Spectrophotometric Analysis in Umbilical Cords of Infants with Meconium Aspiration Syndrome

We compared spectrophotometric analysis of the umbilical cords of infants with meconium aspiration syndrome (MAS) or with meconium-stained amniotic fluid (MSAF) and healthy infants. In a prospective study, 15 infants with MAS and 37 infants with MSAF were enrolled. Twenty healthy infants formed a control group. The absorption peak of umbilical cords with meconium was significantly higher in the infants with MAS or MSAF than in controls. Spectrophotometric analysis of the umbilical cords with meconium may be useful to identify developed neonates with MAS or MSAF.     

ASSOCIATION OF THE ALKALINE PHOSPHATASE OF RABBIT POLYMORPHONUCLEAR LEUKOCYTES WITH THE MEMBRANE OF THE SPECIFIC GRANULES

The localization of alkaline phosphatase in the specific granules of rabbit polymorphonuclear leukocytes was investigated. The results obtained suggest very strongly that alkaline phosphatase is a component of the granule membrane. The enzyme remains attached to the membrane upon disruption of the granules by the use of detergents or by hypotonic shock and subsequent extraction with sodium sulfate, and can be isolated together with fragments of the granule membrane by isopycnic equilibration. Treatment of the granules with high amounts of Triton-X-100, sodium deoxycholate, or hexadecyltrimethylammonium bromide releases the enzyme in soluble form. In polymorphonuclear leukocyte homogenates, lysis of the granules is needed in order to render alkaline phosphatase fully accessible to substrates. This suggests that the catalytic site of the enzyme is exposed at the inner face of the granule membrane.     

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.     

On the mechanism of conversion of dethiobiotin to biotin in Escherichia coli. III. Isolation of an intermediate in the biosynthesis of biotin from dethiobiotin.

A plasma membrane fraction more than 10-fold enriched in 5′-nucleotidase and alkaline phosphatase was prepared from peritoneal polymorphonuclear leukocytes. This fraction was highly enriched in MgATPase and a b-type cytochrome. High NADPH-duroquinone reductase activity was observed in the leukocytes, in addition to a lipid with quinone-like properties, neither of which cofractionated with plasma membrane. Therefore, we propose the possibility that an electron transport chain which functions to produce microbicidal oxygen metabolites is subdivided between the plasma membrane and one of the cytoplasmic membranes in non-phagocytizing cells.     

Microscopic changes induced by the intratracheal inoculation of amniotic fluid and meconium in the lung of neonatal rats

Meconium aspiration syndrome is a major contributor to neonatal respiratory distress in infants and it has been sporadically recognized in neonatal animals. This investigation was designed to study the short and long term effects of meconium and amniotic fluid in the lungs of neonatal rats. Seven-day-old rats (n = 123) divided in three groups were intratracheally inoculated with saline solution, amniotic fluid or meconium. Rats were euthanatized on 1, 3, 7, 14, 28, 56 and 112 postinoculation days (PID) and the lungs were examined by light microscopy. Saline solution did not induce any change while amniotic fluid elicited only a mild foreign body response which disappeared by PID 14. In contrast, meconium induced an exudative alveolitis characterized by recruitment of neutrophilsn in the bronchoalveolar spaces. Meconium also induced atelectasis, hyperinflation and thickening of alveolar septa all of which had disappeared by PID 14. Starting at PID 7, neutrophils were progressively replaced by macrophages, giant cells, and some fibroblasts. There were sporadic foci of mineralization starting at PID 14 and lasting up to PID 112. Some mineralized foci became lined with cuboidal epithelial cells at PID 28. Meconium was slowly degraded but still evident by PID 112. It was concluded that inoculation of meconium in neonatal rats induces acute microscopic changes typical of meconium aspiration syndrome. The long term lesions induced by meconium consisted of persistent multifocal histiocytic alveolitis and bronchiolitis reaction with occasional foci of calcification.     

The effect of Eglin C on the function of human neutrophils in vitro

Eglin C is an inhibitor of polymorphonuclear leukocyte elastase and cathepsin G. Recently, it was suggested that Eglin C may inhibit bacterial clearance in an experimental animal model of pneumonia. Since the phagocytic-bactericidal activity of polymorphonuclear leukocytes is most important in the promotion of bacterial clearance, we determined the effect of Eglin C on a variety of functions of isolated human polymorphonuclear leukocytes such as phagocytic-bactericidal activity, superoxide production, degranulation and chemotaxis. Apart from a partial inhibition of superoxide production, which was shown to be due to a superoxide dismutase-like effect of Eglin C, there was no inhibition of polymorphonuclear leukocyte functions measured. Eglin C can therefore be considered as a protease inhibitor, which does not interfere with the phagocytic-bactericidal activity of human polymorphonuclear leukocytes.     

