Prenatal diagnosis of propionic acidemia by measuring methylcitric acid in dried amniotic fluid on filter paper using GC/MS

Propionic acidemia is a frequent inborn error of metabolism. Methylcitric acid, a key indicator of propionic acidemia, increases in the amniotic fluid of affected fetuses. For prenatal diagnosis, the methylcitric acid in amniotic fluid can be measured by stable-isotope dilution GC/MS. Here, we quantified this indicator in samples of amniotic fluid that had been dried on filter paper and transported at ambient temperatures, and compared the results with data obtained from the original amniotic fluid. We then used the filter-paper method to screen at-risk fetuses and obtained a clear-cut diagnosis in each case.     

Studies on enzymes of nitrogen metabolism, and nitrogen end products in the developing chick (Gallus domesticus) embryo.

1. A total of 450 fertilized eggs were used to study the concentrations of uric acid, urea and ammonia in allantoic and amniotic fluids, and some enzymes of nitrogen metabolism in the liver and kidney during the development of the chick embryo from the 5th to 21st day of incubation. 2. Concentrations of the compounds studied were higher in allantoic fluid. The molar concentration of allantoic uric acid increased steadily with time. The pattern of urea and ammonia in both allantoic and amniotic fluids were the same. 3. Arginase (E.C.3.5.3.1) activity in both embryonic kidney and definitive kidney was higher than that in the liver. The specific activity of arginase (mumole urea formed/hr per g wet wt kidney) dropped during development. 4. Little arginine synthetase activity (argininosuccinate synthetase, E.C.6.3.4.5; and argininosuccinate lyase, E.C.4.3.2.1) was found in kidney, but none in the liver. 5. The complete urea cycle function was absent in both the liver and the kidney of the chick embryo.     

Endotoxins do not influence transplacental transmission of lymphotropic human herpesviruses and human papillomaviruses into amniotic fluid taken from healthy mothers before parturition in Hungary

Pregnant women were examined following healthy pregnancies at term. Amniotic fluids were sampled before arteficial rupture of membranes using closed vacutainer system. Blood samples were also taken from the pregnants simultaneously. Endotoxin concentrations of amniotic fluids were tested by the semiquantitative Limulus amebocyte lysate. Both amniotic fluids and blood samples were tested for the presence of DNA of lymphotropic human herpesviruses. The DNA of human papillomaviruses were tested only in the amniotic fluid samples. One-third of the amniotic fluids tested were found to contain measurable amounts of endotoxin. Lymphotropic herpesvirus DNA was deteced in every fourth amniotic fluid sample and in every 8th blood sample. The prevalence of papillomaviruses was 7 of 96 samples. No significant correlation was found between the presence of endotoxin and viruses in the amniotic fluids. Epstein-Barr virus, human cytomegalovirus and human herpesvirus type 7 were found more frequently in the amniotic fluids than in blood samples (7 to 1). The prevalence of human herpesvirus 6 and 8 was higher in the blood samples than that in the amniotic fluids. The mean weight of the neonates were not impaired significantly by the presence of either viruses or endotoxin. Possible post partum consequences, i.e. partial immunotolerance to viruses is discussed.     

Identification and quantitation of human amniotic fluid components using capillary zone electrophoresis

A capillary electrophoresis method was used to measure albumin, immunoglobulin G (IgG), transferrin, and uric acid in 230 amniotic fluid (AF) samples collected at 15.15 ± 0.06 weeks gestation. Species were quantified by external calibration using thiamine as internal standard. All major components were detected within 10 min. Migration time reproducibility was 3.0% relative standard deviation (RSD) and normalized peak areas were 12% RSD or better at 190 nm from 81 measurements of a pooled AF sample. The separation profile was not affected by 10 h of storage at room temperature or by 10 freeze-thaw cycles, suggesting that frozen AF samples are suitable for protein biomarker studies.     

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.     

Identification of biologic markers of the premature rupture of fetal membranes: proteomic approach

In obstetrics, premature rupture of the membranes (PROM) is a frequent observation which is responsible for many premature deliveries. PROM is also associated with an increased risk of fetal and maternal infections. Early diagnosis is mandatory in order to decrease such complications. Despite that current biological tests allowing the diagnosis of PROM are both sensitive and specific, contamination of the samples by maternal blood can induce false positive results. Therefore, in order to identify new potential markers of PROM (present only in amniotic blood, and absent in maternal blood), proteomic studies were undertaken on samples collected from six women at terms (pairs of maternal plasma and amniotic fluid) as well as on four samples of amniotic fluid collected from other women at the 17(th) week of gestation. All samples (N = 16) were analyzed by two-dimensional (2-D) high-resolution electrophoresis, followed by sensitive silver staining. The gel images were studied using bioinformatic tools. Analyses were focused on regions corresponding to pI between 4.5 and 7 and to molecular masses between 20 and 50 kDa. In this area, 646 +/- 113 spots were detected, and 27 spots appeared to be present on the gels of amniotic fluid, but were absent on those of maternal plasma. Nine out of these 27 spots were also observed on the gels of the four samples of amniotic fluids collected at the 17(th) week of pregnancy. Five of these 9 spots were unambiguously detected on preparative 2-D gels stained by Coomassie blue, and were identified by mass spectrometry analyses. Three spots corresponded to fragments of plasma proteins, and 2 appeared to be fragments of proteins not known to be present in plasma. These 2 proteins were agrin (SWISS-PROT: O00468) and perlecan (SWISS-PROT: P98160). Our results show that proteomics is a valuable approach to identify new potential biological markers for future PROM diagnosis.     

