Decreased amniotic fluid peroxidase in malignancy of the cervix

Studies of amniotic fluid obtained at various times throughout the last trimester of pregnancy from patients with carcinoma of the cervix indicate a complete deficiency of peroxidase activity. Dialysis partially restores the enzyme activity. Cervical epithelial tumor cells apparently release a dialyzable, low molecular weight inhibitory substance directed specifically toward peroxidase. If this proves to be true in future studies, the peroxidase assay could be a sensitive means of differentiating between carcinoma and dysplasia. A peroxidelike substance was also present in the amniotic fluids obtained from patients with carcinoma. Other investigators have demonstrated large amounts of peroxide in malignant tumors. These elevated peroxide levels might well be directly related to deficient peroxidase activity rather than being a result of other abnormal enzyme levels which have been regarded as being principally involved in the metabolism of this highly toxic molecule.     

Use of alcohol oxidase to measure the methanol produced during the hydrolysis of D- and L-methyl-3-hydroxybutyric acid

An enzymatic assay for the measurement of methanol has been developed. The assay uses alcohol oxidase and peroxidase coupled to the oxidation of 2,2'-azino-di-(3-ethyl)-benzthiazoline-6-sulfonic acid as the chromogen. The assay is linear up to 50 nmol of methanol in a 200-microliters sample and sensitive; 1.25 nmol of methanol in a 200-microliters sample can be measured. The assay is rapid and measurements can be made at any convenient time between 15 min and 4 h after initiation of the reaction. The assay shows highest activity with methanol but significant activity with other primary alcohols up to 1-butanol. Little activity is shown with secondary alcohols and diols. We have used this assay to follow the hydrolysis of the two isomers of the methyl ester of 3-hydroxybutyric acid.     

Primary enzyme quantitation using substrates labeled with a second indicator enzyme. I. Elastase determination using peroxidase-labeled elastin

Quantitation of proteolytic enzyme concentration can be accomplished by measuring the release, due to primary enzyme catalysis, of a second enzyme bound to a particulate substrate. As the primary enzyme acts on the substrate, release of the indicator enzyme into the surrounding medium occurs, which in turn can be quantitated colorimetrically, and under suitable reaction conditions the amount of indicator enzyme released is directly proportional to the amount of primary enzyme present. A specific example of such an assay is that for elastolytic activity using powdered elastin labeled with horseradish peroxidase. The detection sensitivity of the system described is 1 ng/ml of pancreatic elastase, and the dynamic range of the assay is 2 orders of magnitude. The reaction time for optimal elastase detection sensitivity is 3 h. For the assay, horseradish peroxidase is coupled to insoluble elastin. Labeled elastin is incubated with varying amounts of pancreatic elastase. The elastase in the test sample solubilizes the elastin and the horseradish peroxidase bound to it. The amount of peroxidase released is then quantified using the colorimetic reaction produced by catalysis of 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonate)-H₂O₂. For a fixed, nonsaturating concentration of elastase, the amount of peroxidase released is proportional to the elastase concentration.     

Maternal or fetal origin of rhesus monkey (Macaca mulatta) amniotic fluid leukocytes can be identified by polymerase chain reaction using the zinc finger Y gene.

