Enzymes In Amniotic Fluid Research Articles

Outcome of non-visualization of fetal gallbladder on second-trimester ultrasound: cohort study and systematic review of literature.

To investigate the ultrasound characteristics and outcome of fetuses with non-visualization of the fetal gallbladder (NVFGB) followed in our tertiary university hospital, and to provide a comprehensive review of the literature on prenatal findings and outcome of NVFGB. NVFGB was defined as non-visualization of the gallbladder on two targeted ultrasound examinations performed within a 1-week period. First, we reviewed the medical records of NVFGB cases managed in our center over a 9-year period. Then, we performed a systematic review of the literature to identify studies on NVFGB. The incidence of chromosomal anomalies, later visualization of the gallbladder, gallbladder agenesis, cystic fibrosis and biliary atresia was assessed in fetuses with isolated and non-isolated NVFGB. The role of hepatic enzyme measurements in the diagnosis of cystic fibrosis and biliary atresia in fetuses with NVFGB was also reviewed. Sixteen cases of NVFGB were followed in our center, in 10 (62.5%) of which it was an isolated finding. The incidence of biliary atresia was 12.5% and that of gallbladder agenesis was 12.5%, while no case of cystic fibrosis was reported. The gallbladder was visualized later in pregnancy or postnatally in 43.8% and 25.0% of cases, respectively. A total of seven studies, including our cohort, involving a total of 280 NVFGB cases, met the inclusion criteria for the systematic review. Overall, 20.5% of fetuses had an associated ultrasound anomaly, and the incidence of chromosomal anomaly in this group was 20.4%. In cases with isolated NVFGB, the incidence of chromosomal anomaly was 1.9%. In fetuses with normal karyotype and isolated NVFGB, the gallbladder was later visualized in 70.4% of cases, while the incidence of gallbladder agenesis, cystic fibrosis and biliary atresia was 25.2%, 3.1% and 4.8%, respectively. In fetuses with non-isolated NVFGB, the incidence of cystic fibrosis and biliary atresia was 23.1% and 18.2%, respectively. The negative predictive value of amniotic fluid enzyme levels for the prediction of severe disease (including biliary atresia or cystic fibrosis) ranged between 94% and 100% when evaluated before 22 weeks' gestation, and dropped to 88% after 22 weeks. In cases with persistent NVFGB, the risk of a severe postnatal condition should be considered. A detailed ultrasound scan should be offered and parents tested for cystic fibrosis gene mutation. An invasive procedure for karyotyping and measurement of liver enzyme concentrations before 22 weeks constitutes a reasonable work-up. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

OP33.07: Evaluation and outcome of prenatal non visualization of gallbladder

Objectives: Nonvisualization of the fetal gallbladder on prenatal ultrasound in the second trimester is uncommon. In most cases, the gallbladder will eventually be detected. Associations with cystic fibrosis, aneuploidy, agenesis of the gallbladder and biliary atresia have been reported. We present our experience and review the literature. Methods: Cases of nonvisualization of the gallbladder were confirmed by 2 examiners on at least 2 occasions and at least a week apart from 18–22 wks. Cases with no additional sonographic malformations were designated as isolated. Further evaluation included follow up scans and a meticulous search for fetal anomalies. All were offered genetic consultation. Cystic fibrosis testing and amniocentesis for karyotype and digestive enzymes in amniotic fluid were performed at the discretion of the referring physician. Results: A total of 20 cases of nonvisualization of the gallbladder were detected. Of these, 5 had additional malformations. In 4 of these cases the associated anomalies were severe, and fetuses were aborted due to aneuploidy or the severity of the associated anomalies. Of the remaining 15 cases, one was aborted due to cystic fibrosis. In all the other 14 cases development was normal with follow up of 4 months to 4 years. Conclusions: When prenatal nonvisualization of the fetal gallbladder is associated with other severe malformation, aneuploidy should be suspected. When isolated, if cystic fibrosis is ruled out, the outcome is good.