Depletion of urate in human nasal lavage following in vitro ozone exposure

Ozone, a strong oxidant present in summer smog, is thought to primarily react with antioxidant molecules found in the epithelial lining fluid of the respiratory tract. In humans, as much as 40% of inhaled ozone can be removed in the nasal cavity where the major extracellular antioxidant has been identified as uric acid. The present study was undertaken to examine urate/oxidant interactions in human nasal lavage fluid following in vitro exposure to ozone at concentrations relevant to the U.K. Lavage fluid was collected from 8 volunteers using a modified Foley catheter which permits prolonged contact of isotonic saline with the anterior nasal cavity. Nasal lavage samples in multiwell plates were exposed to ozone at concentrations of 50, 100 and 250 ppb. Samples were removed at intervals from 15 to 240 min following exposure and assayed for uric acid depletion. Uric acid concentrations in the nasal lavage were found to fall from 8.52 (time zero) to 3.99 μM, 0.05 and 0.07 μM after 240 min at 50, 100 and 250 ppb ozone respectively. At a non-environmentally relevant ozone concentration of 1000 ppb, uric acid was completely depleted after 60 min. Regression analysis showed a linear correlation between rate of loss of urate and ozone concentration (R² = 0.97). A novel, non-invasive technique is described to investigate antioxidant compromise and its importance in individual subjects. We conclude that uric acid in nasal lavage samples is scavenged by ozone in a dose and time dependant manner.     

Amniotic Fluid Cells; Prenatal Sex Prediction and Culture

Sex chromatin studies were carried out on small amounts of amniotic fluid obtained by amniocentesis or from intact amniotic sacs removed at hysterotomy. Provided that satisfactory preparations were obtained the accuracy of fetal sexing was 87%. Nevertheless, in the management of a pregnancy in which there is a risk of a serious X-linked recessive disorder, repeat amniocentesis may be necessary to ensure satisfactory specimens.Of 90 samples of fluid cultured, satisfactory growth was obtained in 49; the success rate was not increased by the addition of stimulants to the culture medium. It is suggested that between the 13th and the 16th week of pregnancy is the optimum time for amniocentesis to obtain cells for culture, since sufficient cells are then present in a small volume of fluid and therapeutic abortion would still be possible once the results were available.     

Amniotic fluid beta-2 microglobulin in normal and complicated pregnancies

Beta-2 microglobulin concentrations were measured in amniotic fluid samples obtained from normal pregnant women at various stages of gestation and complicated pregnancies during weeks 32-42 of gestation by the ELISA method. The concentration of beta-2 microglobulin in amniotic fluid increases markedly up to the 20-24th weeks of pregnancy and reaches a peak during the second trimester, occasionally reaching an eightfold value compared to the maternal serum concentration, while at term the values are similar. The decrease of amniotic fluid beta-2 microglobulin level in the third trimester reflects the maturation of foetal renal tubular function and suggests that this test may be of significance in determining foetal age. Our results revealing elevated concentrations of beta-2 microglobulin in patients with diabetes, toxaemia and placental insufficiency may indicate slower renal maturation of the foetus.     

Variation in some enzymes in amniotic fluid and maternal serum during pregnancy

The following 10 enzymes were assayed in 187 amniotic fluid and maternal serum samples at 15-42 weeks of gestation: alkaline phosphatase, heat-stable alkaline phosphatase (only in amniotic fluid), acid phosphatase, alanine aminotransferase, aspartate aminotransferase, alpha-amylase, gamma-glutamyltransferase, creatine kinase, lactate dehydrogenase, and lysozyme. The normal reference ranges are reported for amniotic fluid and maternal serum enzymes, together with the abnormal values accompanying neural tube defects and EPH-gestosis. The determination of gamma-glutamyltransferase, heat-stable alkaline phosphatase and creatine kinase was found to be of appreciable diagnostic significance in clinical practice.     

