Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis II (Hunter Syndrome)

Abstract

We have developed a tandem mass spectrometry based assay of iduronate-2-sulfatase (IdS) activity for the neonatal detection of mucopolysaccharidosis II (MPS-II, Hunter Syndrome). The assay uses a newly designed synthetic substrate (IdS-S) consisting of α-L-iduronate-2-sulfate, which is glycosidically conjugated to a coumarin and a linker containing a tert-butyloxycarbamido group. A short synthesis of the substrate has been developed that has the potential of being scaled to multigram quantities. Sulfate hydrolysis of IdS-S by IdS found within a 3 mm dried blood spot specifically produces a nonsulfated product (IdS-P) which is detected by electrospray tandem mass spectrometry and quantified using a deuterium-labeled internal standard, both carried out in positive ion mode. Analysis of DBS from 75 random human newborns showed IdS activities in the range of 4.8-16.2 (mean 9.1) μmol/(h L of blood), which were clearly distinguished from the activities measured for 14 MPS-II patients at 0.17-0.52 (mean 0.29) μmol/(h L of blood). The assay shows low blank activity, 0.15 ± 0.03 μmol/(h L of blood). The within-assay coefficient of variation (CV) was 3.1% while the interassay CV was 15%.

Figures

Figure 1

Iduronate-2-sulfatase activities in DBS from 14 MPS II patients (blue points) and 75 random newborns (red points). The range of measured activities for the MPS II patients and random newborns was 0.17–0.52 (average 0.29) and 4.8–16 (average 9.1) μmol/(h L blood), respectively.

Scheme 1

Scheme 2

Scheme 3