RESUMEN
OBJECTIVE: To assess the benefits of using the phenylalanine:tyrosine ratio to screen newborns for phenylketonuria (PKU). SETTING: Data were collected from all newborns in California during a ten month period (n = 404,381). METHODS: Dried blood spot specimens were analysed at nine laboratories. To assure that the results reported from multiple sites were matched accurately, an automated methodology was chosen that included sample processing, analysis, telecommunications, reporting, and information technology. Phenylalanine and tyrosine concentrations were measured independently by continuous flow fluorometry, for which precision, recovery, detection limits, carryover, chemical specificity, reportable range, and number of repeats are reported. RESULTS: In this study, 37% of the newborns were tested at less than 24 hours of age. For this population, using a phenylalanine only cut off of 200 mumol/l, there were 48 recalled infants per case of classic PKU. Using the phenylalanine:tyrosine ratio with a cut off of 1.50, screen positives could be reported with phenylalanine as low as 150 mumol/l and with only 12 recalls per case. CONCLUSIONS: The phenylalanine:tyrosine ratio can be measured accurately at multiple laboratories using two channel chemical analyses. Having applied the methods to the routine clinical screening of a large population, it was confirmed that the clinical sensitivity and specificity of the PKU screening test are higher when the phenylalanine:tyrosine ratio is incorporated into the cut off than when the cut off is based on the phenylalanine concentration alone.
Asunto(s)
Recién Nacido/sangre , Tamizaje Neonatal , Fenilalanina/sangre , Fenilcetonurias/diagnóstico , Tirosina/sangre , California/epidemiología , Cromatografía Líquida de Alta Presión/métodos , Humanos , Laboratorios/normas , Fenilcetonurias/epidemiología , Proyectos Piloto , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
We compared the screening interpretation of fluorometric analytical results for phenylketonuria (PKU) with tandem mass spectrometry (MS/MS) in filter paper blood spots collected from newborns <24 h of age. In MS/MS, both Phe and Tyr are quantified. Two hundred and eight blood spots collected from infants <24 h of age were retrieved from storage from the California newborn screening program. These samples had been categorized on the basis of fluorometric analysis as initial negative, initial positive for hyperphenylalaninemia with negative determination on recall, or initial positive for hyperphenylalaninemia and confirmed on follow up as PKU or variant hyperphenylalaninemia. The retrieved samples were analyzed in a blinded fashion using MS/MS. Correlation analysis of fluorometry vs MS/MS for Phe concentration was high, with a Pearson correlation coefficient of 0.817. When 180 micromol/L was used as the cutoff Phe concentration for MS/MS and 258 micromol/L was used as the cutoff for fluorometry, all infants with confirmed classical PKU and variant hyperphenylalaninemia were detected. MS/MS analysis reduced the number of false-positive results from 91 to 3. Simultaneous quantification of Phe and Tyr by MS/MS with the use of a cutoff Phe/Tyr molar ratio of 2.5 further reduced the number of false positives to 1. Samples from affected infants showed a discernible trend of increasing Phe concentration and Phe/Tyr molar ratio with age of collection. These results demonstrate the utility of MS/MS in the routine PKU screening of early-discharge newborns. MS/MS reduces the false-positive rate of fluorometric screening almost 100-fold because of the improved accuracy and precision of Phe measurement and simultaneous confirmation with the Phe/Tyr molar ratio. In addition to the detection of PKU, MS/MS can also detect other aminoacidopathies and disorders of fatty acid and organic acid metabolism with lower false-positive rates than other methods currently used in newborn screening programs.
