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Technical Briefs |
Departments of1 Chemistry, 3 Pediatrics, and 4 Biochemistry, University of Washington, Seattle, WA; 2 Laboratory of Neurochemistry, Buenos Aires, Argentina;
aaddress correspondence to this author at: Departments of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195; fax 206-685-8665, e-mail gelb{at}chem.washington.edu
Mucopolysaccharidosis type I (MPS-I), caused by a deficiency of 
-L-iduronidase (IDUA; EC 3.2.1.76) activity, can manifest as three major phenotypes, usually defined by clinical criteria: Hurler (severe), Scheie (mild), and Hurler–Scheie (intermediate) syndromes.
IDUA is crucial for degradation of glycosaminoglycans such as dermatan and heparan sulfate. Failure to break down these polysaccharides causes physical changes such as joint stiffness, skeletal abnormalities, and corneal clouding. Hurler syndrome is characterized by valvular heart disease, mental deterioration, and death in childhood. Enzyme replacement therapy has been developed for MPS-I, and bone marrow transplantation is beneficial if performed early (1). Because early detection is necessary for optimum clinical response to therapy, the need for newborn screening of MPS-I is under active discussion.
Lysosomal enzymes can be measured in rehydrated dried blood spots (DBS) (2)(3)(4)(5)(6)(7)(8). Fluorometric, radiometric, and electrospray ionization tandem mass spectrometry (ESI-MS/MS) assays have been developed. The latter offer the capability of assaying the products of several enzymes simultaneously (multiplexing) (8). In this report, we describe an ESI-MS/MS assay that directly measures the reaction velocity of IDUA in rehydrated DBS for the newborn screening of MPS-I. We also show that the assay can be combined with ESI-MS/MS assays of Niemann–Pick type A/B, Krabbe, Gaucher, Pompe, and Fabry diseases (8) for the simultaneous analysis of six lysosomal storage diseases.
All experiments with DBS were conducted in compliance with Institutional Review Board review. All MPS-I-affected patients had been diagnosed previously with established clinical and biochemical procedures. DBS were kept at ambient temperature during shipment (<10 days) and then stored at –20 °C in zip-lock plastic bags (one bag sealed inside of a second bag). Zip-lock bags were kept in a sealed plastic box containing desiccant (anhydrous CaSO4 granules). We developed a practical, low-cost method for synthesizing IDUA substrate (IDUA-S; from commercially available heparin) and the IDUA internal standard (IDUA-IS), which will be published elsewhere.
A 3-mm DBS punch (containing 
3.6 µL of blood) was obtained by use of a standard leather punch and placed in a well of a 96-well plate (F96 MaxiSorp Nunc-Immuno Plate; cat. no. 442404; Nunc Inc.). Elution buffer [40 µL of 50 mmol/L sodium formate, 0.04 mmol/L D-saccharic acid-1,4-lactone (Sigma), pH 2.8; storage at –20 °C (see the note in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue5/)] was added to the well, the plate was capped with a Teflon-lined cover (Cap Mats; cat. no. EK 99116; E & K Scientific), and the plate was shaken gently on an orbital shaker for 10 min at room temperature. To the same well was added 20 µL of 1 mmol/L IDUA-S in water (stored at –20 °C), and the plate was capped and gently shaken for 24 h at 37 °C in a thermostated-air shaker. The reaction was quenched by addition of 200 µL of 85 mmol/L glycine–carbonate buffer, pH 10.5 (6.4 g/L glycine and 27.5 g/L Na2CO3). IDUA-IS was added (20 µL of 10 µmol/L solution in water; storage at –20 °C). The solution was mixed by pipetting the liquid up and down a few times with a multichannel pipettor, and the liquid was applied to a well of a 96-well filter plate (cat. no. F20005; Innovative Microplate) containing C18-silica (see below). The filter plate was attached to a vacuum manifold system (cat. no. MAVM0960R; Millipore Inc.) attached to a water aspirator. After sample loading, 400 µL of 50 mL/L methanol in water was added to wash the solid phase. The manifold was charged with a deep-well receiver plate (96-Well megatiter collective plates, cat. no. 2045; CLP Inc.), and the IDUA-P and IDUA-IS were eluted with one 400-µL portion of 500 mL/L methanol in water. Solvent was removed from the receiver under reduced pressure by use of a vacuum desiccator (typically 1 h at room temperature). To each well we added 70 µL of 5 mmol/L ammonium formate in methanol–chloroform (3:1 by volume), and the sample was infused into the mass spectrometer.
The 96-well filter plate was charged with C18-silica as follows. C18-silica bulk media (1.5 g; cat. no. 377635; Aldrich) was slurried in 10 mL of methylene chloride, and 1 mL of slurry was added to each well. Solvent was removed by suction (filter manifold), and the solid phase was washed with 3 mL of methanol followed by 3 mL of 50 mL/L methanol in water.
