Quantification of glucosylceramide in plasma of Gaucher disease patients

Gaucher disease is a sphingolipidosis that leads to an accumulation of glucosylceramide. The objective of this study was to develop a methodology, based on the extraction, purification and quantification of glucosylceramide from blood plasma, for use in clinical research laboratories. Comparison of the glucosylceramide content in plasma from Gaucher disease patients, submitted to enzyme replacement therapy or otherwise, against that from normal individuals was also carried out. The glucosylceramide, separated from other glycosphingolipids by high performance thin layer chromatography (HPTLC) was chemically developed (CuSO4 / H3PO4) and the respective band confirmed by immunostaining (human anti-glucosylceramide antibody / peroxidase-conjugated secondary antibody). Chromatogram quantification by densitometry demonstrated that the glucosylceramide content in Gaucher disease patients was seventeen times higher than that in normal individuals, and seven times higher than that in patients on enzyme replacement therapy. The results obtained indicate that the methodology established can be used in complementary diagnosis and for treatment monitoring of Gaucher disease patients.

A doença de Gaucher é uma esfingolipidose caracterizada pelo acúmulo de glicosilceramida. O objetivo deste estudo foi desenvolver metodologia baseada na extração, purificação e quantificação da glicosilceramida plasmática a qual possa ser usada em laboratórios de pesquisa clínica. Após o desenvolvimento desta metodologia, foi proposto, também, comparar o conteúdo de glicosilceramida presente no plasma de pacientes com doença de Gaucher, submetidos ou não a tratamento, com aquele de indivíduos normais. A glicosilceramida, separada de outros glicoesfingolipídios por cromatografia de camada delgada de alto desempenho (HPTLC), foi revelada quimicamente (CuSO4/H3PO4) e a respectiva banda foi confirmada por imunorrevelação (anticorpo anti-glicosilceramida humana/anticorpo secundário conjudado à peroxidase). A quantificação do cromatograma por densitometria demonstrou que o conteúdo de glicosilceramida nos pacientes com doença de Gaucher era 17 vezes maior que aquele de indivíduos normais e 7 vezes maior que aquele dos pacientes com doença de Gaucher submetidos a tratamento com terapia de reposição enzimática. Os resultados obtidos demonstram que a metodologia estabelecida pode ser usada como diagnóstico complementar e como monitoração do tratamento de pacientes com doença de Gaucher.

Adaptation of an evaluation method of 5 alpha-reductase type 1 and type 2 enzyme activities in prostate tissue

Objectives: We are proposing the adaptation of a method for the assessment of the activity of 5 alpha-reductase type 1 and type 2 isoenzymes in human prostate tissue to be used in studies of the relationship of enzymatic activity and cancer. Material and Methods: We have been developing a method, based on Thomas et al., 2003 which consists of collecting human prostate samples and performing 5 alpha-reductase activity assessment. We are proposing a method based on samples obtained from prostate biopsies, according to a pilot study developed by Oliveira et al. 2006. We have obtained two samples of the same prostate area, and our idea is to send one of these samples for pathological examination and another for 5 alpha-reductase evaluation. Herein we have showed the feasibility of this assay. Through a thin layer chromatography with 14C-testosterone and NADPH (nicotinamide adenine dinucleotide phosphate) cofactor, we have obtained radioactive spots which were detected by autoradiography. The areas associated with testosterone and its metabolite DHT (dihydrotestosterone) were scrapped and counted with a scintillation chamber. Results: We believe that it is possible to demonstrate the activity of 5 alpha-reductase through the adaptation of this biochemistry method. Conclusions: This method is feasible and can be utilized to assess 5 alpha-reductase type 1 and type 2 activity in prostate tissue samples.