Identification of mucopolysaccharidosis I heterozygotes based on biochemical characteristics of L-iduronidase from dried blood spots.

BACKGROUND: Mucopolysaccharidosis I (MPS I) is a genetic disorder caused by deficiency of L-iduronidase (IDUA) activity. Heterozygote screening is a highly requested service by risk families; however, determination of IDUA activity alone is not sufficient to discriminate between heterozygotes and normal individuals because a significant overlap occurs between them. The aim of this study was to characterize the enzyme eluted from heterozygote's dried blood samples and determine if there are differences with that of normal individuals. METHODS: We determined Km, Vmax and the thermal stability of the enzyme at 50 °C. RESULTS: Vmax from heterozygotes (7.28 ± 2.72 µmol/l blood/h) was significantly different than the obtained in controls (10.52 ± 2.05 µmol/l blood/h), while their Km were similar: 0.633 ± 0.339 mmol/l and 0.672 ± 0.246 mmol/l, respectively. After a 12 h pre-incubation period, IDUA activity in controls was significantly lower compared to heterozygotes. CONCLUSIONS: IDUA eluted from dried blood spots of heterozygotes differs from that of controls in terms of Vmax and thermal stability. These parameters can be used as an important tool for the detection of carriers for MPS I. This is the first report describing a differential behavior of these parameters for a lysosomal enzyme obtained from dried blood.

Optimization of enzymatic diagnosis for mucopolysaccharidosis I in dried blood spots on filter paper.

OBJECTIVES: The aim of this study was to validate an ultramicroassay with a reduced interference of hemoglobin for the enzymatic diagnosis of mucopolysaccharidosis I in dried blood spots on filter paper. DESIGN AND METHODS: A matrix of dried blood was incorporated within the calibration system. In addition, trichloroacetic acid was added to precipitate hemoglobin. Linearity, precision, accuracy and limits of detection and quantification were determined and α-l-iduronidase activity was obtained from 6 patients, 9 heterozygotes, 25 healthy adults and 500 neonates. RESULTS: The ultramicroassay was linear, precise (coefficients of variation less than 10%) and accurate (recovery between 91 and 98%). The interference of hemoglobin was decreased within the hematocrit range of clinical interest: 35-55%. CONCLUSIONS: This ultramicroassay increases in 2.5 times the difference between healthy individuals and patients with respect to the reference assay; optimizing enzymatic quantification and confirmatory biochemical diagnosis for mucopolysaccharidosis I.

Estudio de las enzimas lisosomales N-acetil-a-D-glucosaminidasa y arilsulfatasa A en adultos cubanos / Assay of lysosomal N-acetyl-a-D-glucosaminidase and arylsulfatase A activities in Cuban adults

El objetivo de este estudio fue determinar, en una muestra de individuos cubanos, un rango preliminar de valores normales de actividad específica de N-acetil- a-D-glucosaminidasa y arilsulfatasa A, enzimas deficientes en la mucopolisacaridosis tipo III B y la leucodistrofia metacromática, respectivamente. Se realizó una investigación de corte transversal, en muestras de sangre de 38 individuos adultos. Se realizó la extracción de los leucocitos y se determinó la actividad específica de las enzimas por métodos espectrofotométricos. Todos los participantes fueron caracterizados desde el punto de vista clínico como sanos para ambas enfermedades. Se obtuvieron valores medios de actividad específica de N-acetil-a-D-glucosaminidasa y arilsulfatasa A de 1,52±0,30 y 121,37±20,14 nmol/mg/h, respectivamente. No se encontraron diferencias significativas en relación a las características étnicas para ninguna de las dos enzimas (p<0,05). Este constituye el primer estudio cubano en el cual se publican rangos de actividad específica para estas enzimas en individuos cuya condición de sanos y no relacionados familiarmente con la enfermedad ha sido clínicamente demostrada. El análisis de un mayor número de muestras permitirá establecer los puntos de corte que dotarán al diagnóstico bioquímico de estas enfermedades de una mayor confiabilidad.