P51.09: Amniotic fluid enzyme profile and immunohistochemistry in MMIHS

P51.09: Amniotic fluid enzyme profile and immunohistochemistry in MMIHS

Establishment of reference values of five amniotic fluid enzymes. Analytical performances of the Hitachi 911. Application to complicated pregnancies

Objectives: to evaluate the analytical performances of the Hitachi 911 analyzer (Roche Diagnostics, F) for γ-glutamyl-transferase, 5′nucleotidase, amylase, aspartate-amino-transferase and total-alkaline-phosphatase assay in amniotic fluid. To establish reference intervals for these five enzymes throughout gestation. To determine their antenatal diagnostic value. Design and methods: amniotic fluid samples were collected between 14 and 35 weeks of gestation. Weekly numbers ranged from 31 to 92. Techniques developed for serum enzyme assays were applied to amniotic fluid. Two pools of amniotic fluid containing low and high marker levels were used. Within-day and between-day variations were calculated, together with the limits of detection and linearity. Reference ranges were established on 508 amniotic fluid samples, including 23 samples from pregnancies with chromosomal aberrations, 14 with gastrointestinal tract defects and 5 with gastroschisis. Results: the assay technique for total-alkaline-phosphatase assay had to be adapted to amniotic fluid, but no adaptation was necessary for the other markers. The within-day CV ranged between 2.2 and 11.2% for low-level samples and from 1.1 to 3.4% for high-level samples. The between-day CV ranged from 6.3 to 13.3% for low-level samples and 1.2 to 4.7% for high-level samples. Total-alkaline-phosphatase activity fluctuated throughout gestation. Amylase levels and aspartate-amino-transferase levels increased whereas γ-glutamyl-transferase and 5′nucleotidase levels fell until delivery. All trisomy 18 and trisomy 13 pregnancies, 65% of Down’s syndrome pregnancies and all pregnancies with digestive tract defects were associated with marked changes in the level of at least one enzyme. Conclusion: The Hitachi 911 is suited to rapid, reliable quantification of amniotic fluid enzymes with only minor adaptation. Useful reference intervals can be obtained throughout gestation. Gamma-glutamyl-transferase, 5′nucleotidase, total-alkaline-phosphatase and amylase assay can help to confirm echographic evidence of bowel disorders and thereby improve patient management especially in case of gastroschisis.

Prenatal diagnosis of lysosomal storage diseases.

The prenatal diagnosis of lysosomal storage disorders can be achieved, once the diagnosis is confirmed in the index case, by a variety of techniques including analysis of amniotic fluid, asay of enzymic activity in cultured amniotic fluid cells, cultured chorionic villus cells and by direct assay of activity in chorionic villus samples. These studies can be accompanied by ultrastructural observations which give an independent means of diagnosis. In some instances molecular genetic studies for mutation detection or linkage analysis are appropriate for prenatal diagnosis. Pseudodeficiencies of some of the lysosomal enzymes, which cause no clinical problems, can complicate the initial diagnosis particularly in metachromatic leucodystrophy where the pseudodeficiency is more common than the disease itself. Mutation analysis as well as enzyme assay is necessary not only in the index case but also in the parents before the same techniques are applied to a sample for prenatal diagnosis. A large number of lysosomal storage disorders may present as fetal hydrops and the diagnosis can be established at this late stage by fetal blood sampling and examination by microscopy as well as by biochemical assay of the appropriate enzyme or metabolite in amniotic fluid. All prenatal diagnoses in which an affected fetus is indicated should have confirmation of the diagnosis as soon as possible to reassure anxious parents, and to act as audit of the laboratory's competence to undertake prenatal diagnosis. A combined approach to prenatal diagnosis involving biochemical, molecular genetic and morphological studies is recommended.

Use of amniotic fluid amino acids in prenatal testing for argininosuccinic aciduria and citrullinaemia.

Prenatal testing of 12 pregnancies at risk for argininosuccinic aciduria due to argininosuccinate lyase (ASAL) deficiency and three pregnancies at risk for citrullinaemia due to argininosuccinate synthatase (ASAS) deficiency was performed by metabolite detection in amniotic fluid and measurement of enzyme activity in uncultured and cultured chorionic tissue and in cultured amniocytes. From our data and those of previous studies, amniotic fluid argininosuccinate measurement alone is clearly a reliable and rapid diagnostic test for both severe and mild ASAL deficiency if maternal ASAL deficiency can be excluded. For prenatal diagnosis of ASAS deficiency, however, both measurement of the amniotic fluid citrulline level and enzyme assay should be employed.

