Implementação e padronização de técnicas de dosagem enzimática para detecção de doenças de depósito lisossômico

• Ano de defesa: 2009
• Autor(a) principal: Müller, Karen Barbosa [UNIFESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Diagnóstico; Gota de sangue em papel de filtro; Doença de Gaucher; Doença de Fabry; Doença de Pompe; Mucopolissacaridose tipo I; Doença de depósito de glicogênio tipo II
• Link de acesso: http://repositorio.unifesp.br/handle/11600/9721

Objectives: To implement and to standardize enzymatic activity techniques using dried blood spots on filter paper for the enzymes -galactosidase A, for the diagnosis of Fabry Disease patients; -iduronidase, for the diagnosis of Mucopolysaccharidosis type I patients; -glucosidase and chitotriosidase, for the diagnosis of Gaucher Disease patients and acid -glucosidase, that allows the diagnosis of Pompe Disease patients; besides the enzyme -galactosidase, that allows assessing the quality of the material. In addition, to determine the cut-off value for each enzyme, that will enable the discrimination between healthy subjects and patients. Methodology: 302 volunteers of both gender were selected, with ages between 18 and 82 years old. Peripheral blood was collected in heparin containing tubes and blood spots were prepared according to the protocol. All enzymatic activity techniques carried out in this work were based in the cleavage of a specific synthetic substrate, by the action of target enzymes which then releases a fluorescent molecule that will be detected by the fluorimeter. Results: The values of enzyme activity obtained in this work were the following (mean±standard deviation; minimum and maximum values), expressed in μmol/L/h: -galactosidase (14,09±4,36; 4,04-29,65); -galactosidase A (4,57±1,37; 1,44-10,67); -iduronidase (3,45±1,21; 1,40-7,78); -glucosidase (4,57±1,37; 1,44-10,67); chitotriosidase (12,88±9,84; 0,00-48,07); -glucosidase (5,41±1,74; 2,10-10,65); also expressed in the ratio of neutral -glucosidase/inhibited acid -glucosidase (13,19±4,26; 5,03-28,58); and as % of acid -glucosidase inhibition (70,66±7,60; 36,38-87,08). The sensibility and specificity values obtained with the comparison of leukocytes and dried blood spots results showed that all techniques are reliable and appropriate. Conclusions: The results from this work enabled us to choose the most accurate cut-off values which to distinguish between healthy subjects and patients with Fabry Disease, Mucopolysaccharidosis type I, Gaucher Disease and Pompe Disease. Enzyme activity values are in agreement with the literature data. The use of dried blood spots for lysosomal disease diagnosis can be considered efficient, inexpensive and simple to be performed.