Prenatal diagnosis of hypophosphatasia; genetic, biochemical, and clinical studies.

This report has considered three approaches to the prenatal diagnosis of the severe, early onset form of hypophosphatasia. Two of these approaches, ultrasonography and the determination of the bone/liver isozymes of alkaline phosphatase (ALP) in cultured amniotic fluid cells, have proven useful diagnostically. The third method, assay of the bone/liver isozyme activity or total activity in supernatant amniotic fluid, was not informative for the affected fetus we studies. Failure to visualize a well-defined fetal skull after 16 weeks of pregnancy when the level of alpha-fetoprotein in the amniotic fluid is normal should arouse the suspicion of hypophosphatasia. Because the disease is known to manifest clinical variabiltiy, studies to detect both the biochemical defect as well as the structural manifestations should be considered. The combined use of ultrasonography, analysis of amniotic fluid alpha-fetoprotein, and the measurement of the bone/liver ALP in cultured amniotic fluid cells would appear to be the best approach to the prenatal diagnosis.     

Effect of quercetin on human polymorphonuclear leukocyte lysosomal enzyme release and phospholipid metabolism

Quercetin inhibited in a concentration-dependent manner the release of beta-glucuronidase from human polymorphonuclear leukocytes stimulated with zymosan-activated serum. ³H-arachidonic acid-prelabelled polymorphonuclear leukocytes released ³H-arachidonic acid upon stimulation with zymosan-activated serum and this was associated with a decrease of radioactivity in the phospholipid fraction as determined by thin layer chromatography. Quercetin inhibited the release of ³H-arachidonic acid. These observations suggest that the zymosan-activated serum stimulus activates phospholipase A₂ and that phospholipase A2 is inhibited by quercetin. Thus, quercetin alters polymorphonuclear leukocyte phospholipid metabolism and responses to stimulation.     

Cellular acid phosphatase activity: Correlation of cytochemical and biochemical measurements

Summary Methods for comparing results of cellular acid phosphatase activities obtained by quantitative cytospectrophotometry with those obtained by biochemical analysis are needed to express the cytospectrophotometric data in biochemical units. Since naturally occurring cells have differing amounts of acid phosphatase, enzyme activity was measured cytochemically and biochemically in polymorphonuclear leukocytes and peritoneal and alveolar macrophages from male rats to determine if these measurements permitted construction of a line correlating the two parameters. Cellular acid phosphatase activity, as measured cytospectrophotometrically and biochemically, increased proportionately with polymorphonuclear leukocytes having the lowest activities and alveolar macrophages the highest. These values when subjected to linear regression analysis fixed a line with a correlation coefficient of 0.95 demonstrating that cytochemical and biochemical activities of acid phosphatase activity can be correlated using naturally occurring cells.     

NADPH oxidizing activity in rabbit polymorphonuclear leukocytes: localization in azurophilic granules

Rabbit polymorphonuclear leukocyte granules were submitted to zonal fractionation through a discontinuous sucrose gradient. Distribution of azurophilic and specific granules, enzymatically characterized by peroxidase and alkaline phosphatase respectively, was as reported by others. NADPH oxidizing activity was associated with azurophilic granules. 3-Amino-1H-1, 2,4-triazole stimulated NADPH oxidation by azurophilic granules and inhibited peroxidase. Relationships between peroxidase and NADPH oxidizing activity are discussed.     

Subcellular localization and properties of adenosine diphosphatase activity in human polymorphonuclear leukocytes

Adenosine diphosphatase (ADPase) activities were studied in human polymorphonuclear leukocytes with a recently developed radio-assay. The neutrophils were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation. The sucrose density gradient fractions were assayed for ADPase activity and for principal organelle marker enzymes. ADPase activity was distributed between the plasma membrane, specific granule and soluble fractions. The plasma membrane and specific granule activities had similar kinetic and inhibitor properties but the cytosolic enzyme was clearly different. Studies with the non-penetrating inhibitor diazotized sulphanilic acid and measurements of latent activity indicate that plasma membrane ADPase activity is located on the external aspect to the cell. Its possible role in inhibiting platelet aggregation is discussed. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (mU/mg protein) of ADPase activity, in contrast to those of alkaline phosphatase, were similar in all three groups. This result, together with fractionation experiments and inhibition studies strongly suggests that ADPase activity is not attributable to neutrophil alkaline phosphatase.     