Leukocytes can be found in substantial numbers within the intrauterine tissues and amniotic fluid of women, and play a central role in the pathophysiology of infection-related preterm labor by their production of proinflammatory mediators. It remains unclear whether these leukocytes represent a fetal immune response, a maternal response, or a combination of the two. The objective of this study was to develop a test in the rhesus monkey (Macaca mulatta) suitable for determining the percentage of male fetal cells present in a population of leukocytes recovered from blood or amniotic fluid. We found inadequate specificity for rhesus monkey cells using commercial human Y-chromosome paint kits (fluorescence in situ hybridization (FISH)). Human-specific primers for the repetitive Y chromosome DYZ-1 locus employed in the polymerase chain reaction (PCR) produced an unacceptable percentage of false positives. However, we successfully developed a PCR-based test using rhesus-specific primers for the zinc finger Y (ZFY) locus. Densitometry of PCR products from known ratios of male and female adult peripheral leukocytes generated a linear standard curve which provided quantitative results and required only 400 cells per sample. The rhesus beta globin (RBG) gene served as an internal control. The PCR test correctly discriminated the sex of peripheral leukocytes in 20 adult males, 20 adult females, two male fetuses, and one female fetus. Serial samples of amniotic fluid from four chronically catheterized rhesus monkeys bearing male fetuses were used to confirm the utility of this assay for quantifying fetal cells in amniotic fluid. In conclusion, we have developed a PCR test which is suitable for distinguishing male from female cells in adult and fetal blood and in amniotic fluid, which lends itself to a variety of diagnostic and biologic applications in the rhesus monkey and potentially in other nonhuman primates.     

The localization of trehalase in polyacrylamide gels with eugenol by a coupled enzyme assay

Methods for the localization of trehalase (α, α′-trehalose 1-d-glucohydrolase, EC 3.2.1.28) activity following electrophoresis in nondenaturing polyacrylamide gels have been developed, in which trehalose hydrolysis is coupled to oxidation of the peroxidase substrate eugenol (2-methoxy-4-allyl phenol) by the use of glucose oxidase (EC 1.1.3.4), and peroxidase (EC 1.11.1.7) as ancillary enzymes. The basis for this procedure stems from the fact that free radicals of eugenol which are generated during the coupled trehalase assay condense to form a white precipitate whose location in the gel may be determined by densitometric scanning and whose surface area is a linear function of the enzyme level subjected to electrophoresis.     

Colorimetric coupled enzyme assay for gamma-glutamyltransferase activity using glutathione as substrate

A colorimetric coupled enzyme assay for the determination of gamma-glutamyltransferase (GGT) activity using glutathione as substrate is described. The cysteine released from glutathione upon sequential action of GGT and leucine aminopeptidase is spectrophotometrically detected through its reaction with ninhydrin at 100 degrees C in acidic conditions. The method was applied to the determination of the activity of both bovine kidney and human serum GGT. In the described assay conditions with final GGT concentrations ranging from 0.18 to 4 mU/ml, a linear relationship between produced cysteine and incubation times up to 90 min was observed. When a standard chromogenic assay for GGT using L-gamma-glutamyl-3-carboxy-4-nitroanilide as substrate and the proposed assay were applied on the same serum sample a linear relationship between the two method was observed. Since the use of GSH as substrate, the proposed method can be usefully adopted for enzymological studies on GGT-related enzymes, a class of enzymes which is still waiting to be characterized.     

Automated continuous-flow colorimetric determination of glutathione peroxidase with dichloroindophenol

Automation of the glutathione peroxidase enzyme assay has been problematical. Although such methods have been reported, they do not give equivalent results to the standard manual assay, wherein glutathione oxidation is coupled to NADPH oxidation via glutathione reductase. We report here the development of a fully automated, continuous-flow, colorimetric method for glutathione peroxidase assays in which glutathione oxidation is monitored by its effect on the reaction of glutathione with the colorimetric reagent 2,6-dichloroindophenol. This method has a linear response to glutathione peroxidase over an 800-fold range of enzyme concentrations. Results of assays done by this method in erythrocyte and plasma samples correlate well with the standard manual coupled assay (r = 0.997 and 0.923, respectively), with no evidence of systematic errors. The assay works equally well with hydrogen peroxide or cumene hydroperoxide as substrate and shows the same selectivity toward glutathione S-transferases as the standard coupled assay. The within-day repeatability and the between-day reproducibility were estimated as 1.1 to 6.4% and 1.3 to 7.1% (relative standard deviation), respectively. This method is suitable for enzyme determinations in whole blood, erythrocytes, plasma, and serum from rats, rabbits, monkeys, and humans.     

