RESUMO
We describe a microfluorometric method for determination of carnosinase activity in dried blood spots on filter paper. The carnosinase in discs punched from such dried blood specimens is activated with cadmium at 30 degrees C. After incubation with the substrate carnosine at the same temperature, histidine is determined fluorometrically. We compare the results of this method with those of a fluorometric method using liquid blood and assess the stability of the enzyme in dried blood on filter paper. The presence of erythrocytes has no effect on the activity. The method may be useful in early detection of carnosinase deficiency and certain other metabolic disorders.
Assuntos
Manchas de Sangue , Dipeptidases/sangue , Adulto , Cádmio/farmacologia , Dipeptidases/deficiência , Eritrócitos/enzimologia , Feminino , Humanos , Recém-Nascido , Masculino , Microquímica , Valores de Referência , Espectrometria de FluorescênciaRESUMO
A fluorometric assay for blood galactose and galactose-1-phosphate has been modified and improved to shorten the analysis time and to increase sensitivity above other published methods. The method may be useful as a quantitative screening or routine clinical test to detect infants suspected of having a defect of galactose metabolism. It can also be used to monitor blood galactose or galactose-1-phosphate levels in children with galactosemia who are on a lactose-free diet.
Assuntos
Manchas de Sangue , Galactose/análise , Galactosefosfatos/sangue , Hexosefosfatos/sangue , Fosfatase Alcalina/farmacologia , Fluorometria , Galactose Desidrogenases/farmacologia , Galactosemias/diagnóstico , Humanos , Recém-Nascido , Microquímica/métodosRESUMO
A very simple fluorescent spot screening test for the detection of elevated creatine kinase activity in dried blood specimens is described. The assay uses reagents commercially available in kit form and modifies the procedure for use with whole blood spotted on filter paper. Additional amounts of diadenosine pentaphosphate had to be added to the reaction mixture to inhibit the excess adenylate kinase present in erythrocytes. The sensitivity and reliability of the test is demonstrated. This qualitative assay has the potential for use as a mass neonatal screening test for Duchenne muscular dystrophy.
Assuntos
Ensaios Enzimáticos Clínicos/métodos , Creatina Quinase/sangue , Distrofias Musculares/diagnóstico , Fluorescência , Humanos , Lactente , RiscoRESUMO
We describe a fluorescent spot test for detecting alpha 1-antitrypsin activity in dried-blood specimens. The eluate of a blood disc is mixed with carbobenzoxy-L-arginine-7-amino-4-methylcoumarin amide and trypsin at the appropriate pH. In the absence of alpha 1-antitrypsin, aminomethylcoumarin, a strongly fluorescent compound, is released. The reaction mixture, when spotted on chromatography paper and viewed under ultraviolet light, exhibits a bright fluorescence only in the case of specimens with alpha 1-antitrypsin deficiency. alpha 1-Antitrypsin activity so estimated correlated well with quantitative assays of dried-blood spots and serum. The procedure is simple and inexpensive, and has the potential for use as a screening test.
Assuntos
alfa 1-Antitripsina/análise , Adulto , Criança , Humanos , Recém-Nascido , Hepatopatias/sangue , Hepatopatias/genética , Métodos , Fenótipo , Enfisema Pulmonar/sangue , Enfisema Pulmonar/genética , Risco , Fumar , Manejo de Espécimes , alfa 1-Antitripsina/genética , Deficiência de alfa 1-AntitripsinaRESUMO
We describe a microfluorometric method for determination of arginase activity in dried blood spots on filter paper. The arginase in discs punched from such dried blood specimens is activated by preincubation with Mn2+ at 37 degrees C. After incubation with substrate at the same temperature, urea is determined fluorometrically by oxidation of NADH to NAD+ in a coupled kinetic reaction. We compare the results of this method with those of a colorimetric method involving liquid blood samples, and assess the stability of the enzyme in dried blood on filter paper. The presence of serum has no effect on the activity. This method may be useful in the early detection of arginase deficiency and certain hematological disorders.
Assuntos
Arginase/sangue , Adulto , Coleta de Amostras Sanguíneas , Ensaios Enzimáticos Clínicos , Estabilidade de Medicamentos , Doenças Hematológicas/diagnóstico , Doenças Hematológicas/genética , Humanos , Recém-Nascido , Doenças do Recém-Nascido/diagnóstico , Papel , Valores de Referência , Espectrometria de Fluorescência/métodosRESUMO
We describe a fluorometric micromethod for measuring adenosine deaminase activity in dried blood spots on filter paper. Earlier methods require venipuncture and preparation of washed erythrocytes; in the present method, whole capillary blood, spotted on filter paper and mailed (dried) to a central laboratory, is used. The stability of the enzyme in dried blood on filter paper was assessed. The results were compared with those of a spectrophotometric method. The presence of serum appears not to affect the estimation of the activity and the method may be useful in early detection of severe combined immunodeficiency disease and hereditary hemolytic anemia.
Assuntos
Adenosina Desaminase/sangue , Nucleosídeo Desaminases/sangue , Adenosina Desaminase/deficiência , Adulto , Estudos de Avaliação como Assunto , Feminino , Filtração , Hemoglobinometria , Humanos , Recém-Nascido , Masculino , Métodos , Microquímica , Valores de Referência , Xantina OxidaseRESUMO
A simple fluorescent spot screening test has been developed for the identification of individuals with arginase deficiency (hyperargininemia). The assay is based on the coversion of arginine to ornithine and urea by arginase present in 1/8 inch disc of dried blood on filter paper. The enzyme activity is visually estimated by the oxidation of NAD-H to NAD+ in a coupled kinetic reaction. In the absence of the enzyme, there is no oxidation of the NAD-H and consequently no loss of fluorescence. The screening assay has been used to identify successfully both heterozygous and homozygous arginase-deficient crabeater macaques (M. fascicularis) as well as three patients with hyperargininemia. This test can be used to screen large numbers of patients with mental retardation or seizure disorders rapidly to determine the frequency of this disorder more precisely.
Assuntos
Hiperargininemia , Programas de Rastreamento , Erros Inatos do Metabolismo/epidemiologia , Animais , Fluorescência , Humanos , Macaca fascicularisRESUMO
A simple microfluorometric procedure for the measurement of protoporphyrin in erythrocytes (EPP) is described. The technique, which can be performed in less than 4 minutes with portable equipment, utilizes 40 mul of capillary blood and only one reagent (94 percent acidified ethanol) for the extraction. The results obtained by this micromethod are in close agreement with those of the ethyl acetate/hydrochloric acid double-extraction methods. The EPP concentration increases markedly when the blood lead level is increased, and the test can be used to screen children for lead poisoning. This new micromethod also gives reproducible results when dried blood spots on filter paper are used instead of liquid blood.
