplication and foetal death. It would appear that the amniotic fluid of sheep has a similar inhibitory effect as that previously identified in humans.