Detection of Lysosomal Storage Disorders Using Tandem Mass Spectrometry
Barcenas, Mariana Natali
MetadataShow full item record
Lysosomal storage disorders is a group of diverse autosomal recessive metabolic diseases. These disorders are caused by a deficiency in an enzyme necessary for the catabolic degradation of proteins, glycolipids, and glycosaminoglycans in the lysosome. Although these disorders can be caused by different mutations of different enzymes, they all manifest themselves through the abnormal accumulation of material within the affected cells. For decades lysosomal storage disorders were considered interesting neurodegenerative fatal disorders with no possibility for treatment. A lot of progress has been made in understanding these diseases and the enzymes behind the malfunction as well as in treatments for some of these disorders. Treatments are more successful when initiated before symptoms manifest; therefore early diagnosis is desired. Tandem mass spectrometry has become a powerful tool in clinical analysis. It is also used in newborn screening laboratories for inborn error detection. Our work focuses on developing assays to detect lysosomal storage diseases in newborns by measuring substrate concentrations and measuring enzyme activity using tandem mass spectrometry as a quantification tool. A new direct assay for palmitoyl protein thioestarase 1 was developed for the clinical diagnosis of infantile neuronal ceroid lipofuscinosis. Dried blood spots (DBS) are incubated in an assay cocktail containing detergent, synthetic peptide substrate and internal standard at 37 ºC for 10 hours. Product and internal standard are detected by electrospray tandem mass spectrometry in positive ion mode using selected reaction monitoring. A new method for determination of sulfatide levels in blood for the detection of metachromatic leukodystrophy (MLD) was also attempted. Measurement of urinary sulfatide levels is used as a baseline method to distinguish healthy individuals from affected MLD individuals. In an effort to easily include our assay with other newborn blood spot screening methods, sulfatides were extracted from dried urine and blood spots using organic solvent and subsequently analyzed using ultra high performance liquid chromatography tandem mass spectrometry. An acceptable separation of sulfatide levels between MLD and non-MLD dried blood spots however was not obtained; because MLD and non-MLD affected individuals had measurable levels of sulfatide. On the other hand, sulfatide levels in dried urine from MLD affected individuals were elevated when compared to non- MLD individuals. A multiplex assay using a single DBS was developed to screen for 6 disorders: Fabry, Pompe, Niemann-Pick A/B, Krabbe, Gaucher, and mucopolysaccharidosis type I. Samples were analyzed using flow injection tandem mass spectrometry. We demonstrate substantial differences in enzyme activity between blanks and blood punches for all six disorders studied. A new cassette of substrates was introduced to improve assay performance. New reagents for mucopolysaccharidoses type II and type VI were synthesized to improve assay performance. Results show that the new reagents have enhanced mass spectrometry sensitivity and thus, a lower detection limit.
- Chemistry