ABSTRACT
BACKGROUND: There are 45 known genetic diseases that impair the lysosomal degradation of macromolecules. The loss of a single lysosomal hydrolase leads to the accumulation of its undegraded substrates in tissues and increases of related glycoconjugates in urine, some of which can be detected by screening of free oligosaccharides (FOS) in urine. Traditional 1-dimensional TLC for urine oligosaccharide analysis has limited analytical specificity and sensitivity. We developed fast and robust urinary FOS and glycoaminoacid analyses by MALDI-time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry for the diagnosis of oligosaccharidoses and other lysosomal storage diseases. METHODS: The FOS in urine equivalent to 0.09 mg creatinine were purified through sequential passage over a Sep-Pak C18 column and a carbograph column and were then permethylated. MALDI-TOF/TOF was used to analyze the permethylated FOS. We studied urine samples from individuals in 7 different age groups ranging from 0-1 months to ≥ 17 years as well as urine from known patients with different lysosomal storage diseases. RESULTS: We identified diagnostic urinary FOS patterns for α-mannosidosis, galactosialidosis, mucolipidosis type II/III, sialidosis, α-fucosidosis, aspartylglucosaminuria (AGU), Pompe disease, Gaucher disease, and GM1 and GM2 gangliosidosis. Interestingly, the increase in urinary FOS characteristic of lysosomal storage diseases relative to normal FOS appeared to correlate with the disease severity. CONCLUSIONS: The analysis of urinary FOS by MALDI-TOF/TOF is a powerful tool for first-tier screening of oligosaccharidoses and lysosomal storage diseases.
Subject(s)
Lysosomal Storage Diseases/diagnosis , Lysosomal Storage Diseases/urine , Oligosaccharides/urine , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Adolescent , Aspartylglucosaminuria/diagnosis , Aspartylglucosaminuria/urine , Child , Child, Preschool , Female , Fucosidosis/diagnosis , Fucosidosis/urine , Gangliosidoses, GM2/diagnosis , Gangliosidoses, GM2/urine , Gangliosidosis, GM1/diagnosis , Gangliosidosis, GM1/urine , Gaucher Disease/diagnosis , Gaucher Disease/urine , Glycogen Storage Disease Type II/diagnosis , Glycogen Storage Disease Type II/urine , Humans , Infant , Infant, Newborn , Male , Mannosidase Deficiency Diseases/diagnosis , Mannosidase Deficiency Diseases/urine , Mucolipidoses/diagnosis , Mucolipidoses/urineABSTRACT
The oligosaccharidoses are a group of metabolic disorders resulting from a deficiency in enzymes responsible for the catabolism of protein bound oligosaccharides and are typified by the accumulation of corresponding sugars in the urine. Screening is typically accomplished using thin layer chromatography. However, analyte specificity can be a problem and thus complicate interpretation of results. For this reason we developed a mixed mode liquid chromatography tandem mass spectrometry assay for the screening of the oligosaccharidoses which potentially mitigates many of the problems associated with thin layer chromatography. Samples from patients previously diagnosed with I-Cell disease, mannosidosis, Pompe, galactosialidosis, and fucosidosis were derivatized with 3-methyl-1-phenyl-2-pyrazolin-5-one and subjected to analysis by liquid chromatography tandem mass spectrometry. Results were compared to normal control samples. Preliminary results suggest that each oligosaccharidoses produces a unique selected reaction monitoring fingerprint and that the developed method may be an effective screening and diagnostic tool for these disorders.
Subject(s)
Chromatography, High Pressure Liquid/methods , Oligosaccharides/urine , Tandem Mass Spectrometry/methods , Antipyrine/analogs & derivatives , Antipyrine/chemistry , Edaravone , Fucosidosis/diagnosis , Fucosidosis/metabolism , Glycogen Storage Disease Type II/diagnosis , Glycogen Storage Disease Type II/metabolism , Humans , Lysosomal Storage Diseases/diagnosis , Lysosomal Storage Diseases/metabolism , Mannosidase Deficiency Diseases/diagnosis , Mannosidase Deficiency Diseases/metabolism , Mucolipidoses/diagnosis , Mucolipidoses/metabolismABSTRACT
Lysosomal alpha-mannosidase (EC 3.2.1.24) is a major exoglycosidase in the glycoprotein degradation pathway. A deficiency of this enzyme causes the lysosomal storage disease, alpha-mannosidosis, which has been described in humans, cattle, domestic cats and guinea pigs. Recently, great progress has been made in studying the enzyme and its deficiency. This includes cloning of the gene encoding the enzyme, characterization of mutations related to the disease, establishment of valuable animal models, and encouraging results from bone marrow transplantation experiments.
