Lack of change in the composition of fetal lamb lung surfactant during gestation

Fetal surfactant from lamb lung fluids collected daily from day 114 to day 146 of gestation, was isolated by centrifugation (pellet material) and further purified by sucrose density gradient centrifugation. The concentration of the pellet material from lung fluid (crude surfactant) increased from day 125 till day 135 and fluctuated strongly from that period onwards, whereas lung fluid secretion increased linearly until a few days before parturition. The pellet phospholipid composition changed with gestational age, suggesting biochemical maturation of the surfactant-producing system. The purified surfactant fraction, of which approximately 85% was phosphatidylcholine, did not change however from day 122 onwards except for a small increase in the percentage of phosphatidylglycerol. Alveolar wash surfactant or the lamellar body material, isolated from fetal lungs at different gestational ages had the same composition as surfactant from lung fluids. Only the composition of lamellar bodies of '125 day' lungs differed slightly from that of the lung fluid surfactant. The similar characteristics of all purified surfactant fractions throughout gestation indicate that, in the fetal lamb, lung maturation is associated with an increase in surfactant production no significant changes in phospholipid composition.     

Effects of prenatal ethanol exposure on the lungs of postnatal lambs

Prenatal ethanol exposure increases collagen deposition and alters surfactant protein (SP) expression and immune status in lungs of near-term fetal sheep. Our objectives were to determine 1) whether these prenatal effects of repeated gestational ethanol exposure persist after birth and 2) whether surfactant phospholipid composition is altered following prenatal ethanol exposure. Pregnant ewes were chronically catheterized at 90 days of gestational age (DGA) and given a 1-h daily infusion of ethanol (0.75 g/kg, n = 9) or saline (n = 7) from 95 to 135 DGA; ethanol administration ceased after 135 DGA. Lambs were born naturally at full term (146 ± 0.5 DGA). Lung tissue was examined at 9 wk postnatal age for alterations in structure, SP expression, and inflammation; bronchoalveolar lavage fluid was examined for alterations in surfactant phospholipid composition. At 134 DGA, surfactant phospholipid concentration in amniotic fluid was significantly reduced (P < 0.05) by ethanol exposure, and the composition was altered. In postnatal lambs, there were no significant differences between treatment groups in birth weight, postnatal growth, blood gas parameters, and lung weight, volume, tissue fraction, mean linear intercept, collagen content, proinflammatory cytokine gene expression, and bronchoalveolar lavage fluid surfactant phospholipid composition. Although SP-A, SP-B, and SP-C mRNA levels were not significantly different between treatment groups, SP-D mRNA levels were significantly greater (P < 0.05) in ethanol-treated animals; as SP-D has immunomodulatory roles, innate immunity may be altered. The adverse effects of daily ethanol exposure during late gestation on the fetal lung do not persist to 2 mo after birth, indicating that the developing lung is capable of repair.     

Developmental changes of free amino acids in bovine fetal fluids with gestational age and the interrelationships between the amino acid concentrations in the fluid compartments.

Thirty ninhydrin-positive compounds were determined in the sera, amniotic fluid, stomach content and allantoic fluid from 29 bovine fetuses of various gestational ages. Fetal serum was found to contain about 3-fold higher concentrations of free amino acids (FAA) than maternal serum, and allantoic fluid contained about 3-fold higher concentration of FAA than fetal serum. Decreasing concentrations of FAA were found in serum as a function of the crown-rump length for the amino acids taurine, aspartic acid, threonine, serine, ornithine and lysine. Decreasing concentrations of FAA in allantoic fluid were found for threonine, alanine and ornithine, whereas increasing concentrations were found for phosphoserine and methionine as a function of the crown-rump length. Correlations were found between the concentrations of most amino acids in amniotic fluid and stomach content, but fewer correlations were found between the other fluid compartments. The transport of amino acids between compartments is discussed.     

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.     

Eustachian tube surfactant is different from alveolar surfactant: determination of phospholipid composition of porcine eustachian tube lavage fluid.

