Valores de referencia de la actividad enzimática alfa-glucosidasa ácida linfocitaria / Reference values of intralymphocyte acid alpha-glucosidase activity

Introducción. La enfermedad de Pompe, también denominada déficit de maltasa ácida o glucogenosis tipo ii, es un trastorno metabólico autosómico recesivo caracterizado por un acúmulo anormal de glucógeno lisosomal, causado por la deficiencia de la enzima α-glucosidasa ácida (GAA). Según la edad de inicio y el grado de afectación orgánica, la enfermedad de Pompe se suele subdividir en 2 tipos: neonatal (infantile-onset) y tardío (late-onset). El diagnóstico clínico se confirma mediante la ausencia virtual (forma infantil) o marcada reducción (forma del adulto) de la actividad enzimática GAA en diferentes muestras biológicas. Material y métodos. El objetivo de este trabajo consiste en establecer los valores de referencia para la actividad enzimática GAA intralinfocitaria que constituye el método gold standard para el diagnóstico de la enfermedad. Resultados. Los resultados obtenidos en una población de sujetos control difieren de los publicados en otros trabajos, lo que refleja la importancia de establecer valores de referencia dentro de cada laboratorio (AU)

Introduction. Pompe disease also called acid maltase deficiency or glycogenosis type ii is an autosomal recessive metabolic disorder characterized by an abnormal accumulation of lysosomal glycogen caused by deficiency of the enzyme acid α-glucosidase (GAA). According to the age of onset and the extent of organ involvement, Pompe disease is usually divided into 2 subtypes: Neonatal (infantile-onset) and adult (late-onset). The clinical diagnosis is confirmed by the virtual absence (infantile form) or markedly reduced (adult form) GAA enzyme activity in different biological samples. Material and methods. The aim of this work is to establish reference values for intralymphocyte alpha-glucosidase acid activity, which is the 'gold standard' method for the diagnosis of Pompe disease. Results. The results obtained in a population of control subjects are different from those published in other studies. This reflects the importance of establishing reference values within each laboratory (AU)

Biomarkers in Lysosomal Storage Diseases.

A biomarker is generally an analyte that indicates the presence and/or extent of a biological process, which is in itself usually directly linked to the clinical manifestations and outcome of a particular disease. The biomarkers in the field of lysosomal storage diseases (LSDs) have particular relevance where spectacular therapeutic initiatives have been achieved, most notably with the introduction of enzyme replacement therapy (ERT). There are two main types of biomarkers. The first group is comprised of those molecules whose accumulation is directly enhanced as a result of defective lysosomal function. These molecules represent the storage of the principal macro-molecular substrate(s) of a specific enzyme or protein, whose function is deficient in the given disease. In the second group of biomarkers, the relationship between the lysosomal defect and the biomarker is indirect. In this group, the biomarker reflects the effects of the primary lysosomal defect on cell, tissue, or organ functions. There is no "gold standard" among biomarkers used to diagnosis and/or monitor LSDs, but there are a number that exist that can be used to reasonably assess and monitor the state of certain organs or functions. A number of biomarkers have been proposed for the analysis of the most important LSDs. In this review, we will summarize the most promising biomarkers in major LSDs and discuss why these are the most promising candidates for screening systems.

Catepsina K como biomarcador de afectación ósea en la enfermedad de Gaucher tipo 1 / Cathepsin K as a biomarker of bone involvement in type 1 Gaucher disease

