title: A practical fluorometric assay method to measure lysosomal acid lipase activity in dried blood spots for the screening of cholesteryl ester storage disease and Wolman disease.
abstract: Fluorometric measurements of 4-methylumbelliferone (4-MU) are generally used to screen lysosomal storage diseases (LSDs) using dried blood spots (DBSs). However, in DBS, it is difficult to measure lysosomal acid lipase (LAL) activity due to the influence of other lipases in whole blood. Recently, Hamilton used a fluorometric enzyme assay with 4-MU derivatives to measure the LAL activity in DBS. This method requires mercury chloride as stopping reagent, and the fluorescence intensity of 4-MU was measured at an acidic pH. We report a revised method to measure the LAL activity without using toxic mercury chloride and to measure the fluorescence intensity of 4-MU at a basic pH. For this measurement, we established a more practical method that does not require mercury chloride. The LAL activity in DBS was measured in 51 normal controls, seven obligate carriers and seven patients with CESD. The average LAL activities ± SD in the DBS from the normal, obligate carriers and CESD patients were 0.68 ± 0.2 (range: 0.3-1.08), 0.21 ± 0.1 (range: 0.11-0.41) and 0.02 ± 0.02 (range: 0-0.06) nmol/punch/h, respectively. There was a significant difference between the normal and the CESD. Our method does not require toxic mercury chloride and is an appropriate revised enzyme assay using DBS for screening patients with CESD.
authors.affiliation: Advanced Clinical Research Center, Southern TOHOKU Research Institute for Neuroscience, Fukushima, Japan.
authors.forename: Takenori
authors.initials: T
authors.lastname: Dairaku
authors.affiliation: Division of Biochemistry, Core Research Facilities, The Jikei University School of Medicine, Tokyo, Japan.
authors.forename: Takeo
authors.initials: T
authors.lastname: Iwamoto
authors.affiliation: Advanced Clinical Research Center, Southern TOHOKU Research Institute for Neuroscience, Fukushima, Japan.
authors.forename: Minami
authors.initials: M
authors.lastname: Nishimura
authors.affiliation: Advanced Clinical Research Center, Southern TOHOKU Research Institute for Neuroscience, Fukushima, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Endo
authors.affiliation: Department of Pediatrics, The Jikei University School of Medicine, Tokyo, Japan; Department of Gene Therapy, The Jikei University School of Medicine, Tokyo, Japan.
authors.forename: Toya
authors.initials: T
authors.lastname: Ohashi
authors.affiliation: Advanced Clinical Research Center, Southern TOHOKU Research Institute for Neuroscience, Fukushima, Japan. Electronic address: yosh@sepia.ocn.ne.jp.
authors.forename: Yoshikatu
authors.initials: Y
authors.lastname: Eto
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000097:blood; D002219:Carbamates / Q000737:chemistry; D016022:Case-Control Studies; D015217:Cholesterol Ester Storage Disease / Q000097:blood* / Q000175:diagnosis; D059788:Dried Blood Spot Testing / Q000379:methods*; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000737:chemistry; D057230:Limit of Detection; D002787:Sterol Esterase / Q000037:antagonists & inhibitors / Q000097:blood*; D013830:Thiadiazoles / Q000737:chemistry; D015223:Wolman Disease / Q000097:blood* / Q000175:diagnosis
chemical_list: D015415:Biomarkers; D002219:Carbamates; D013830:Thiadiazoles; C576036:lalistat 2; D006923:Hymecromone; D002787:Sterol Esterase
keywords: 4-MU; 4-MUP; 4-Methylumbelliferone; 4-methylumbelliferone; 4-methylumbelliferyl-palmitate; CESD; Cholesteryl ester storage disease; DBS; Excitation wavelength; FWHM; Full width at half maximum; LAL; LSD; Lysosomal acid lipase; WD; Wolman disease; cholesteryl ester storage disease; dried blood spots; full width at half maximum; lysosomal acid lipase; lysosomal storage disorders