Phosphatidylcholine (PC) is the main phospholipid in lung surfactant and, more specifically, dipalmitoyl PC (PC16:0/16:0) is the major surface-active component. Several studies have tentatively shown that eustachian tube lavage fluid (ETLF) contains surface-active material. The aim of the present study was to determine, using electrospray ionization mass spectrometry, whether the phospholipid molecular species composition of ETLF is similar to that of lung surfactant. PC was the main component of both ETLF and bronchoalveolar lavage fluid (BALF). The concentration of phosphatidylethanolamine was higher and phosphatidylglycerol was undetectable in ETLF compared with BALF. The molecular species composition of PC in ETLF was notably different from that of BALF, palmitoyloleoyl PC being the major component. Importantly, given its predominance in BALF PC, the concentration of PC16:0/16:0 was low in ETLF. As expected on the basis of this molecular species composition of PC, ETLF did not generate low surface tension values under dynamic compression in a pulsating bubble surfactometer.We conclude that the surfactant in ET is different from lung surfactant, and that low surface tension is not a major determinant of ETLF function.     

Interaction of plasma apolipoproteins with lipid monolayers

The monolayer technique has been used to study the interaction of lipids with plasma apolipoproteins. Apolipoprotein C-II and C-III from human very low density lipoproteins, apolipoprotein A-I from human high density lipoproteins and arginine-rich protein from swine very low density lipoproteins were studied. The injection of each apoprotein underneath a monolayer of egg phosphatidyl[¹⁴C]choline at 20 mN/m caused an increase in surface pressure to approximately 30 mN/m. With apolipoprotein C-II and apolipoprotein C-III there was a decrease in surface radioactivity indicating that the apoproteins were removing phospholipid from the interface; the removal of phospholipid was specific for apolipoprotein C-II and apolipoprotein C-III. Although there was a removal of phospholipid from the monolayer, the surface pressure remained constant and was due to the accumulation of apoprotein at the interface. The rate of surface radioactivity decrease was a function of protein concentration, required lipid in a fluid state and, of the lipids tested, was specific for phosphatidylcholine. Cholesterol and phosphatidylinositol were not removed from the interface. The addition of 33 mol% cholesterol to the phosphatidylcholine monolayer did not affect the removal of phospholipid by apolipoprotein C-III.The addition of phospholipid liposomes to the subphase greatly facilitated the apolipoprotein C-II-mediated removal of phospholipid from the interface.     

Fetomaternal adrenomedullin levels in diabetic pregnancy.

We investigated whether maternal and fetoplacental adrenomedullin, a newly discovered hypotensive peptide involved in the insulin regulatory system, is modified in diabetic pregnancy. We studied its correlation with pregnancy complications associated with this disease. Thirty-six pregnant women with diabetes (13 with type I and 23 with gestational diabetes mellitus) and in 40 uncomplicated pregnancies were included. 10 out of 36 diabetic pregnancies were complicated by gestational hypertension. In each woman, adrenomedullin concentration in maternal and fetal plasma and in amniotic fluid was assessed by specific radioimmunoassay. We found that overall mean amniotic fluid adrenomedullin concentration was higher (p < 0.05) in diabetic (14.7 +/- 1.6 fmol/ml) than in uncomplicated pregnancies (10.8 +/- 0.9 fmol/ml), whereas no differences were present in maternal and fetal plasma adrenomedullin levels between diabetic and uncomplicated pregnant women. High levels of amniotic fluid adrenomedullin were found in both type I and gestational diabetes mellitus pregnancies (13.7 +/- 1.4 and 15.6 +/- 2.2 fmol/ml, respectively). Diabetic pregnancies complicated by gestational hypertension showed lower (p < 0.05) amniotic fluid adrenomedullin concentrations than normotensive diabetic patients. These findings suggest that placental adrenomedullin production is upregulated in diabetic pregnancy, and it may be important to prevent excessive vasoconstriction of placental vessels.     

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.     