Determination of myrosinase (thioglucoside glucohydrolase) activity by a spectrophotometric coupled enzyme assay

The hexokinase/glucose-6-phosphate dehydrogenase coupled enzyme system was used to assay for plant thioglucoside glucohydrolase (myrosinase, EC 3.2.3.1) by measuring the rate of glucose released during hydrolysis of glucosinolates. This coupled assay was compared with two other assays for myrosinase: a pH-stat assay that measures the rate of acid released during glucosinolate hydrolysis, and a spectrophotometric assay in which the decrease in the absorbance at 227.5 nm is used to measure the disappearance of the substrate, 2-propenylglucosinolate (DSA assay). The coupled and pH-stat assays were found to give comparable activities and were linear with enzyme concentration over the range 0 to 30 micrograms. The DSA assay gave lower myrosinase activity in comparison to the coupled and pH-stat assays. This is due to the lower concentrations of substrate and activator (ascorbate) which must be used in the assay. The DSA assay was found to give a nonlinear relationship with enzyme concentration over the range 2 to 30 micrograms. For these reasons this assay was found to be unsatisfactory. The coupled assay was found to be more sensitive and more widely applicable than the pH-stat assay as a routine continuous assay for myrosinase activity.     

Capillary electrophoresis analysis of biofluids with a focus on less commonly analyzed matrices

The analysis by capillary electrophoresis of less commonly analyzed biofluids is reviewed. The sample matrices considered include airway surface fluid, sputum, synovial fluid, amniotic fluid, saliva, cerebrospinal fluid, aqueous humor, vitreous humor, and sweat. Many of the techniques used in the analysis of abundant and commonly tested biofluids such as plasma or urine can be applied to these other matrices, e.g. sample extraction prior to analysis. However, for some of these alternative biofluids the available sample amounts are only in the nanoliter or low microliter range, which places constraints on the sample preparation options which are available. For such samples, direct sample injection may be necessary, possibly coupled with on-capillary concentration or derivatization approaches. Particular attention is paid in this review to analyses where the sample is directly injected onto the separation capillary or where minimal sample preparation is performed.     

Purification of mouse alpha1-fetoprotein and preparation of specific peroxidase conjugates for its cellular localization.

Mouse alpha1-fetoprotein (AFP) was isolated from amniotic fluid by immunoadsorbent columns and preparative electrophoresis. Specific antibodies were isolated from monospecific hyperimmunsera by use of immunoadsorbents, and subsequently coupled with horseradish peroxidase. At the light microscopical level, purified antibody-peroxidase conjugates were used for the cellular localization of AFP in fetal liver by direct and indirect staining methods. Fixatives containing ethanol or aldehydes were tried for antigen staining. Prior to immunocytological reactions, endogenous peroxidases were inhibited by hydrogen peroxide.     

Argininosuccinic aciduria: prenatal studies in a family at risk.

We have monitored two successive pregnancies in a family which we found to be at risk for argininosuccinic aciduria. We measured argininosuccinic acid (ASA) concentrations in amniotic fluid and utilized an indirect assay of ASA lyase activity in cultured amniotic fluid cells. The assay procedure is based on the uptake of 14C from [14C]citrulline and of [3H]leucine into protein. ASA was easily measured in amniotic fluid from the first fetus at risk, whereas none was detectable in control fluids. Amniotic fluid cells cultured from this fetus had only 5.5% of control ASA lyase activity. The pregnancy was terminated, and hepatic ASA lyase activity in the fetus was shown to be about 1.3% of control values. In addition, eight fetal tissues were analyzed for ASA, and all had significant accumulation. ASA was not detected in amniotic fluid from the second fetus at risk, and ASA lyase activity in cultured cells was 80% of control activity. Enzymatic analysis of erythrocyte lysate confirmed the diagnosis of an unaffected child (ASA lyase = 46% of control) and indicated heterozygosity. Thus, we provide further evidence that argininosuccinic aciduria can be diagnosed successfully in utero by indirect assay of ASA lyase activity in cultured amniotic fluid cells. In addition, high amniotic fluid ASA concentrations provide strong adjunctive evidence for such a prenatal determination, and may prove to be sufficient for diagnosis.     