Modulation of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) acetyltransferase in human polymorphonuclears. The role of cyclic AMP-dependent and phospholipid sensitive, calcium-dependent protein kinases

In order to characterize the mechanism of activation of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (EC 2.3.1.67) which is the limiting step in the regulation of the synthesis of the potent inflammatory mediator 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; homogenates from human polymorphonuclear leukocytes were incubated in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase and in the presence of a partially purified phospholipid sensitive, calcium-dependent protein kinase (PrKC). The first kinase was found to enhance up to 3-fold acetyltransferase activity in a dose- and time-dependent manner. In homogenates from PMN previously stimulated with complement-coated zymosan particles, the decay of acetyltransferase activity was partially prevented by the addition of soybean trypsin inhibitor and almost completely inhibited when the homogenates were supplemented with inhibitors of alkaline phosphatase such as 50 mM KF and 100 microM paranitrophenylphosphate. Under these conditions it was possible to initiate the decay of acetyltransferase activity by adding an excess of alkaline phosphatase. Preincubation of PMN with 12-O-tetradecanoylphorbol-13-acetate previous or simultaneously to the addition of ionophore A23187 reduced the increase in acetyltransferase produced by ionophore A23187, whereas the generation of superoxide anions was enhanced. Addition of partially purified PrKC to homogenates from ionophore A23187-stimulated PMN, reduced acetyltransferase activity by 63%, whereas only a 16% inhibition was observed on homogenates from resting PMN. These data indicate the modulation of acetyltransferase activity in human polymorphonuclear leukocytes by a phosphorylation-dephosphorylation mechanism linked to cyclic AMP-dependent protein kinase. Phospholipid sensitive, calcium-dependent protein kinase seems not to be involved in the mechanism of activation, but, most probably, in the generation of negative activation signals.     

Biochemical characteristics of amniotic and allantoic fluid in late gestational mares

The purpose of this study was to determine certain clinically important parameters of amniotic and allantoic fluid. Amniocentesis was performed on 10 normal mares in late gestation (323.8±10.2 d) and fluid was collected from both amniotic and allantoic cavities. Compared to amniotic fluid, allantoic fluid had significantly higher values of specific gravity, sorbitol dehydrogenase, total bilirubin, gamma glutamyltransferase, creatinine, phosphorus, total protein, and globulin. However, allantoic fluid had significantly lower values for sodium, chloride and alkaline phosphatase than amniotic fluid.     

Purification and properties of alkaline phosphatase from human polymorphonuclear leukocytes

A procedure for the purification of alkaline phosphatase from human polymorphonuclear leukocytes is described, involving enzyme solubilisation with Triton X-100 and chromatography on DEAE-Sepharose CL 6B and Cibacron Red F = B-Sepharose 4B. The final enzyme preparation was 244-fold purified and was shown to be capable of hydrolysis of a wide range of phosphorylated substates.     

The urinary excretion of pregnanediol during pregnancy determined by gas-liquid chromatography. II. Its relation with other parameters controling pregnancy (author's transl)]

The relation between the urinary pregnanediol determined by gas-liquid chromatography during pregnancy, with ultrasonic findings (biparietal diameter, thickness and echorrefringency of the placenta and the appreciated quantity of amniotic fluid), with biochemical parameters of control of pregnancy (beta-glucoronidase, total and thermostable alkaline phosphatase), with amnioscopic findings, and with several studied parameters of the amniotic fluid by amniocentesis, were studied. A good relation exists with the parameters which denote placentary insufficiency and fetal risk (irregularity in the homogeneity of the placenta, elevation of the total and thermostable alkaline phosphatase, positive amnioscopy) and also with those that are related with fetal maturity (biparietal diameter, shake test, organge cells, creatinine in amniotic fluid, etc.). These results indicate that the determination of urinary pregnanediol can be one of the tests controlling the normal and pathological pregnancies, being useful in the detection of fetal risk and the diagnosis of the intrauterine fetal maturity.     

A method for continuous monitoring of meconium in the amniotic fluid during labour

In about 10% of pregnancies overall, the fetus discharges meconium (its bowel contents) into the amniotic fluid during labour. In about 10% of cases where meconium is passed, the fetus gasps, inhaling the sticky meconium into the upper respiratory tract. After birth, the meconium blocks the air passages in the lungs, impairing gas exchange — meconium aspiration syndrome (MAS). Up to 20% of infants suffering from MAS die and recently published studies have shown a long-term effect of MAS in causing cough and wheeze. The risk of meconium aspiration is thought to be increased by intrauterine hypoxia. At present, meconium is only noticed at birth or occasionally when amniotic fluid leaks past the presenting part of the fetus. A method has been developed which measures absolute meconium concentration with a 99% prediction interval of ± 30 gl⁻¹; allows monitoring of the rate of appearance of meconium linearly with a nonlinearity of 5%, and differentiates between meconium and blood. The method uses the ratio of the intensity of back-scattered light from the amniotic fluid at 700 and 415 nm, the latter being near the peak of light absorption by meconium and the former a reference value. The ratio is also affected by the presence of blood. However, blood has specific absorption peaks at 540 and 575 nm from which it can be detected (the presence of blood is also a significant abnormality, and is relatively uncommon). The measurement method could easily be integrated into an optical sensor mounted onto an intrauterine probe. The measurement of back-scattered light at 415, 540 and 700 nm would allow continuous monitoring of meconium which could provide valuable information for the study of the pathophysiology of meconium passage in utero during labour and allow preventative measures to be developed.     