Amniotic fluid levels of tumor necrosis factor-alpha and soluble tumor necrosis factor receptor 1 before and after the onset of labor in normal pregnancies.

Tumor necrosis factor-alpha (TNF-alpha) is present in human placental and uterine cells and promotes the regulation of trophoblast growth and invasion. Tumor necrosis factor receptor 1 (TNF-R1) is a receptor for TNF-alpha, and soluble TNF-R1 (sTNF-R1) is present in amniotic fluid after receptor shedding. We evaluated whether amniotic fluid TNF-alpha and sTNF-R1 levels during labor differed from those before the onset of labor in normal pregnancies. This study enrolled 34 Japanese women experiencing normal pregnancies with single fetuses who had no infection. Of these subjects, 17 went into labor and had subsequent term deliveries (the labor group), and the other 17 underwent cesarean section without labor (the nonlabor group). The average gestational age at entry was 38-39 weeks of gestation. Maternal ages and gestational ages did not differ significantly between the two groups. Amniotic fluid was collected and the TNF-alpha and sTNF-R1 levels were determined by the ELISA method. Each of these levels was compared between the two groups. There was a significant increase in amniotic fluid TNF-alpha levels in the labor group. However, amniotic fluid sTNF-R1 levels did not differ significantly between the two groups. Amniotic fluid TNF-alpha may promote the onset of labor at term and/or term labor contributing to subsequent delivery may induce the production and secretion of TNF-alpha into the amniotic cavity. There was no pregnancy-associated increase in receptor shedding or cell apoptosis at the onset of labor.     

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.     

The origin and fate of hyaluronan in amniotic fluid

The mechanisms which regulate the steady-state concentration and molecular weight of hyaluronan in the amniotic fluid of sheep at different gestational ages have been investigated. An attempt to trace the origin of the polysaccharide has been made by analyses of various fetal fluids (amniotic fluid, allantoic fluid, tracheal fluid, urine, and serum). The fate has been studied by injection of radioactively labelled hyaluronan into the amniotic cavity and following the tracer in fetal tissues and fluids. The concentration of hyaluronan in amniotic fluid varies considerably but is in the order of 5 mg/l at mid-pregnancy and decreases to 1 mg/l in late pregnancy. The polysaccharide has a Mr-distribution with a weight-average in the order of 10(6) at 10 to 13 weeks of gestation which decreases to 10(5) closer to term. Calculations show that urine contributes 0.1 and 0.5 mg of low-molecular (Mr = 10(4) hyaluronan per day in mid- and late pregnancy, respectively, and the lung 10-20% of that amount in the form of high-molecular weight polymer (Mr greater than 10(6). The hyaluronan disappears from the amniotic cavity by bulk flow due to fetal swallowing. It is taken up and degraded in the fetal intestine. Molecules of Mr = 10(3) can pass the intestinal barrier. Calculations show that about 0.5 mg and 1.0 mg of hyaluronan is eliminated per day from the amniotic fluid at 12 and 17 weeks of gestation, respectively. Thus, the higher rate of elimination and the relatively high urinary contribution in more mature fetuses explain the low concentration and Mr of amniotic hyaluronan in late gestation, whereas a slower elimination combined with a relatively larger contribution of high molecular weight hyaluronan both from lung and urine and possibly from other sources are responsible for the higher concentration and Mr of the compound in early pregnancy.     

Enzyme-histochemically detectable glucose-6-phosphatase is present in chorion laeve trophoblasts of human fetal membranes

The purpose of the present study was to demonstrate the presence of glucose-6-phosphatase (G6Pase) in fetal membranes from various gestational ages (20-40 weeks of gestation). Ultrastructural enzyme-histochemical analysis of G6Pase was performed using cerium and lead as capturing agents. Precipitates indicating G6Pase activity were present mainly in the endoplasmic reticulum and partly in the nuclear envelope of chorion laeve trophoblasts, but absent in amniotic epithelial cells. Stringent histochemical control experiments performed ensured specific detection of G6Pase activity. The results indicate that histochemically detectable G6Pase is present in the chorion laeve trophoblasts of human fetal membranes. This enzyme may have some physiological significance in carbohydrate metabolism in human fetal membranes and regulation of amniotic fluid glucose concentration.     