Catecholamines and DOPA in the amniotic fluid and amnion of the chick embryo

Identification and quantitative fluorimetric assay have been made on the content of DOPA, dopamine, noradrenaline and adrenaline in the amniotic fluid and amnion of the developing chick embryos. Significant increase in the content of DOPA, noradrenaline and adrenaline in the amniotic fluid was observed between the 6th and the 13th days of incubation; dopamine content sharply decreases at the 13th day. The content on amines in the amnion tissue remained essentially constant throughout the investigated period. The role of catecholamine in amniotic fluid in regulation of contractile activity of amniotic membrane in the developing chick embryo is discussed.     

Glial origin of rapidly adhering amniotic fluid cells.

Rapidly adhering cells (RA cells) from the amniotic fluid of a pregnancy with fetal anencephaly were investigated by immunofluorescence assay with an antiserum against glial cells. After 24 hours'' cultivation a high proportion of the cells showed positive glial-specific fluorescence, whereas no staining was seen in cells from samples of normal amniotic fluid. At the 24th week the mother was delivered of a stillborn infant with anencephaly. Immunofluorescence staining of RA cells with glial-specific antiserum may be used for the differential diagnosis of fetal abnormalities associated with a high alpha-fetoprotein concentration in amniotic fluid.     

Metabolic response to starvation in late pregnant rats. II. Fetal response

Effect of prolonged maternal fasting on the fetal liver and heart glycogen and triglyceride content and on concentration of glucose, urea, uric acid and alpha amino-nitrogen in the amniotic fluid has been studied in rats. The animals were divided into four groups: fed (control), fasted for one day (from 20 to 21 day of pregnancy), fasted for two days (from 19 to 21 day) and fasted for three days (from 18 to 21 day). Maternal fasting for two and three days resulted in reduction in fetal growth. The fetal liver glycogen content was reduced already after one day of fasting, stabilized after two days and then further decreased after three days. The fetal heart glycogen content was reduced only after three days of fasting. The fetal liver triglyceride content increased gradually during the first two days of fasting and then stabilized. The content of triglycerides in the heart was elevated after two and three days of food deprivation. The amniotic fluid glucose concentration decreased after one day of fasting and then stabilized. Fasting did not effect the concentration of the nitrogenous compounds in the amniotic fluid. It is concluded that maternal fasting affects markedly metabolism of energy substrates stored in the fetal liver and the heart and the composition of the amniotic fluid.     

Glycosaminoglycan composition of human amniotic fluid

The glycosaminoglycan composition of human amniotic fluid between 12–21 weeks gestation has been studied by Dowex column chromatography coupled with enzymatic analyses of the specific glycosaminoglycan in each column fraction. The total uronic acid recovered from the columns consisted of “glycopeptides” (7%), hyaluronic acid (34%), nonsulfated chondroitin (14%), chondroitin-4-sulfate (13%), chondroitin-6-sulfate (20%), dermatan sulfate (5%), and heparan sulfate (6%). Based on these studies a simple screening procedure was devised to detect increased quantities of heparan sulfate and dermatan sulfate in 5–10-ml samples of amniotic fluid and tested in the antenatal diagnosis of Hurler and Hunter's syndrome. A false negative result was recorded in a Hunter fluid obtained early gestation and a false positive result recorded in a normal fluid obtained at weeks. These data suggest that the time in gestation when amniotic fluid is sampled for chemical analysis is an important variable affecting glycosaminoglycan composition in both normal and pathological pregnancies.     

Standardization criteria for the study of fetal lung maturity by means of gas-liquid chromatography of the fatty acid content of the amniotic fluid]

Gas-chromatographic analysis of the fatty acids (P/S ratio) in 10 samples of amniotic fluid and 10 samples of the pellets obtained after centrifugation of amniotic fluid at 3500 X g for 60 minutes were carried out to evaluate the effects of contaminants that might be present in amniotic fluid. The P/S ratio is used as an index of the degree of maturity of the fetal or neonatal lung. We propose a standard procedure of centrifugation for 60 minutes at 3500 X g followed by extraction and gas-chromatography as a rapid, valid way to measure the P/S ratio.     

Soluble Leukocyte-Associated Ig-Like Receptor-1 in Amniotic Fluid Is of Fetal Origin and Positively Associates with Lung Compliance

