RESUMO
Quantitative and qualitative analysis of alkaline phosphatases (AP) was performed on amniotic fluid in 59 normal pregnancies and 14 Down's syndrome (DS) pregnancies at 16, 18 and 19 weeks of gestation. In DS cases, intestinal and placental isoenzyme levels were significantly reduced (P<0.001) and the AP electrophoretic pattern was seen to be modified on polyacrylamide gel electrophoresis. A unique component was detected. After extraction and purification of the abnormal isoenzyme, peptide fragments obtained after cyanogen bromide cleavage indicated a hybrid heterodimeric AP composed of intestinal and tissue non-specific subunits, as evaluated by SDS polyacrylamide gel electrophoresis.
Assuntos
Fosfatase Alcalina/metabolismo , Líquido Amniótico/enzimologia , Síndrome de Down/enzimologia , Intestinos/enzimologia , Adulto , Amniocentese , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Isoenzimas/metabolismo , Gravidez , Segundo Trimestre da GravidezAssuntos
Fosfatase Alcalina/biossíntese , Osso e Ossos/enzimologia , Síndrome de Down/enzimologia , Intestinos/enzimologia , Fígado/enzimologia , Neutrófilos/enzimologia , Adolescente , Fosfatase Alcalina/sangue , Criança , Síndrome de Down/sangue , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Estabilidade Enzimática , Feminino , Temperatura Alta , Humanos , Isoenzimas/biossíntese , Isoenzimas/sangue , MasculinoRESUMO
We report the case of a healthy young man presenting with atypical neutrophil alkaline phosphatase (NAP) and reduced neutrophil chemotactic activity, but with no susceptibility to infection. NAP activity was low, kinetic parameters were modified and immunoreactive properties and subcellular distribution were abnormal. Neutrophil morphology was normal. A similar pattern was observed in the patient's healthy brother. The profile of the observed anomalies offers some similarity to that previously described in patients with chronic myelogenous leukaemia. However, in the present case, the NAP deficiency with impaired neutrophil function was present in two brothers with no haematological symptoms and is probably related to a non-acquired neutrophil abnormality. This observation of a primary NAP variant reinforces the hypothesis of a direct link between NAP activity and functional properties of neutrophils.
Assuntos
Fosfatase Alcalina/deficiência , Neutrófilos/enzimologia , Neutrófilos/fisiologia , Adulto , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/química , Núcleo Celular/enzimologia , Quelantes , Quimiotaxia de Leucócito , Citoplasma/enzimologia , Dimerização , Ácido Edético/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Microscopia Eletrônica , Neuraminidase/farmacologia , Neutrófilos/ultraestrutura , Ureia/farmacologiaRESUMO
A kinetic parameter (the Vm/Km ratio), an index of neutrophil alkaline phosphatase catalytic efficiency, was studied in 36 women with normal pregnancies at 16-20 weeks' gestation. On each blood sample, determinations were achieved on enzyme extracted from maternal granulocytes by monitoring the phosphohydrolytic activity at 400 nm on a spectrophotometer equipped with software for computation of kinetic parameters. Infant sex was recorded at the delivery for all women included in this study. The recent introduction of NAP as a marker for some pathological pregnancies requires a better knowledge of the behaviour of that enzyme in physiological conditions. Data reported focus attention on fetal sex. It appears to be one of the factors involved in variations of kinetic parameters observed in maternal NAP. Sex-linked differences in placental maturation could explain these results.
Assuntos
Fosfatase Alcalina/sangue , Idade Gestacional , Neutrófilos/enzimologia , Caracteres Sexuais , Adulto , Feminino , Humanos , Cinética , Masculino , Gravidez , Análise de RegressãoRESUMO
Intensive studies have been conducted so far on biochemical markers available for screening of chromosome defects in obstetrical monitoring. In this paper we report further data on two protein phosphatases: alkaline phosphatase (a marker of cell maturation) and phosphotyrosine phosphatase (a marker of cell proliferation) assayed in cultured amniotic cells from fetuses with trisomy 18 at 15 weeks of gestation. Comparison with normal fetal cells showed a different behaviour for each enzyme: alkaline phosphatase was very significantly lowered while phosphotyrosine phosphatase remained a normal levels. These results provide a further enlargement of the field of biochemical markers used in the screening tests of trisomy 18.