Alterations in biliary lipids of mice during dehydrocholic acid feeding

Mice were fed a lithogenic diet consisting of Purina chow and 0.5% dehydrocholic acid (DHA group). Controls received Purina chow. Every 2 wk for 20 wk animals were killed, and biliary phospholipid, cholesterol, and bile salt concentrations were determined, as well as the extent of gallstone formation. With time there was a gradual, significant decline in the concentration and the relative composition of phospholipid in both groups compared with initial values. There was a significant increase in biliary cholesterol concentration and relative amount in the DHA group compared with the control. No significant differences were found in the relative amounts of bile salt or phospholipid between the two groups. Feeding DHA resulted in an increased concentration of bile salts and the sum of measured lipid compared with controls. After 8 wk, gallstones were found in approximately 60% of autopsied animals and correlated with increased cholesterol concentration. Our data support the hypothesis that there is a component of cholesterol secretion that may not be bile salt- or phospholipid-dependent. Our data also suggest that biliary phospholipid secretion decreases with age.     

Sodium dodecyl sulfate-disc gel electrophoresis patterns of bovine lung surfactant

Bovine lung surfactant material, isolated by three steps of centrifugation from lung washings, had a phospholipid: protein ration of 4.98+/-S.D. 0.62 mumol/mg. Sucrose continuous density gradient centrifugation showed only one visible band at a density of 1.081 g/ml. Sodium dodecyl sulfate-disc gel electrophoresis patterns of the apoprotein extracted from this material revealed that the main component (70-80%) of the protein subunits of bovine lung surfactant material has a molecular weight of 36 000. It was suggested that the apoprotein of bovine lung surfactant material exists as a combination of a few protein subunits, most of which have a molecular weight of 36 000.     

Nanosecond time-resolved emission anisotropy of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in human amniotic fluid

The fluorescence emission anisotropy of 1,6-diphenyl-1,3,5-hexatriene in human amniotic fluid has been studied using nanosecond time-resolved emission techniques. These studies demonstrate that the previously reported decrease in the steady state emission anisotropy, 〈r〉, with gestational age is due to a change in the rate of rotational motion of the probe. The emission anisotropy decays to a limiting value (r_∞) greater than zero, suggesting a hindered rotation of the probe, and this is independent of gestational age. The decay function for the emission anisotropy of amniotic fluids from 17, 29, 40 and 41 weeks in gestational age can be best expressed as a single exponential plus a constant term, with rotational correlation times varying from 17 ns to 2.2 ns, respectively. The zero time emission anisotropy remains approx. 0.30 for both early and late gestational times.     

Prenatal diagnosis of sex in bovine fetuses by amniocentesis

The sex of 1, 056 bovine fetuses was diagnosed by cytogenetic analysis of amniotic cells collected surgically from cows made pregnant as a result of embryo transfer. The fetuses ranged in gestational age from 7 to 22 wk at the time of amniocentesis. Amniotic cells were cultured for 5 to 30 d to obtain a sufficient number of cells for cytogenetic analysis. The growth rate of 819 samples was examined in detail. On average, amniotic cells from pregnancies that were 7 or 8 wks old required about 13 d to reach a cell concentration sufficient for analysis, whereas those from pregnancies that were 10 wk old or more required only 10 d or less to reach that concentration. Final disposition of 325 pregnancies subjected to amniocentesis was unequivocally confirmed. Of these, the sex of 302 fetuses (93%) was determined with a male: female ratio of 51:49. The gender of 213 of 220 fetuses (96.8%) was correctly dignosed, as verified by examination of either 33 intentionally induced abortuses or of 187 live calves. Thirteen percent (29) of 216 pregnancies that were allowed to proceed to term ended in spontaneous abortion, a rate only about 5% higher than that reported to occur normally in embryo transfer pregnancies. The remaining 53 pregnancies were induced to abort, but it was not possible to recover and verify the sex of those fetuses. The capability to diagnose fetal sex in utero yielded other useful information as well. For example, the sex ratio of 1,056 fetuses during development from 7 to 15+ wk of gestation was found to be the same (51:49) as it was at calving. Finally, amniocentesis to determine prenatal sex provides time to alter the sex ratio of a calf crop. It may ultimately prove valuable as an adjunct to genetic engineering of cattle to identify presumptive transgenic calves in utero.     

