Development of New Biochemical Tests Using UPLC-Tandem Mass Spectrometry in the Patients with Metachromatic Leukodystrophy : 탠덤 질량분석기를 이용한 이염색백색질장애의 새로운 생화학적 검사법 개발

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의과대학 의학과
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서울대학교 대학원
Tandem mass spectrometryDried blood spotMetachromatic leukodystrophyArylsulfatase AGenetic mutation
학위논문 (박사)-- 서울대학교 대학원 : 의학과, 2014. 2. 송정한.
Introduction: Metachromatic leukodystrophy (MLD) is a genetic autosomal recessive disease caused by a deficiency in arylsulfatase A (ARSA). Accumulated sulfatides can be detected in the urine and detection of
sulfatiduria is a useful test for diagnosis and monitoring. To our knowledge, no studies have explored the accumulation of sulfatides in dried blood spots (DBSs). We performed gene mutation analysis in patients with MLD, and developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for detection of ARSA activity in leukocytes and for measurement of sulfatides in DBSs.
Methods: Gene mutation analysis was performed by direct sequencing in 12 patients suspected of MLD. We revised an enzyme assay using a natural substrate and quantified the reaction product using UPLC-MS/MS. In
sulfatides detection, DBSs were eluted with internal standards. After preparation, samples were analyzed on a UPLC system. Quantification was achieved by multiple reaction monitoring using a MS/MS. We evaluated the precision, linearity, and ion suppression of the method and analyzed sulfatide concentrations in DBS specimen from MLD group (n = 12) and normal controls (n = 124).
Results: Gene mutation analysis confirmed 7 patients with MLD, two patients with MLD-pseudodeficiency (PD), one PD patient, and two obligate heterozygotes. In addition, a novel pathogenic variant was found. In the enzyme assay, we were able to detect substrates, products, and internal standard from a complex mixture. The amount of product obtained was proportional to the leukocyte volume and the incubation time. There was a good correlation between enzyme activities measured by employing the MS/MS and spectrophotometric methods. In sulfatides detection, species subjected to collision-induced dissociation readily fragmented to produce an intense ion at m/z 96.8 (HSO4-). The precision of low and high concentration controls ranged from 5.4% to 19.9 %, and the sulfatides produced linear responses. Molecular species of sulfatides were barely detected in DBSs from normal individuals. By contrast, there was a marked increase in several molecular species of sulfatides in DBSs isolated from MLD patients.
Conclusions: Gene mutation analysis can clearly be used to support the diagnosis of MLD. In addition, we found that the UPLC-MS/MS method for ARSA activity was acceptable. Moreover, simultaneous detection of multiple sulfatide species using UPLC-MS/MS could be successfully applied to DBS analysis. These methods will provide fast and effective screening and monitoring tools for the diagnosis and treatment of MLD.
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College of Medicine/School of Medicine (의과대학/대학원)Dept. of Medicine (의학과)Theses (Ph.D. / Sc.D._의학과)
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