Low molecular mass proteins and urinary enzymes in amniotic fluid of healthy pregnant women at progressive stages of gestation

Objectives: Amniotic fluid α 1-microglobulin ( α 1-m) and β 2-microglobulin ( β 2-m) levels, as well as N-acetyl-β-D-glucosaminidase (NAG) and alanine aminopeptidase (AAP) activities, were measured in the course of uncomplicated pregnancies to assess their variations as gestation progresses. Design and Methods: Samples were obtained from 141 healthy pregnant women divided into three groups on the basis of gestational stage. Quantitative estimation of proteins was performed immunometrically and enzyme activities were determined spectrophotometrically. Results: It was found that, during pregnancy, α 1-m and β 2-m concentrations as well as AAP activity significantly decrease, although their reduction patterns vary. Controversial results were found for NAG activity: the normalization of values for amniotic fluid creatinine significantly changed the reduction pattern of this enzyme. No statistically significant differences were found between male and female fetuses for amniotic fluid values of the biochemical substances studied. Conclusions: The behavior observed for α 1-m, β 2-m, and AAP might be linked to the progressive development and maturation of fetal renal tubular function. Amniotic fluid total NAG activity seems not to depend only on fetal urinary excretion.

Electrolytes and Biochemical Enzymes in Amniotic and Allantoic Fluid of the Equine Fetus during Late Gestation

Electrolytes and Biochemical Enzymes in Amniotic and Allantoic Fluid of the Equine Fetus during Late Gestation

Amniotic fluid digestive enzymes: Diagnostic value in fetal gastrointestinal obstructions

The diagnostic value of amniotic fluid gamma-glutamyl-transpeptidase (GGTP) and intestinal alkaline phosphatase (iALP) was evaluated in 55 patients who underwent amniocentesis for karyotyping because fetal gastric or small bowel dilatation had been detected by ultrasound. Gastrointestinal malformation was confirmed in 46 cases and there was no gastrointestinal anomaly in nine cases. Prenatal ultrasound was suggestive of gastroduodenal dilatation in 34 cases (group I) and small bowel dilatation in 21 cases (group II). In group I, amniotic fluid GGTP above the 99th percentile was 71 per cent sensitive and 100 per cent specific for a true anatomical defect of the digestive tract (mainly duodenal atresia). In group II, high levels of GGTP and/or iALP were 69 per cent sensitive and 83 per cent specific for a fetal digestive tract anomaly. In other words, when digestive tract dilatations were diagnosed by prenatal sonography, abnormal amniotic fluid enzyme activities were strongly suggestive of such an anomaly, the possibility of which was not precluded by normal amniotic fluid iALP and GGTP activities. But amniotic fluid digestive enzyme activities do not help in defining the prognosis.

Tubular proteins and enzyme content in the amniotic fluid

Amniotic fluid is the product of many substances and fetal urine is considered to be one of the principal components. Only a few reports have been published describing the concentration of microglobulins and urinary enzymes in the amniotic fluid. We determined the levels of alpha 1-m, beta 2-m, AAP and NAG, in 154 samples of amniotic fluid (103 early determinations and 51 late determinations) as a function of gestational age. We observed a statistically significant decrease in concentration of alpha 1-m ( P < 0.001), beta 2-m ( P < 0.01) and AAP ( P < 0.001) when early and late amniotic fluid samples were compared. A statistically significant increase of NAG ( P < 0.01) and creatinine ( P < 0.01) was also found. A significant correlation was observed between alpha 1-m and beta 2-m, and between AAP and NAG, respectively. The potential role of urinary enzyme and microglobulin determination in amniotic fluid as an index of fetal kidney development, is discussed.

New Insights into the Pathogenesis of Cystic Fibrosis

Cystic fibrosis (CF) is the most frequent inheritable disease with a lethal course during childhood. The characteristic high viscosity of the mucoid secretion products in the lungs, pancreas, and gut cause plugging and secondary damage of these organs. In the past 20 years effective treatment of intestinal obstruction in the neonatal period and the infections of the lungs has improved the prognosis significantly. Many patients will reach adulthood in the near future. In the past 10 years new insights into the cause of the disease changed diagnostic procedures and, it is to be hoped, soon also treatment. The first development was the estimation of brush-border enzymes in amniotic fluid. With this method prenatal diagnosis is possible in the 17th-18th week of pregnancy. The recent discovery of the gene on chromosome 7 and its structure is the most important breakthrough. At the same time the process of Cl- transport across the mucosal membrane of many types of epithelium was subject to investigation by several laboratories. We have studied the transport of ions in the small and large intestines of CF patients. The effect of all three types of intracellular signal transfer is abnormal, although the second messengers themselves (cAMP, cGMP, and Ca2+) are present. Evidence is found for K+ instead of Cl- secretion after addition of secretagogues.