Amniotic fluid uric acid levels determined by reversed-phase liquid chromatography with spectrophotometric and electrochemical detection

A rapid and precise, reversed-phase high-performance liquid chromatographic method for amniotic fluid uric acid is described. Detection of uric acid and other naturally occurring constituents is based on UV absorption at a wavelength of 280 nm and direct electrochemical oxidation at a potential of +0.800 V. The total analysis time is short (20 min) and the assay requires only filtration of the samples.Uric acid levels were determined in 14 samples of amniotic fluid obtained during the 15th to 24th week of gestation. Results ranged from 0.897 to 4.39 mg per 100 ml of amniotic fluid.     

The measurement of phosphatidate phosphohydrolase in human amniotic fluid.

Phosphatidate phosphohydrolase (EC 3.1.3.4) activity can be found in late gestational human amniotic fluid and is thought to originate in type II alveolar cells of the fetal lungs where it plays an important role in lung surfactant synthesis. In the present study, phosphatidate phosphohydrolase activity was detected and characterized in a 105 000 X g pellet of amniotic fluid using either [32P]phosphatidate or a water-soluble analog, 1-O-hexadecyl-rac-[2-(3)H]glycerol 3-phosphate as substrate. With either substrate, enzyme activity was optimal at pH 6.0. The soluble analog was hydrolyzed with a Km value of 163 micrometer and a V of 30 nmole/min per mg of protein, and offered several advantages over phosphatidate as a substrate for assaying phosphatidate phosphohydrolase in amniotic fluid. Using the synthetic analog, phosphatidate phosphohydrolase activity was measured in the 700 X g supernatant fraction of 30 human amniocentesis samples and compared with another index of fetal lung maturity, the phosphatidylcholine/sphingomyelin ratio. The results suggest that the new phosphohydrolase assay may be clinically useful in the assessment of fetal lung development.     

Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma,Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases

Background

Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate.

Methods

We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples.

Results

Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR.

Conclusions

We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and the prevention of preterm delivery.     

羊水胆碱酯酶凝胶圆盘电泳改良法及其产前诊断神经管缺损的应用

本文在羊水胆碱酯酶凝胶圆盘电泳中,改用亚铁氰化铜的棕色显带沉淀反应,使显带比原法清晰,温培时间由12小时以上缩短为3小时。测定了210例正常的和19例神经管缺损畸胎的妊娠羊水,其阴性和阳性期望率分别达97.1％和100％。     

An enzyme linked immunosorbent assay (ELISA) for plasma medroxyprogesterone acetate (MPA).

A single extraction fixed antigen enzyme-linked immunosorbent assay (ELISA) that can be completed in less than 24 h is described for the measurement of medroxyprogesterone acetate (MPA) in plasma. MPA is covalently coupled to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to a standard 96-well microtitre plate where it competes with MPA in the extracted plasma sample for goat anti-MPA. Antibody binding to the solid phase is determined via binding of a horse-radish peroxidase second antibody which reacts colorimetrically with its substrate. The reaction is stopped by addition of 1.25 M H2SO4 and absorbance read at 492 nm. All steps except for sample addition and extraction can be performed on an automatic ELISA processing machine. The assay is sensitive, specific and precise, with intra- and inter-assay coefficients of variation of less than 10 and 15%, respectively. Assay sensitivity is 0.08 ng/ml. The assay follows established methodology for other assays in this laboratory which assists standardization, cost structure and sample throughput and thus is a useful alternative to radioimmunoassays for the determination of MPA in plasma.     