Asunto(s)
Tamizaje Neonatal/métodos , Fenilalanina/sangre , Fenilcetonurias/sangre , Tirosina/sangre , Recolección de Muestras de Sangre , Reacciones Falso Positivas , Fluorometría , Humanos , Recién Nacido , Espectrometría de Masas/métodos , Factores de TiempoRESUMEN
STUDY OBJECTIVE: To evaluate the safety and pharmacokinetics of recombinant, soluble human CD4 (rCD4) in subjects with the acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. The protein rCD4 binds to envelope protein, gp120, of the human immunodeficiency virus (HIV) and blocks HIV infection of CD4 lymphocytes in vitro. DESIGN: Phase 1 trial with dose escalation. SETTING: Two university-affiliated hospital clinics. SUBJECTS: Of 42 subjects enrolled, 29 had AIDS and 13 had AIDS-related complex. INTERVENTIONS: The rCD4 was administered by rapid intravenous infusion on day 1, followed by a 3-day washout, then once a day for 10 days, followed by a 7-day washout, and then three times a week for 8 weeks. Doses of 1, 10, 30, 100, and 300 micrograms/kg body weight per day of rCD4 were administered intravenously to 6 subjects at each dose level. Twelve additional patients received 300 micrograms/kg.d of rCD4: 6 by intramuscular and 6 by subcutaneous injection. All subjects were monitored for toxicity. Immunologic and virologic variables were also monitored. MEASUREMENTS AND MAIN RESULTS: Administration of rCD4 was not associated with important toxicity as determined by clinical monitoring or by serum chemistry, hematologic, or immunologic variables. No subjects required dose reduction or discontinuation of therapy due to rCD4-related toxicity. No consistent or sustained changes in CD4 lymphocyte populations or HIV antigen levels were observed. The volume of distribution of rCD4 was small, and clearance remained constant over the dose range studied. The bioavailability of intramuscular injection and subcutaneous injection was 51% and 45%, respectively. CONCLUSIONS: At the dose levels used in this study, rCD4 appears safe and well tolerated. Serum concentrations of rCD4 were achieved that were comparable to concentrations shown to have antiviral activity in vitro. Further studies are indicated to determine whether rCD4 or related molecules will be useful in treating HIV infection.
Asunto(s)
Complejo Relacionado con el SIDA/terapia , Síndrome de Inmunodeficiencia Adquirida/terapia , Antígenos CD4/efectos adversos , Complejo Relacionado con el SIDA/sangre , Síndrome de Inmunodeficiencia Adquirida/sangre , Adulto , Anciano , Anticuerpos/análisis , Disponibilidad Biológica , Antígenos CD4/administración & dosificación , Antígenos CD4/farmacocinética , Esquema de Medicación , Evaluación de Medicamentos , Femenino , Antígenos VIH/sangre , Semivida , Pruebas Hematológicas , Humanos , Inyecciones Intramusculares , Inyecciones Intravenosas , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/farmacocinética , SolubilidadRESUMEN
Vancomycin 'Vancocin HCl' is a potent antimicrobial used most frequently in the treatment of gram positive septicemias. Ototoxicity has occurred at serum levels of 80 micrograms/mL. A rapid, sensitive assay for vancomycin that utilizes high-pressure liquid chromatography with ultraviolet detection, and requires only 100 microL of serum, is presented. The method shows excellent linearity and precision throughout the therapeutic and toxic range.
Asunto(s)
Vancomicina/sangre , Cromatografía Líquida de Alta Presión/métodos , Trastornos de la Audición/inducido químicamente , Humanos , Vancomicina/efectos adversosRESUMEN
We report a study motivated by a report that guanidinosuccinate is formed by transamidination from arginine to aspartate by perfused liver [J. Clin. Invest 57, 807 (1976)]. We prepared viable liver cells and incubated them with [14C]arginine labeled at the guanidino carbon and aspartate labeled at the methylene groups with tritium. A diacetyl-reacting band, similar to that reported with the perfusate in the above reference, was obtained by column chromatography. This band did not give a Sakaguchi reaction and contained no measurable tritium or 14C. Thus it did not derive from aspartate or arginine. On electrophoresis at pH 5.0, the diacetyl-reacting material moves to the cathode, guanidinosuccinate to the anode. The absorption spectrum of the diacetyl-reacting band showed a double peak, with maxima at 539 and 432 nm; guanidinosuccinate has only one maximum, at 533 nm. The diacetyl reagent reacts with sulfhydryl compounds and polypyrroles (e.g., bilirubin) to produce blue colors with significant absorbance in the 432-nm range. We saw no evidence for guanidinosuccinate formation by transamidination in these experiments with viable liver cells.
Asunto(s)
Guanidinas/biosíntesis , Hígado/metabolismo , Succinatos/biosíntesis , Animales , Arginina/metabolismo , Ácido Aspártico/metabolismo , Colorimetría , Masculino , Ratas , EspectrofotometríaRESUMEN
Measurement of argininosuccinase (I; EC 4.3.2.1) activity is useful in following the course of disease in hepatitis and in screening for the genetic defect, argininosuccinic aciduria. Methodology is proposed for a novel procedure for the determination of I in serum and erythrocytes. In the procedure, fumarate, generated in the reaction, is assayed by conversion to malate with fumarase, determining the malate enzymatically with malate dehydrogenase, and estimating the NADH formed spectrofluorometrically. By this procedure, the enzyme activity in serum from normal individuals is less than 11 mumol/liter of erthrocytes/per hour. The correlation coefficient between results by this method and by the colorimetric method, which measures the arginine generated in the reaction, is +0.97 for serum and +0.98 for erythrocytes. The proposed procedure has a relatively low initial blank, requires less serum, and is completed faster.