Amniotic fluid enzymes in pregnancies with trisomy 21

The activities of a range of microvillous enzymes in amniotic fluid from normal pregnancies (n = 213) and those complicated by trisomy 21 (n = 26) were compared in a prospective study. Using a centrifugal analyser, the activities of leucine aminopeptidase (LAP), gamma glutamyl transferase (GGT), aspartate transferase (AST), and isoenzymes of alkaline phosphate (ALP) were measured in amniotic fluid alongside alpha fetoprotein (AFP) levels. Of the markers studied, LAP was found to be the most reliable indicator of trisomy 21. Using levels below the 5th percentile, LAP showed sensitivity 73 per cent, specificity 94 per cent, and predictive value positive 63 per cent. Although these tests would not replace karyotyping in all cases, the measurement of LAP could be useful as a rapid initial screening test, particularly when amniocentesis is performed for indications other than chromosomal abnormalities.

Vasopressin is metabolized by a trypsinlike enzyme in guinea pig amniotic fluid.

We have demonstrated that arginine vasopressin (AVP) is degraded to desglycinamide AVP by a trypsinlike enzyme found in guinea pig amniotic fluid. Incubation of [3H]AVP with guinea pig amniotic fluid in vivo or in vitro produced a metabolite that comigrated on high-pressure liquid chromatography with desglycinamide AVP in three different buffer systems. Also, AVP antisera that cross-reacted with standard desglycinamide AVP could detect this amniotic fluid metabolite. Because the enzyme responsible for the cleavage of glycinamide from AVP was likely to be trypsin, experiments with aprotinin, a trypsin inhibitor, were conducted. Results demonstrated that the production of the amniotic fluid AVP metabolite could be completely blocked in the presence of the trypsin inhibitor. In addition, examination of amniotic fluid collected from fetuses in the second half of gestation (term = 68 days) showed that AVP could not be metabolized to desglycinamide AVP until after 52 days of gestation. In conclusion, AVP appears to be metabolized by a trypsinlike enzyme in amniotic fluid, and because trypsin is a general proteolytic enzyme, the amniotic compartment may also serve as a clearance site for other proteins.

Prenatal diagnosis of cystic fibrosis: experience of two complementary methods.

Cystic fibrosis (CF; McKusick 21970) is a common and severe autosomal recessive single gene disorder affecting one in 2000–2500 Caucasians. A prenatal test for one in four at-risk pregnancies is based on reduced levels of microvillar intestinal enzymes (MVE) in amniotic fluid (AF) due to intestinal obstruction in affected fetuses (Brock, 1983).

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.

Enzyme analysis of amniotic fluid for prenatal diagnosis of cystic fibrosis in high-risk pregnancies.

We determined the activity concentrations of alkaline phosphatase (ALP), ALP isoenzymes, gamma-glutamyltransferase (GGT), and alpha-glucosidase (AGL) in 1200 unselected amniotic fluids and in amniotic fluids from 40 pregnancies at high risk for cystic fibrosis (CF). From the results we established the normal range and CF-predictive cutoff values for these enzymes in the second trimester of pregnancy. In all predicted normal pregnancies that went to term, normal children were born. Among the predicted affected pregnancies, 14 were terminated and two went to term, one resulting in a CF-affected child and the other in a healthy child. Evidence for CF was found in all 13 aborted fetuses examined (the parents of one refused to allow autopsy). We noted no differences in the amniotic fluid enzyme activities for the Arab and various Jewish ethnic groups living in Israel. We conclude that prenatal diagnosis of CF among the Israeli population at risk for CF is feasible by means of a reliable, fast, and economic test in the second trimester of pregnancy.

Microvillar enzyme assays in amniotic fluid and fetal tissues at different stages of development.