Assuntos
Fosfatase Alcalina/metabolismo , Âmnio/enzimologia , Cromossomos Humanos Par 18/genética , Proteínas Tirosina Fosfatases/metabolismo , Trissomia/genética , Amniocentese , Âmnio/citologia , Divisão Celular , Células Cultivadas , Feminino , Marcadores Genéticos , Idade Gestacional , Humanos , Cariotipagem , Gravidez , Segundo Trimestre da GravidezRESUMO
Alkaline phosphatase concentrations are known to increase in blood neutrophils of normal pregnant women. The main kinetic parameters of this enzyme were analysed and compared in a group of 30 women with normal pregnancies and a group of 11 women whose fetuses had trisomy 21 (Down's syndrome = DS). The subjects were studied at an identical stage of gestation. Significant changes occurred in thermal stability and urea resistance in cases of DS pregnancies. We also investigated the inactivation constants for two chemicals: L-p-bromotetramisole, an uncompetitive inhibitor, and sodium thiophosphate, a competitive inhibitor. Ki measured for the two inhibitors were found to be significantly lower in cases of pathological pregnancies. The patterns observed in inhibition constants extend the biochemical characteristics of the atypical isoenzyme expressed in neutrophils of women with DS pregnancies.
Assuntos
Fosfatase Alcalina/antagonistas & inibidores , Síndrome de Down/sangue , Inibidores Enzimáticos/farmacologia , Neutrófilos/efeitos dos fármacos , Complicações na Gravidez/sangue , Gravidez/sangue , Adulto , Ligação Competitiva , Osso e Ossos/enzimologia , Estudos de Casos e Controles , Feminino , Humanos , Rim/enzimologia , Fígado/enzimologia , Idade Materna , Neutrófilos/enzimologia , Fosfatos/farmacologia , Gravidez de Alto Risco , Tetramizol/análogos & derivados , Tetramizol/farmacologiaRESUMO
Immunoreactivity, cytochemical, immunocytochemical characteristics and subcellular distribution of neutrophil alkaline phosphatase (NAP) were investigated in blood and/or smear samples from 18 women aged 23-46 years (mean 32.5 years) with trisomy 21 fetuses (17-21 weeks) and 28 women aged 20-42 years (mean 31 years) with normal fetuses (17-22 weeks). Immunochemical NAP investigations were carried out in 8 pathological and 8 normal pregnancies; cytochemical and immunocytochemical procedures were carried out in 18 pregnant women with trisomy 21 fetuses and 28 controls. NAP from women with trisomy 21 fetuses is characterized by: (1) a significant decrease in reactivity with anti-liver-type alkaline phosphatase (AP) and anti-NAP antisera; (2) low or very slight reactivity with antiplacental or anti-intestinal antibodies; (3) marked dispersion of NAP lead citrate reaction products or anti-NAP antibody colloidal gold-labelling in neutrophil cytoplasms, as detected by electron microscopy. This subcellular AP distribution (extramembranous) is different from that of normal NAP sites associated with plasma membrane, nuclear membrane and secretory vesicles. The NAP immunochemical and cytochemical characteristics suggest that neutrophils of a woman with a trisomy 21 fetus contain two AP isoenzymes: the liver/bone type and an atypical AP.
Assuntos
Fosfatase Alcalina/sangue , Fosfatase Alcalina/imunologia , Síndrome de Down/imunologia , Neutrófilos/enzimologia , Neutrófilos/imunologia , Complicações na Gravidez/imunologia , Adulto , Síndrome de Down/enzimologia , Feminino , Humanos , Imuno-Histoquímica , Cariotipagem , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Neutrófilos/ultraestrutura , Gravidez , Complicações na Gravidez/enzimologia , Segundo Trimestre da GravidezRESUMO
There were controversial data concerning localization of alkaline phosphatase (AP) in neutrophil nuclei under physiological conditions. In this context, the AP pattern has been determined on nuclei preparations from normal human neutrophils. Blood cells were isolated from 10 healthy adults and from 3 women in the third trimester of an uncomplicated pregnancy. Purity of nuclear suspension was checked by electron microscopy and assay of organelle marker enzymes. Electron microscope cytochemistry and immunocytochemistry studies were carried out on WBC. Enzyme characterization was performed by the usual biochemical procedures. AP was found in nuclear preparations from four of ten normal controls. When present, AP was detected in approximately two-thirds of the nuclei examined, representing an average of 20% of the total cell activity. Conversely, a large amount of nucleus-bound enzyme (55% of total AP activity) was recognized in all pregnant women samples. Biochemical and immunological characteristics clearly differentiate AP forms in the two groups of subjects. Normal controls have an heterogeneous enzyme pattern. AP positive preparations contain a mixture of isoenzymes: a prominent heat labile form and a relatively heat stable minor component. The heat stable fraction displays some properties similar to those previously described in leukocyte AP. Pregnant women express a unique very heat labile isoenzyme identical in its main characteristics to the early placental type.