Activation of p44/p42 mitogen-activated protein kinase limits triiodothyronine-stimulated alkaline phosphatase activity in osteoblasts

It has been shown that thyroid hormone stimulates the activity of alkaline phosphatase, a marker of mature osteoblast phenotype, in osteoblasts. In the present study, we investigated whether p44/p42 mitogen-activated protein (MAP) kinase is involved in the thyroid hormone-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. Triiodothyronine (T(3)) markedly induced the phosphorylation of p44/p42 MAP kinase. PD98059 and U0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly enhanced the T(3)-induced alkaline phosphatase activity in a dose-dependent manner. The phosphorylation of p44/p42 MAP kinase induced by T(3) was reduced by U0126. These results strongly suggest that p44/p42 MAP kinase takes part in the thyroid hormone-stimulated alkaline phosphatase activity in osteoblasts and that p44/p42 MAP kinase plays an inhibitory role in the thyroid hormone-effect.     

Prenatal diagnosis in 200 pregnancies with a 1-in-4 risk of cystic fibrosis

Summary Prenatal diagnosis of cystic fibrosis was performed in 200 pregnancies with a 1-in-4 risk, and was based on significant modifications in amniotic fluid taken at 17, 18, 19 weeks of pregnancy, of six enzymatic assays: gamma-glutamyl-transpeptidase, aminopeptidase M, and alkaline phosphatase (total and isoenzymes). On the basis of normal values, normal outcome was predicted in 135 pregnancies reaching term, all the babies were normal. On the basis of significantly abnormal enzymatic values, an affected fetus was predicted in 56 pregnancies, 53 were terminated, and 3 went to term; the infants were affected. There were discrepancies in enzymatic values in nine cases, in eight cases normal outcome was predicted, six babies were normal and two were affected; in one case an affected baby was predicted, the pregnancy went to term and the baby is normal. Criteria giving evidence for cystic fibrosis in fetuses have been described: macroscopic observation of a typical meconium ileus, significant increase of albumin content in the meconium, and PAS-positive mucus-like material in some pancreatic acini. Using these criteria, diagnosis of cystic fibrosis has been confirmed in all the examined fetuses. The recurrence rate of cystic fibrosis was 22.5% in 147 diagnoses in which the index case had cystic fibrosis without a history of meconium ileus at birth, but was 47.5% when the index case had meconium ileus. The results of the study suggest that prenatal diagnosis of cystic fibrosis can be performed with an accuracy of 98%.     

Prenatal diagnosis of cystic fibrosis. Analytical evaluation of microvillar enzyme determinations in amniotic fluid

The activity of gamma-glutamyltranspeptidase and total alkaline phosphatase and its isoenzymes has been determined in 261 amniotic fluid samples taken from pregnant women with known normal outcome and in 30 amniotic fluid samples from pregnant women with a 1:4 risk for cystic fibrosis (CF). Preliminarily, 114 amniotic fluid samples were assayed in parallel in three different laboratories, and a good correlation was found even though different assays were used. From the results obtained in control amniotic fluids, normal range and CF-predictive cutoff values were established. No false-negative results were found in this study. Among the predicted affected pregnancies 7 were terminated, and 3 went to term: 1 resulting in a CF-affected child and the other 2 in healthy children. CF was confirmed in all the aborted fetuses. In 1 case the results were inconclusive. In this study numerical results obtained for samples with a 1:4 risk of CF analyzed in the three laboratories were always virtually identical.     

Highly sensitive chemiluminescence method for determining myeloperoxidase in human polymorphonuclear leukocytes.

Myeloperoxidase activity was assayed by a chemiluminescence method, using a cypridina luciferin analog as a chemiluminescence probe, after extraction from peripheral human polymorphonuclear leukocytes. The chemiluminescence method was based on the detection of 1O2 generated by myeloperoxidase-catalyzed HOBr formation followed by the interaction of HOBr with H2O2 at pH 4.5. With this method, myeloperoxidase in less than 100 polymorphonuclear leukocytes could be detected and myeloperoxidase in 10(6) polymorphonuclear leukocytes would be calculated to be 14.4 pmol. Eosinophil extract, which contains eosinophil peroxidase, catalyzed 1O2 generation to a great extent, compared with the polymorphonuclear leukocyte extract at pH 4.5. Myeloperoxidase activity in extract of neutrophil fraction could be greatly influenced by eosinophil contamination.