Fundamento y objetivo: La enfermedad de Gaucher es un trastorno hereditario, que se origina como consecuencia del déficit de la actividad β-glucocerebrosidasa ácida, responsable de la degradación de glucosilceramida hasta ceramida y glucosa. Aunque el trastorno de base es fundamentalmente hematológico, el hueso es la segunda estructura más frecuentemente afectada. La catepsina K (CATK) es una enzima implicada en el proceso de remodelado óseo, habiéndose propuesto que la determinación de sus concentraciones séricas podría aportar información complementaria a la de otros biomarcadores. Pacientes y métodos: Se realizó un estudio en 20 controles sanos y 20 pacientes con enfermedad de Gaucher tipo 1, de las comunidades autónomas de Andalucía y Extremadura. Se determinaron como biomarcadores de remodelado óseo la bone alkaline phosphatase (B-ALP, «fosfatasa alcalina ósea»), el amino-terminal propeptide of procollagen type 1 (P1NP, «propéptido aminoterminal del procolágeno 1»), la β-Cross Laps, carboxy-terminal telopeptide of collagen type 1 (CTx, «fracción β del colágeno tipo 1») y CATK por técnicas de electroquimioluminiscencia y enzimoinmunoanálisis. Resultados: Existe un incremento en los niveles de CATK y las ratios CATK/P1NP y CATK/B-ALP en los pacientes con Gaucher tipo 1 respecto a la media obtenida en el grupo control. Por otro lado, considerando la existencia o no de manifestaciones óseas en el grupo de pacientes, la CATK y la ratio CATK/P1NP muestran niveles medios superiores en aquellos pacientes con daño óseo respecto a los que no lo presentan. Conclusiones: Aunque los estudios radiológicos constituyen el gold-standard para el seguimiento de enfermedad ósea en pacientes con enfermedad de Gaucher tipo 1, debe considerarse la utilidad de la CATK como posible indicador de daño óseo en estos pacientes. Asimismo, este parámetro puede utilizarse en la monitorización del tratamiento de la enfermedad ósea (AU)

Background and objective: Gaucher disease is an inherited disorder caused by deficit of acid β-glucocerebrosidase, responsible for the degradation of glucosylceramide to ceramide and glucose. Although the disorder is primarily hematologic, bone is the second most commonly affected structure. Cathepsin K (CATK) is an enzyme involved in bone remodelling process. It has been proposed that determination of its serum concentrations may provide additional information to other biomarkers. Patients and methods: The study included 20 control subjects and 20 Gaucher type 1 patients from Andalusia and Extremadura regions. We analyzed the biomarkers of bone remodelling: the bone alkaline phosphatase (B-ALP), the N-terminal propeptide of type 1 procollagen (P1NP), the β carboxyterminal telopeptide of type 1 collagen (CTx) and the CATK through electrochemiluminescence and immunoassay techniques. Results: There is an increase in levels of CATK, CATK/P1NP and CATK/B-ALP ratios in type 1 Gaucher patients compared to the control group. Considering the existence of skeletal manifestations in the patient group, the CATK and CATK/P1NP ratio showed higher levels in patients with bone damage compared to those without it. Conclusions: Although imaging studies are the gold standard for monitoring bone disease in type 1 Gaucher patients, the utility of CATK should be considered as a possible indicator of bone damage in these patients. Furthermore, this parameter can be used in the monitoring of the treatment of bone pathology (AU)

[Cathepsin K as a biomarker of bone involvement in type 1 Gaucher disease]. / Catepsina K como biomarcador de afectación ósea en la enfermedad de Gaucher tipo 1.

BACKGROUND AND OBJECTIVE: Gaucher disease is an inherited disorder caused by deficit of acid ß-glucocerebrosidase, responsible for the degradation of glucosylceramide to ceramide and glucose. Although the disorder is primarily hematologic, bone is the second most commonly affected structure. Cathepsin K (CATK) is an enzyme involved in bone remodelling process. It has been proposed that determination of its serum concentrations may provide additional information to other biomarkers. PATIENTS AND METHODS: The study included 20 control subjects and 20 Gaucher type 1 patients from Andalusia and Extremadura regions. We analyzed the biomarkers of bone remodelling: the bone alkaline phosphatase (B-ALP), the N-terminal propeptide of type 1 procollagen (P1NP), the ß carboxyterminal telopeptide of type 1 collagen (CTx) and the CATK through electrochemiluminescence and immunoassay techniques. RESULTS: There is an increase in levels of CATK, CATK/P1NP and CATK/B-ALP ratios in type 1 Gaucher patients compared to the control group. Considering the existence of skeletal manifestations in the patient group, the CATK and CATK/P1NP ratio showed higher levels in patients with bone damage compared to those without it. CONCLUSIONS: Although imaging studies are the gold standard for monitoring bone disease in type 1 Gaucher patients, the utility of CATK should be considered as a possible indicator of bone damage in these patients. Furthermore, this parameter can be used in the monitoring of the treatment of bone pathology.