Biological evidence that separate hypothalamic hormones release the follicle stimulating and lutneinizing hormones

The first $\text{in}\text{utero}$ diagnosis of Sandhoff's disease was made in an at-risk fetus by the demonstration of deficient β-N-acetyl-hexosaminidase A and B activities in amniotic fluid components the day of amniocentesis. These enzymatic deficiencies were determined by enzyme assay and electrophoresis using 4-methylumbelliferyl-β-N-acetyl-glucosaminide as substrate. The concentrations of the neutral glycosphingolipids were quantified in amniotic fluid; the level of the glycosphingolipid substrate, globoside, was markedly increased in amniotic fluid from the at-risk fetus compared to that of fetal controls. In addition, ultrastructural examination demonstrated pathologic glycosphingolipid accumulation in uncultured amniotic cells. These enzymatic, chemical and ultrastructural procedures provided the rapid and accurate $\text{in}\text{utero}$ diagnosis Sandhoff's disease within three days of amniocentesis. The $\text{in}\text{utero}$ diagnosis was confirmed by the marked deficiencies of β-N-acetyl-hexosaminidase A and B in plasma and various tissues from the aborted fetus. These findings indicated that maternal hexosaminidases do not cross the fetal-placental barrier.     

Multi-step reactions on microchip platform using nitrocellulose membrane reactor

A straightforward and effective method is presented for immobilizing enzymes on a microchip platform without chemically modifying a micro-channel or technically microfabricating a column reactor and fluid channel network. The proposed method consists of three steps: the reconstitution of a nitrocellulose (NC) membrane on a plane substrate without a channel network, enzyme immobilization on the NC membrane, and the assembly of another substrate with a fabricated channel network. As a result, enzymes can be stably and efficiently immobilized on a microchip. To evaluate the proposed method, two kinds of enzymatic reaction are applied: a sequential two-step reaction by one enzyme, alkaline phosphatase, and a coupled reaction by two enzymes, glucose oxidase and peroxidase, for a glucose assay.     

An inhibition enzyme immunoassay for myelin basic protein

An improved Enzyme Immunoassay for Myelin Basic Protein is described. Myelin Basic Protein covalently attached to glass balls, and Myelin Basic Protein in samples compete with each other for binding of a peroxidase conjugated anti Myelin Basic Protein antibody. The peroxidase activity on the balls is then inversely proportional to the amount of Myelin Basic Protein in the sample. A detection limit of 0.6 ng/ml is demonstrated for diluent or spinal fluid. For plasma a dilution step increases this to 1.8 ng/ml. Both the coated balls and the peroxidase conjugate are stable for long periods. The assay requires no expensive equipment. Although the assay appears to be valid for subcellular fractions spinal fluid and plasma, successful detection of Myelin Basic Protection peptides in clinical samples may require careful selection of suitable antisera. The assay would be very suitable for eventual use with an appropriate monoclonal antibody.     

Rapid prenatal testing for human beta-glucuronidase deficiency (MPS VII)

Prenatal diagnosis for the lysosomal storage disorders is typically achieved by enzymatic analysis of the relevant lysosomal enzyme in cultured amniocytes or chorionic villi. While prenatal diagnosis of some genetic diseases can be done by analysis of pertinent metabolites in amniotic fluid, there are few data regarding prenatal diagnosis of lysosomal disorders by enzyme analysis of amniotic fluid. Prenatal diagnosis by enzyme analysis of amniotic fluid has the potential advantage of providing a more rapid prenatal test result. In this study we describe an assay for the prenatal diagnosis of the mucopolysaccharidosis beta-glucuronidase deficiency (MPS VII; MIM #253220) using amniotic fluid and we confirm its reliability in detecting an affected fetus in an at-risk pregnancy by enzyme analysis of cultured amniocytes and fetal fibroblasts. Because MPS VII is rare and few instances of prenatal diagnosis for this and nearly all other lysosomal disorders have been accomplished by enzyme analysis of amniotic fluid, confirmation of results obtained from enzyme analysis of amniotic fluid should be carried out by enzyme or mutation analysis using cultured amniocytes or chorionic villus specimens.