Comparison of the specific binding of cortisol in human amnion, amniotic fluid, and plasma

The purpose of this study was to compare the specific cortisol-binding protein found associated with human amnion with specific cortisol binding in human amniotic fluid and plasma. The electrophoretic mobility on polyacrylamide gels of the specific cortisol binding in amnion, amniotic fluid, and maternal plasma was identical. The influence of pH on cortisol binding activity was similar in all tissues and the cortisol binding was immunoprecipitable by a polyclonal antibody raised against human corticosteroid-binding globulin. The interaction of the cortisol binding protein with concanavalin A was studied in preterm amniotic fluid, term amniotic fluid, term amnion, and plasma from pregnant women at term and women under oral contraceptive treatment. Binding to concanavalin A was similar in term amnion and term amniotic fluid but was less than that found with both preterm amniotic fluid and term plasma. These results indicate that the cortisol binding protein associated with human amnion has similar characteristics to plasma corticosteroid-binding globulin, but that its state of glycosylation appears to be more like that of the cortisol binding protein in term amniotic fluid rather than in plasma.     

Appearance of a brain specific antigen (th S-100 protein) during human foetal development

—The concentration of a protein specific to brain, the S-100 protein, was measured in various regions of the human foetal brain at gestational ages ranging from 10 weeks until term. The relative increase in concentrations of the S-100 protein during development of the human foetal brain proceeded in a caudal-rostral fashion. This observation is emphasized by the delayed appearance of the S-100 protein in the frontal cerebral cortex until the 30th week of gestation.     

Intestinal and renal origin of trehalase activity in rabbit amniotic fluid

To utilize specific fetal markers in amniotic fluid for prenatal detection of fetal anomalies, it is necessary to determine the precise tissue origin of these markers. In rabbit fetuses, we distinguished between intestinal and renal forms of trehalase (alpha,alpha'-trehalose-1-D-glucohydrolase, EC 3.2.1.28) in amniotic fluid on the basis of differences in net electric charges. Trehalase was solubilized from purified brush-border membranes of fetal rabbit kidney and intestine by Triton X-100 treatment, whereas the trehalase activity in amniotic fluid was soluble. The kinetic properties of trehalase from intestine, kidney and amniotic fluid were very similar. The Mr of the soluble amniotic fluid trehalase was between 72,600 and 66,300 from hydrodynamic parameters, depending on the amount of sugar bound to the enzyme, and 48,500 by radiation inactivation, a method which detects only the protein part of the enzyme. For membrane-bound trehalase from kidney and intestine in situ the radiation inactivation method also gave a molecular size of around 49,000. Isoelectric focusing of freshly solubilized membranes allowed us to distinguish between renal and intestinal forms of trehalase in rabbit fetuses on the basis of different isoelectric points. Each trehalase form was also present in the amniotic fluid but in varying proportions depending on the gestational age at which the amniotic fluid was collected. The results suggest that early in gestation amniotic fluid trehalase activity originates exclusively from the fetal kidney but that more and more intestinal enzyme is released into the amniotic cavity as the fetus develops. Similar results were also obtained when ion-exchange chromatography was used to separate the various trehalase forms. The development of trehalase activity in rabbit fetal kidney and intestine correlates well with its occurrence in the amniotic fluid; trehalase activity in the kidney develops early in gestation whereas the intestinal trehalase activity develops just before term.