Glucosa tetrasacárido como biomarcador diagnóstico de la enfermedad de Pompe: estudio en 35 pacientes / Tetra-saccharide glucose as a diagnostic biomarker for Pompe disease: A study with 35 patients

Fundamento y objetivos: La enfermedad de Pompe es un trastorno originado por la deficiencia de la enzima alfa glucosidasa ácida (GAA). En esta afección se produce un acúmulo de glucógeno lisosomal en diferentes tejidos, estando especialmente implicados los músculos esquelético y cardíaco. El diagnóstico de confirmación se realiza mediante identificación del déficit de GAA. Existen, además, otros biomarcadores diagnósticos secundarios, como la glucosa tetrasacárido (Glc4), que se muestra elevada en orina de estos pacientes. Así, con este trabajo queremos poner de manifiesto la utilidad de la Glc4 como biomarcador diagnóstico para la enfermedad de Pompe en sus diferentes formas de presentación, utilizando un método de high-performance liquid chromatography (HPLC, «cromatografía líquida de alta resolución») con detección ultravioleta (UV) adaptado para nuestro estudio. Pacientes y métodos: Hemos analizado un total de 75 individuos: 40 controles sanos y 35 pacientes diagnosticados de enfermedad de Pompe. Se han recogido muestras de orina de 24 h de todos ellos y se han determinado sus niveles de Glc4 mediante HPLC/UV. Resultados: La evaluación de la Glc4 urinaria muestra una gran capacidad de discriminación entre individuos sanos/enfermos. Además, los resultados obtenidos nos han permitido establecer el nivel de decisión o punto de corte más apropiado para la identificación de los enfermos. Coclusiones: Los niveles de Glc4 urinarios se encuentran elevados en los pacientes con enfermedad de Pompe, y aunque se encuentran incrementados en otras dolencias, la existencia de un déficit de GAA, junto a una clínica compatible, proporcionan una alta sensibilidad para el diagnóstico de esta grave enfermedad (AU)

Background and objectives: Pompe disease is a disorder originating from an acid alpha-glycosidase (AAG) enzyme deficiency. This disease produces an accumulation of lysosomal glycogen in different tissues, whereby the skeletal and heart muscles are especially involved. The established diagnosis is achieved through the identification of the AAG deficiency. There are also other secondary diagnostic biomarkers, such as tetra-saccharide glucose (Glc4), which shows high levels in the urine of these patients. In this study it is highlighted the usefulness of Glc4 as a diagnostic biomarker for Pompe disease in its different forms of presentation, using a high-performance liquid chromatography with ultraviolet detection (HPLC/UV) adapted to the study. Patients and methods: A total of 75 individuals have been analyzed: 40 healthy controls and 35 patients diagnosed with Pompe disease. Twenty-four hour samples of urine were collected from all of the patients and their Glc4 levels were determined by means of PLC/UV. Results: The evaluation of the urinary Glc4 shows a high discrimination ability between healthy/sick individuals. In addition, the results obtained have allowed to establish the most appropriate level of decision or cut-off point for the identification of sick people. Conclusions: Glc4 urinary levels are found to be high in patients suffering from Pompe disease and even though increased levels are also found in other conditions, the existence of a AAG deficiency together with a compatible clinical symptoms, prove very helpful for a correct diagnosis of this serious disease (AU)

[Tetra-saccharide glucose as a diagnostic biomarker for Pompe disease: a study with 35 patients]. / Glucosa tetrasacárido como biomarcador diagnóstico de la enfermedad de Pompe: estudio en 35 pacientes.