A systematic study of microvillar enzyme activities in the amniotic fluid in correlation with their values in different fetal tissues during development has been undertaken. Microvillar enzymes appeared in the amniotic fluid at the time of disappearance of the anal membrane, 12-13 weeks, and declined from the 18th week until the 24th week. The study of fetal tissues and fluids has shown that gamma-glutamyltranspeptidase is mainly of liver origin. The significant decrease of the activities of these amniotic fluid enzymes has been the basis of prenatal diagnosis of cystic fibrosis. These assays may be useful for the diagnosis of certain digestive tract abnormalities at later stages of pregnancy.

Microvillar enzymes in amniotic fluid: Considerations for the prenatal diagnosis of cystic fibrosis

The activity of gamma-glutamyl transpeptidase (GGT) and the proportion of alkaline phosphatase (ALP) activity that remains in cell-free amniotic fluid (AF) after inhibition by amino acids ("residual ALP activity") have been evaluated as possible prenatal diagnostic tests for cystic fibrosis. A total of 1511 AFs were examined. In 1435 "reference" AFs (excluding those from pregnancies with a known fetal abnormality and those with a known one in four risk of cystic fibrosis) at 14-24 weeks' gestation, the mean residual ALP activity in the presence of 2.5 mmol/L L-phenylalanine was 32% (median, 28%) and in 10.0 mmol/L L-homoarginine it was 70% (median, 72%). AFs were arbitrarily considered to be "abnormal" if the residual activity was greater than 50% in L-phenylalanine and less than 50% in L-homoarginine. An abnormal residual ALP activity pattern was found in nine of 27 pregnancies which resulted in a neural tube (or other developmental) defect and in five of the 10 pregnancies with a chromosomal abnormality; a further 23 (1.6%) abnormal patterns occurred in the 1435 reference AFs from pregnancies that were believed to have had a normal outcome. However, of an additional 23 AFs that were sampled at the 25th week of pregnancy or later, 13 had an abnormal residual activity pattern; the outcome at term in each case was normal. The residual activity of ALP in the presence of either inhibitor did not change with increasing gestational age. However, the absolute amount of ALP that was inhibited by L-phenylalanine (the so-called "phe-inhibitable activity") was greatest at 18 weeks. GGT activity decreased with increasing length of gestation. Of the AF samples from 16 pregnancies at one in four risk for cystic fibrosis that were obtained at 18 weeks' gestation, 11 AFs had a normal residual ALP activity pattern, normal GGT and normal phe-inhibitable ALP activity. Of these 11, five have come to term and the infants are not affected by cystic fibrosis. Three of the 16 AFs had an abnormal residual ALP activity pattern, low GGT and low phe-inhibitable ALP activity; these pregnancies were assessed to be affected by cystic fibrosis and termination was chosen in each case. The two remaining AFs had borderline, paradoxical GGT and residual ALP activity, and initially could not be classified clearly into either of the two groups; however, phe-inhibitable ALP activity was low in each. These pregnancies are continuing.

Gestational Age and Renin Converting Enzyme and Kallikrein-Like Activity in Amniotic Fluid

Electrolytes, angiotensin-converting enzyme and kallikrein-like activity, renin activity, active and inactive renin and angiotensinogen concentrations were determined in amniotic fluid from normotenslve pregnant women during early (17–20 weeks) and late (35–40 weeks) stage of pregnancy.Sodium and protein concentrations were lower whilst potassium and creatinine were higher in late pregnancy. ACE activity was lower by 85% at late stage when compared to early stage. Active renin concentration and renin substrate decreased by 60% and 45% respectively in late pregnancy. Gestational age had no significant influence on renin activity, inactive renin concentration and the ratio total/active renin. Kallikrein activity was lower by 70% in late pregnancy. These results demonstrate that increasing gestational age is associated with consistant decreases in the concentration of most components of the renin-angiotensin system and in kallikrein activity

Enzyme activities in amniotic fluid and maternal blood in sheep, before and after induced foetal death and abortion.

Abstract Tissue enzymes, LDH, AST, ALT, AP, TACP and CK, were measured in amniotic fluid collected from ten chronically-catheterized (intra-amniotic) ewes from 90 to 105 days of gestation until abortion occurred or, in five ewes, the lambs were removed by hysterotomy. Bacterial contamination occurred either during or after surgery in six of the ewes; in eight ewes a test inoculum of Staphylococcus aureus was infused into the amniotic sac. All six enzymes increased in activity in the amniotic fluid just before foetal death or abortion. However, with the exception of one ewe in which amniotic fluid enzyme activity reached very high values, there were no parallel changes in the maternal plasma.