The aim of this study was to determine, in a sample of Cuban individuals, a preliminary range of normal values of N-acetyl-a-D-glucosaminidase and arylsulfatase A activity, enzymes that are deficient in mucopolysaccharidosis type III B and metachromatic leukodystrophy, respectively. A cross-sectional research was conducted. Blood samples were obtained out of 38 adult individuals. The leucocytes were extracted and the specific enzymatic activity was assayed by spectrophotometric methods. All participants were characterized from the clinical point of view as healthy for both diseases. Average values of N-acetyl-a-D-glucosaminidase and arylsulfatase A specific activities of 1.52 ± 0.30 and 121.37 ± 20.14 nmol/mg/h, respectively were obtained. There were no significant differences related to ethnicity for any of the two enzymes (p <0.05). This is the first Cuban study in which ranges of activity for these enzymes have been reported in healthy individuals whose healthy and non-familiarly related with the disease status have been clinically demonstrated. The analysis of more samples will establish the cutoff points that will increase the reliability of the biochemical diagnosis of these diseases.

An enzyme immunoassay for determining 17alpha-hydroxyprogesterone in dried blood spots on filter paper using an ultramicroanalytical system.

BACKGROUND: 17alpha-hydroxyprogesterone has been used for the diagnosis of congenital adrenal hyperplasia (CAH) in the newborn period. METHODS: A simple and rapid competitive ultramicro ELISA assay based on competition between 17-OHP-alkaline phosphatase conjugate and 17-OHP in blood specimens for a limited number of binding sites on specific polyclonal rabbit anti-17-OHP antibodies, has been developed for the measurement of 17-OHP in dried blood spots on filter paper. The assay buffer contains danazol to displace 17-OHP from steroid-binding proteins. RESULTS: The 17-OHP assay was completed in 3 h, with a measuring range of 10-250 nmol/l. The intra- and inter-assay CV were 5.5-8.2% and 6.4-9.1%, respectively, depending on the 17-OHP concentrations. The recovery ranged from 98-103%. Of 3750 newborn samples collected on filter paper, 903 from the national neonatal screening program were analyzed, and the mean 17-OHP concentration was 12.2 nmol/l. Our assay showed high Pearson and concordance correlations with the commercially available ICN Neoscreen ELISA 17alpha-hydroxyprogesterone kit. CONCLUSIONS: The analytical performance characteristics of our 17-OHP Neonatal UMELISA suggest that it can be used for the neonatal screening of CAH.

Qualitative colorimetric ultramicroassay for the detection of biotinidase deficiency in newborns.

BACKGROUND: We describe a simple qualitative visual ultramicroassay based on the colorimetric method introduced by Heard et al. for the detection of biotinidase deficiency in dried blood samples spotted on filter paper. METHODS: The assay uses 3-mm discs of dried blood on Schleicher and Schuell 903 filter paper and ultramicrovolumes of each reagent. Ten thousand newborn samples from the National Screening Program for the Detection of Phenylketonuria were evaluated. RESULTS: The ultramicroassay shows a good reproducibility. The lower detection limit is around 2% of the mean normal activity. We found one sample with the absence of enzymatic activity, another that was between 10% and 30%, and 10 with activity levels <40%. There was coincidence of our results with those obtained by the conventional colorimetric method that uses B-PAB as substrate. CONCLUSIONS: The qualitative colorimetric ultramicroassay does not require special laboratory equipment and it is suitable for the neonatal screening of biotinidase deficiency.

Ensayo cuantitativo para la determinación de la actividad hidrolítica de la enzima biotinidasa / Quantitative assay to determine the hydrolytic activity of biotinidase

El presente trabajo describe la estandarización de un ensayo diseñado para cuantificar la actividad hidrolítica de la enzima biotinidasa en muestras de suero humano, el cual se basa en el método de Wolf y colaboradores, que emplea el N-biotinil-p-aminobenzoico (BPABA) como sustrato. Con la adición de detergentes a la solución de nitrito de sodio, se eliminan las burbujas de nitrógeno formadas durante la reacción de diazotación y se mejora la precisión del ensayo. Se observó que la congelación-descongelación de las muestras de suero no afecta la actividad hidrolítica de la enzima biotinidasa. La evaluación de la interferencia de fármacos en el ensayo mostró que con el sulfametoxasol/trimetoprim y la procaína/benzilpenicilina hay desarrollo de color en ausencia del sustrato BPABA. Los valores promedio de actividad hidrolítica de la biotinidasa, obtenidos en un grupo de 205 niños sanos, fue de 7,04 ñ 2,2 nmol/min/ml. No se encontraron diferencias estadísticamente significativas al evaluar la actividad de la enzima por sexo, raza y grupos de edad. Este ensayo se puede emplear en la determinación de la actividad hidrolítica de la enzima biotinidasa en muestras de suero humano y, específicamente, en la confirmación de los casos detectados por los programas de tamizaje neonatal para la deficiencia de biotinidasa