title: New substrates and enzyme assays for the detection of mucopolysaccharidosis III (Sanfilippo Syndrome) types A, B, C, and D by tandem mass spectrometry.
abstract: The clinical phenotype of Sanfilippo Syndrome is caused by one of four enzyme deficiencies that are associated with a defect in mucopolysaccharide metabolism. The four subtypes (A, B, C, and D) are each caused by an enzyme deficiency involved in the degradation of heparan sulfate. We have developed a highly efficient synthesis of the substrates and internal standards required for the enzymatic assay of each of the four enzymes. The synthesis of the substrates involves chemical modification of a common intermediate. The substrates and internal standards allow the measurement of the enzymes relevant to heparan N-sulfatase (type A); N-acetyl-α-glucosaminidase (type B); acetyl-CoA:α-glucosamide N-acetyltransferase (type C); and N-acetylglucosamine 6-sulfatase (type D). The internal standards are similar to the substrates and allow for the accurate quantification of the enzyme assays using tandem mass spectrometry. The synthetic substrates incorporate a coumarin moiety and can also be used in fluorometric enzyme assays. We confirm that all four substrates can detect the appropriate Sanfilippo Syndrome in fibroblast lysates, and the measured enzyme activities are distinctly lower by a factor of 10 when compared to fibroblast lysates from unaffected persons.
authors.affiliation: Department of Chemistry, Bagley Hall, University of Washington, Box 351700, Seattle, Washington 98195-1700, USA.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Wolfe
authors.forename: Farideh
authors.initials: F
authors.lastname: Ghomashchi
authors.forename: Tim
authors.initials: T
authors.lastname: Kim
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Abam
authors.forename: Martin
authors.initials: M
authors.lastname: Sadilek
authors.forename: Rhona
authors.initials: R
authors.lastname: Jack
authors.forename: Jerry N
authors.initials: JN
authors.lastname: Thompson
authors.forename: C Ronald
authors.initials: CR
authors.lastname: Scott
authors.forename: Michael H
authors.initials: MH
authors.lastname: Gelb
authors.forename: Frantisek
authors.initials: F
authors.lastname: Turecek
mesh_terms: D006801:Humans; D009084:Mucopolysaccharidosis III / Q000175:diagnosis*; D012015:Reference Standards; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*