The soluble form of the inhibitory immune receptor leukocyte-Associated Ig-like Receptor-1 (sLAIR-1) is present in plasma, urine and synovial fluid and correlates to inflammation. We and others previously showed inflammatory protein expression in normal amniotic fluid at term. We hypothesized that sLAIR-1 is present in amniotic fluid during term parturition and is related to fetal lung function development. sLAIR-1 was detectable in all amniotic fluid samples (n=355) collected during term spontaneous deliveries. First, potential intra-uterine origins of amniotic fluid sLAIR-1 were explored. Although LAIR-1 was expressed on the surface of amniotic fluid neutrophils, LAIR-1 was not secreted upon ex vivo neutrophil stimulation with LPS, or PMA/ionomycin. Cord blood concentrations of sLAIR-1 were fourfold lower than and not related to amniotic fluid concentrations and placentas showed no or only sporadic LAIR-1 positive cells. Similarly, in post-mortem lung tissue of term neonates that died of non-pulmonary disorders LAIR-1 positive cells were absent or only sporadically present. In fetal urine samples, however, sLAIR-1 levels were even higher than in amniotic fluid and correlated with amniotic fluid sLAIR-1 concentrations. Second, the potential relevance of amniotic fluid sLAIR-1 was studied. sLAIR-1 concentrations had low correlation to amniotic fluid cytokines. We measured neonatal lung function in a convenient subset of 152 infants, using the single occlusion technique, at a median age of 34 days (IQR 30-39). The amniotic fluid concentration of sLAIR-1 was independently correlated to airway compliance (ρ=0.29, P=.001). Taken together, we show the consistent presence of sLAIR-1 in amniotic fluid, which originates from fetal urine. Concentrations of sLAIR-1 in amniotic fluid during term deliveries are independent from levels of other soluble immune mediators. The positive association between concentrations of amniotic fluid sLAIR-1 and neonatal lung compliance suggests that amniotic fluid sLAIR-1 may be useful as a novel independent marker of neonatal lung maturation.     

Identification of phospholipid platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in human amniotic fluid and urine

Human amniotic fluid and fetal urine were examined for the presence of phospholipid platelet-activating factor (PAF). PAF was detected in lipid extracts of some samples of amniotic fluid obtained from women in labor but it was undetectable in samples of amniotic fluid obtained before the onset of labor. PAF was identified by chromatographic mobility, platelet aggregation and chemical modifications. LysoPAF was also present in amniotic fluid at higher concentrations than those of PAF. Both PAF and lysoPAF were identified also in newborn and adult urine.     

Amniotic fluid testosterone and testosterone glucuronide levels in the determination of foetal sex

Unconjugated testosterone levels were assayed in 351 amniotic fluid samples obtained at 15-19 weeks gestation. The median values for unconjugated testosterone in the 166 female foetuses and 185 male foetuses were 137 and 712 pmol/l respectively. Sixteen amniotic fluid samples from male foetuses had unconjugated testosterone levels lower than the highest female unconjugated testosterone value (361 pmol/l). Testosterone glucuronide was measured in amniotic fluid from 48 female and 55 male foetuses. There was a significant sex difference in the median values of testosterone glucuronide between female (median 160 pmol/l, range 64-465 pmol/l) and male (median 817 pmol/l, range 68-3707 pmol/l) amniotic fluid specimens (P less than 0.001). Of the sixteen male foetuses with amniotic fluid unconjugated testosterone levels in the female range, 12 had amniotic fluid testosterone glucuronide levels within the male testosterone glucuronide range of values. Hence used in conjunction with unconjugated testosterone, testosterone glucuronide increased the predictive accuracy of foetal sexing from 95.4 to 98.9%. Testosterone sulphate was measured in 24 female and 25 male amniotic fluid samples. There was no Testosterone sulphate was measured in 24 female and 25 male amniotic fluid samples. There was no significant difference between female (median 2591 pmol/l) and male (median 2964 pmol/l) testosterone sulphate levels.     

Maternal cell contamination in amniotic fluid samples as a consequence of the sampling technique

Maternal cell contamination in amniotic fluid samples is easily detected by in situ hybridization if the karyotype of the fetus differs from the karyotype of the mother. One out of two amniotic fluid samples appears to contain more than 20% maternal cells. Bloody samples often contain even more than 50% maternal cells. Maternal cells can also be identified on the basis of their nuclear morphology. Maternal cell contamination is regularly observed in pregnancies with an anterior placenta, whereas it is rare in posterior placenta pregnancies. The maternal cells are therefore thought to be artificially introduced into the amniotic fluid sample, as a result of placental bleeding during amniocentesis. The implications of maternal cell contamination for prenatal diagnosis using uncultured amniotic fluid samples are discussed.     

Antenatal diagnosis of glutaric acidemia

Two pregnancies at risk for glutaric acidemia were monitored. In one, in which the fetus was not affected, glutaric acid was not detected in the amniotic fluid at amniocentesis (15 weeks) and the glutaryl-CoA dehydrogenase activity of cultured amniotic cells was normal. In the other, a marked elevation of glutaric acid in the amniotic fluid, together with deficiency of glutaryl-CoA dehydrogenase in amniotic cells, prompted termination of the pregnancy, and studies on the abortus confirmed the diagnosis of glutaric acidemia. Glutaric acidemia, is, thus, another inborn error of metabolism which can be diagnosed in utero.