Dissertation
Dissertation > Medicine, health > Internal Medicine > Systemic disease > Genetic diseases

Establishment and Clinical Application of Enzyme Activity Detection Method of LSDs

Author HanYaZuo
Tutor LuoXiaoPing
School Huazhong University of Science and Technology
Course Pediatrics
Keywords lysosomal storage disorders mucopolysaccharidosis enzyme assaysynthetic fluorescent substrate
CLC R596
Type Master's thesis
Year 2013
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ObjectiveLysosomal storage disorders(LSDs)are a group of genetic disorders that result from adisorder of lysosomal catabolism. At present, some fifty different LSDs have beenidentified. Hematopoietic stem cell transplantation and enzyme replacement therapy areavailable for a number of LSDs, therefore early diagnosis and timely treatment is critical,especially for patients the irreversible damage has not yet happen, then a positivetherapeutic effect can be obtained. The purpose of this research:1. To establish enzymology diagnosis methods of LSDs and the normal reference range ofour lab;2. To measure the specific enzyme activity of clinical suspected patients;3. To summarize the clinical manifestations of LSDs.Methods1.Objects:(1)Case group:According to the clinical manifestations and auxiliary examination,we identified10cases of suspected MPS at the outpatient of Pediatric endocrinology andmetabolism in Tongji hospital from April2011to September2012, including8males and2females, ages spanned from3to9years.(2)Control group: The blood specimens used toestablish the normal range were from the clinical laboratory of Tongji hospital, with no ageand gender differences. Each of the lysosomal enzymes should be measured in20-40normal random samples. All blood samples(including patients and control group) should be extracted of the leukocytes and plasma on the same day, and immediately used for detectionor stored at-80℃.2.Methods:The whole blood was about2ml of EDTA anticoagulated, and the leukocyteswere isolated with erythrocyte pyrolysis buffer and PBS buffer. Homogenates wereprepared by sonication of cell material in water. The protein concentration was determinedby using the BCA kit. Enzyme activity assays were measure using synthetic fluorescentsubstrate with the exception MPSⅥ and MLD were colorimetric method. Eleven commomLSDs enzymes were included: α-L-iduronidas(eMPSⅠ),iduronate-2-sulphate sulphatase(MPSⅡ),α-N-acetylglucosaminidas(eMPSⅢB),galactose-6-sulphate sulphatas(eMPSⅣA),β-galactosidase(MPSⅣB),ASB(MPSⅥ),β-glucuronidase(MPSⅦ),α-Mannosidase,ASA(MLD),hexosaminidase(Sandhoff),β-glucosidase(Gaucher).Results1. We established the fluorimetric assay for11common LSDs enzymes in the leukocytes orplasma and the spectrophotometry for ASAand ASB.2. We established the normal reference range of11common LSDs enzymes in our lab, suchas α-L-iduronidase42.62±14.04nmol/h/mg.Pro (n=24)iduronate-2-sulphate sulphatase415.6±124.8nmol/4h/ml (n=40)α-N-acetylglucosaminidase14.50±5.306nmol/h/ml (n=40)galactose-6-sulphate sulphatase223.9±68.43nmol/17h/mg.Pro (n=40)β-galactosidase148.6±29.76nmol/h/mg.Pro (n=40)ASB150.3±45.46nmol/h/mg.Pro (n=20)β-glucuronidase25.91±11.92nmol/h/ml (n=33)386.1±99.04nmol/h/mg.Pro (n=41)α-Mannosidase30.03±10.52nmol/h/ml (n=42)ASA171.4±44.37nmol/17h/mg.Pro (n=20)Hexosaminidase1844±444.3nmol/h/mg.Pro (n=39)β-glucosidase8.79±2.19nmol/h/mg.Pro (n=35) 3. In10cases of clinical suspected MPS,4cases were MPSⅡ,1case was MPSⅣA,1casewas MPS Ⅶ,1case was ML.Conclusion1. We established the fluorimetric assay for LSDs enzymes in the leukocytes or plasma andthe spectrophotometry for ASAand ASB.2. We established the normal reference range of11common LSDs enzymes in our lab.3. In10cases of clinical suspected MPS,4cases were MPSⅡ,1case was MPSⅣA,1casewas MPS Ⅶ,1case was ML.4. The definitive diagnosis of LSDs requires close collaboration between laboratoryspecialists and pediatrician.The pediatrician must select the appropriate auxiliaryexamination on the basis of a comprehensive evaluation of clinical symptoms,such asenzyme assay, peripheral blood smear, radiation/neurophysiology examination, biopsy andso on. So according to clinical symptoms and auxiliary examination, pediatrician choosewhich kinds of lysosomal enzymes should be detected.

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