BACKGROUND AND OBJECTIVES: Pompe disease is a disorder originating from an acid alpha-glycosidase (AAG) enzyme deficiency. This disease produces an accumulation of lysosomal glycogen in different tissues, whereby the skeletal and heart muscles are especially involved. The established diagnosis is achieved through the identification of the AAG deficiency. There are also other secondary diagnostic biomarkers, such as tetra-saccharide glucose (Glc4), which shows high levels in the urine of these patients. In this study it is highlighted the usefulness of Glc4 as a diagnostic biomarker for Pompe disease in its different forms of presentation, using a high-performance liquid chromatography with ultraviolet detection (HPLC/UV) adapted to the study. PATIENTS AND METHODS: A total of 75 individuals have been analyzed: 40 healthy controls and 35 patients diagnosed with Pompe disease. Twenty-four hour samples of urine were collected from all of the patients and their Glc4 levels were determined by means of HPLC/UV. RESULTS: The evaluation of the urinary Glc4 shows a high discrimination ability between healthy/sick individuals. In addition, the results obtained have allowed to establish the most appropriate level of decision or cut-off point for the identification of sick people. CONCLUSIONS: Glc4 urinary levels are found to be high in patients suffering from Pompe disease and even though increased levels are also found in other conditions, the existence of a AAG deficiency together with a compatible clinical symptoms, prove very helpful for a correct diagnosis of this serious disease.

Detection of c. -32T>G (IVS1-13T>G) mutation of Pompe disease by real-time PCR in dried blood spot specimen.

BACKGROUND: Pompe disease, or acid maltase deficiency, is a genetic muscle disorder caused by mutations in the gene encoding the acid alpha-glucosidase (GAA) enzyme, which is essential for the degradation of glycogen to glucose in lysosomes. The wide clinical variability is resulted from genetic heterogeneity, and many different mutations of the GAA gene have been reported. Some of these mutations are associated with specific phenotypes, such as the c. -32T>G (IVS1-13T>G) mutation seen in late-onset Pompe disease. METHODS: We used a real-time PCR, after genomic DNA extraction isolated from DBS (dried blood spots) and PCR amplification. RESULTS: Our results successfully detected in controls and patients have been 100% concordant with sequencing results. CONCLUSIONS: This assay combines simple sample processing and rapid analysis and it allows to detect the patients with a milder form and slower progression of this disease with a high reliability.

La actividad ß-galactosidasa como índice de control de calidad de la muestra de sangre seca recogida sobre papel / Beta-galactosidase activity as an index of quality control of dried blood sample collected on paper

Las enfermedades de depósito lisosomal son errores congénitos del metabolismo originados por la deficiencia hereditaria de hidrolasas lisosomales, causando un acúmulo progresivo de moléculas complejas que no pueden degradarse. El diagnóstico definitivo de estas patologías consiste en la determinación de la actividad enzimática específica en leucocitos, fibroblastos u otros tipos celulares, además del diagnóstico molecular. Estos métodos son complejos y presentan incomodidades para los pacientes. En los últimos años, la determinación de la actividad enzimática en muestras de sangre seca recogida sobre papel ha facilitado enormemente tanto la toma de muestras, su envío a los centros de referencia, así como la manipulación de las mismas. En este trabajo, presentamos la determinación de la actividad enzimática β-galactosidasa en muestras de sangre seca recogida sobre papel, como un parámetro de gran utilidad que nos asegura la estabilidad de la muestra remitida a los laboratorios para el diagnóstico de cualquier tipo de enfermedad lisosomal (AU)

The lysosomal storage diseases are inborn errors of the metabolism originated by the hereditary deficiency of lysosomal hydrolases, which cause a progressive accumulation of complex molecules that cannot be degraded. Besides the molecular diagnosis, the definitive diagnosis of these pathologies consists of the determination of specific enzymatic activities in leukocytes, fibroblasts or other types of cells. These methods are complex and may be inconvenient for the patients. In the last few years, the determination of enzymatic activity in dried blood specimen (DBS) has facilitated sampling, their shipment to the reference laboratories, as well as their manipulation. In this work, we present the determination of the enzymatic β-galactosidase activity in DBS as a very useful parameter to ensure the stability of the sample sent to the laboratories for the diagnosis of any lysosomal disease (AU)