Asunto(s)
Eritrocitos/enzimología , Liasas/sangre , Animales , Arginina , Cromatografía por Intercambio Iónico , Fumarato Hidratasa , Fumaratos , Humanos , Cinética , Miocardio/enzimología , NAD , Espectrometría de Fluorescencia , Succinatos , PorcinosRESUMEN
The transport of cyclic adenosine 3', 5'-monophosphate in corn coleoptile segments is very rapid. The linear velocity of basipetal transport is 183 millimeters per hour, while the velocity of acropetal transport is 79 millimeters per hour. Transport velocity as well as intensity thus appear to be polar in the corn coleoptile. Application of metabolic inhibitors such as cyanide, ouabain, and 2,4-dinitrophenol increase rather than decrease the velocity and intensity of transport. The mechanism of transport in light of these data is discussed.
RESUMEN
Preincubation of apical segments of etiolated peas (Pisum sativum L.) in indole-3-acetic acid (IAA) results in an inhibition of the incorporation of [(3)H] thymidine ([(3)H]TdR) into DNA. Preincubation in IAA for 4 h led to an inhibition of [(3)H]TdR incorporation only at the highest concentration of IAA tested (10(-4) M). A 20-h preincubation in various concentrations of IAA resulted in a bimodal dose response curve. High concentrations of IAA (10(-4) M) inhibited incorporation by ca. 50%, as did concentrations of about 10(-6)M, but 10(-5) M IAA did not inhibit this incorporation. The absorption of [(3)H]TdR was not affected by preincubation in IAA for either 4 or 20 h. When the apical segments were cut into two portions, the hook with the shoot apex, and the portion remaining below the apical hook, preincubation in IAA for 20 h gave different results for the upper and lower portions of the apical segments. In the lower portion, concentrations of about 5×10(-6) M gave a slight increase in [(3)H]TdR incorporation and 10(-4) M IAA inhibited DNA synthesis. In the upper portion, IAA pretreatment for 20 h resulted in a bimodal dose response curve which was very similar to that found initially for the entire apical segment. Thus the effect of pretreatment of apical segments with IAA depends upon the physiological status of the tissue. The rapidly expanding cells in the lower portion of the apical segments respond to IAA differently than the cells of the upper portion which are principally quiescent.
RESUMEN
The uptake and accumulation of exogenous indoleacetic acid-(14)C by intact rice coleoptiles were examined. The absorption of exogenous indoleacetic acid was controlled by phytochrome, while the subsequent accumulation of this indoleacetic acid in various portions of the coleoptile was complex, and the effect of red light in this system was small compared to the alteration of the uptake of indoleacetic acid by red light. The absorption of indoleacetic acid exhibited two phases: the first occurring during the first 3-hour portion of the incubation was an inhibition, while the second was a promotive effect at about the 5th hour of incubation. Both of these effects were red, far redreversible, implicating phytochrome in this effect. Neither the destruction nor the immobilization of this exogenous indoleacetic acid apeared to be greatly affected by red light irradiation. The principal interaction between phytochrome and indoleacetic acid appears to occur during the absorption of exogenous indoleacetic acid. This effect may be related to the control by phytochrome of the amount of auxin which diffuses from coleoptile tips.
RESUMEN
The conversion of tryptophan-(14)C to indoleacetic acid-(14)C in cucumber hypocotyls occurred under both sterile and non-sterile conditions. This conversion was not reduced under sterile conditions. The growth response of cucumber hypocotyl segments to exogenously supplied tryptophan was almost as great under sterile conditions as when contaminating micro-organisms were present. These data are consistent with the hypothesis that tryptophan is a normal precursor of indoleacetic acid in cucumber tissues. The conversions of tryptamine-(14)C and indoleethanol-(14)C to indoleacetic acid-(14)C also occurred under both sterile and non-sterile conditions. Indoleethanol-(14)C was formed from tryptamine-(14)C. Hypocotyl segment growth responses to tryptamine and to indoleethanol were not decreased under sterile conditions.