Assuntos
Fosfatase Alcalina/análise , Núcleo Celular/enzimologia , Isoenzimas/análise , Neutrófilos/enzimologia , Terceiro Trimestre da Gravidez/sangue , Adulto , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/farmacocinética , Núcleo Celular/ultraestrutura , Feminino , Histocitoquímica , Humanos , Imuno-Histoquímica , Isoenzimas/metabolismo , Isoenzimas/farmacocinética , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Neutrófilos/ultraestrutura , GravidezRESUMO
Assays of neutrophil phosphotyrosine phosphatase activity and determination of haematological parameters were performed on 12 trisomic 21 probands without any clinical or biological symptom of other evolutive disease. Haematological studies showed the two main classical abnormalities: the existence of a macrocytosis and an enhanced lymphocyte count. Of interest are the very reduced rates of phosphotyrosine phosphatase activity found in granulocytes from these patients. This defect in protein phosphatase can be considered as an additional enzymatic change extending the list of modified factors recognized at molecular and cellular levels in subjects whose risk of leukaemia is significantly increased.
Assuntos
Síndrome de Down/sangue , Síndrome de Down/enzimologia , Proteínas Tirosina Fosfatases/sangue , Adolescente , Adulto , Contagem de Células Sanguíneas , Eritrócitos/patologia , Feminino , Hemoglobinas/análise , Humanos , Contagem de Leucócitos , Masculino , Neutrófilos/enzimologiaRESUMO
In cultured amniotic cells from fetuses with Edward's syndrome (trisomy 18), the activities of two protein phosphatases, alkaline phosphatase and phosphotyrosine phosphatase, were measured. Comparison with normal fetal cells showed a different behavior for each enzyme. Alkaline phosphatase was significantly lowered while phosphotyrosine phosphatase remained at normal levels. The interest of these enzyme assays in the screening procedure of this severe chromosome defect is discussed.
Assuntos
Fosfatase Alcalina/metabolismo , Líquido Amniótico/citologia , Cromossomos Humanos Par 18 , Proteínas Tirosina Fosfatases/metabolismo , Trissomia , Fosfatase Alcalina/análise , Amniocentese , Líquido Amniótico/enzimologia , Feminino , Humanos , Gravidez , Segundo Trimestre da Gravidez , Proteínas Tirosina Fosfatases/análise , Valores de ReferênciaRESUMO
Biochemical, cytochemical characteristics and electron microscopy subcellular distribution of neutrophil alkaline phosphatase (NAP) were analysed in blood and/or smear samples from 39 trisomy 21 patients (Down's syndrome) aged 11.5-18 years (mean 15.5 years) and 55 normal subjects aged 12-20.5 years (mean 17 years). All patients were karyotyped. NAP cytochemical procedures were carried out on all subjects; biochemical NAP determinations were made in 10 patients and 20 controls; ultrastructural electron microscopy of AP was performed in three patients and four normal subjects. Neutrophil alkaline phosphatase from patients with trisomy 21 displayed the following changes: (1) a significant increase of enzyme activity, (2) a high thermal lability of enzyme. Electron microscope morphology exhibited large deposits of NAP reaction product associated with the plasma membrane and intracellular main organelles, like phosphasomes. The NAP biochemical and cytochemical characteristics suggest that trisomy 21 neutrophils contain a non-specific AP isoenzyme, closely related to the early placental form.
Assuntos
Fosfatase Alcalina/sangue , Síndrome de Down/enzimologia , Neutrófilos/enzimologia , Adolescente , Adulto , Criança , Síndrome de Down/patologia , Temperatura Alta , Humanos , Microscopia Eletrônica , Neutrófilos/ultraestruturaRESUMO
An electrophoretically slow-moving alkaline phosphatase was found in the serum of a 17-year-old patient with Down's syndrome. Immunological and biochemical studies suggested that this abnormal enzyme pattern consisted of a complex of liver/bone isoenzyme with kappa-type immunoglobulins A and G.
Assuntos
Fosfatase Alcalina/química , Síndrome de Down/patologia , Adolescente , Síndrome de Down/enzimologia , Humanos , Imunoglobulina A/química , Imunoglobulina G/química , Cadeias kappa de Imunoglobulina/química , Isoenzimas , Substâncias Macromoleculares , MasculinoRESUMO
In vitro effects of uric acid (2-6-8 trioxypurine) on purified human neutrophil alkaline phosphatase were studied. A marked activation of the enzyme catalyzed reaction is observed. This activation is dose and pH dependent and not influenced by dialysis. Uric acid can form a stable complex with alkaline phosphatase, whose biochemical characteristics (heat stability, reactivity towards some inhibitors) are significantly modified.