We used a Sciex API-III Plus tandem quadrupole instrument operating in positive multiple-reaction monitoring mode. Instrument settings are given in the online Data Supplement. The parent ions for IDUA-P and IDUA-IS (m/z 489 and 494, respectively) were isolated and subjected to collision-induced dissociation. The fragment ions analyzed were m/z 409 and 414 derived from IDUA-P and IDUA-IS, respectively (Fig. 1A
). We calculated the amount of product by comparing the ion peak intensities of IDUA-P with IDUA-IS.
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We used a 5-mm diameter DBS for the five different lysosomal enzyme assays as described previously (8). The sample was processed by solid-phase extraction using silica gel as described (8). A second 3-mm diameter DBS was used to assay IDUA as described above. The eluate from the C18-silica gel plate was combined with the eluate from the silica gel plate (from the five-plex assay), and the solvent was removed under reduced pressure. The residue was taken up in 200 µL of 5 mmol/L ammonium formate in methanol–CHCl3 (3:1 by volume). The sample was infused into the mass spectrometer.
IDUA-S was readily prepared from commercially available heparin. Incubation with IDUA present in DBS leads to enzymatic release of the iduronyl group to produce the 6-sulfated anhydromannose-containing product IDUA-P (Fig. 1A
). IDUA-IS is chemically identical to IDUA-P but is 5 Da heavier because of the presence of 5 deuterium atoms in the benzoyl group. IDUA-P and IDUA-IS are separately detected and quantified by ESI-MS/MS as their fragment ions after collision-induced elimination of SO3 (80-Da mass difference; Fig. 1A
). The 6-sulfate group present in the anhydromannosyl residue leads to a better substrate for IDUA, and thus a more sensitive assay [Ref. (9) and our unpublished data]. IDUA-S also contains a hydrophobic 1,6-diaminohexyl linker capped with a benzoyl group. The former allows for sample clean-up before ESI-MS/MS (see below), and the latter provides an additional hydrophobic moiety and a practical and inexpensive heavy-isotope tag.
To remove the buffer salts, which are present in relatively high concentrations, we used a simple solid-phase extraction step that is appropriate for high-throughput analyses. The presence of the hydrophobic benzoylated 1,6-diaminohexyl linker of IDUA-P and IDUA-IS allows these compounds to be retained on reversed-phase C18-silica gel, whereas the buffer salts pass through during the wash with 50 mL/L methanol in water. IDUA-P and IDUA-IS are then eluted with a single wash step with 500 mL/L methanol in water as the eluant.
The amount of IDUA-P increased linearly with reaction time from 0 to 30 h (Fig. 1 in the online Data Supplement); we therefore chose 24 h for assays. The amount of IDUA-P formed at 24 h increased as the concentration of IDUA-S increased from 0 to 0.5 mmol/L, and saturation was not achieved with 0.5 mmol/L substrate; thus, Km >0.5 mmol/L (Fig. 2 in the online Data Supplement) An IDUA-S concentration of 0.33 mmol/L was chosen for all assays.
As indicated in Fig. 3 of the online Data Supplement, the IDUA in DBS is stable for at least 4 years. IDUA activity in 14 patients [range, 0.007–0.11 µmol · h–1 · (L blood)–1; mean, 0.066 µmol · h–1 · (L blood)–1; median, 0.065 µmol · h–1 · (L blood)–1] was well below the range of activities in samples obtained from 48 unaffected newborns [4.54–12.6 µmol · h–1 · (L blood)–1; mean (median), 7.87 (7.22) µmol · h–1 · (L blood)–1; Fig. 1B
]. IDUA activity in the four MPS-I carriers was intermediate [1.07–1.48 µmol · h–1 · (L blood)–1; mean (median), 1.32 (1.36) µmol · h–1 · (L blood)–1], but more study will be needed to determine whether the values for carriers overlap IDUA activity values in unaffected newborns.
We have assayed five lysosomal enzymes in DBS by ESI-MS/MS with five substrates (8). We mixed the reaction mixtures from these assays with that from the MPS-I assays and analyzed all six reactions by ESI-MS/MS in multiple-reaction monitoring mode. The activities for the six enzymes measured in a single DBS (1 of the 48 newborn samples from Fig. 1B
) were as follows: Fabry, 6.99 µmol · h–1 · (L blood)–1; Gaucher, 9.04 µmol · h–1 · (L blood)–1; Krabbe, 2.73 µmol · h–1 · (L blood)–1; MPS-I, 9.4 µmol · h–1 · (L blood)–1; Niemann-Pick type A/B, 2.68 µmol · h–1 · (L blood)–1; and Pompe, 1.51 µmol · h–1 · (L blood)–1. All of these activities are in the ranges typical of healthy individuals (Fig. 1B
) (8).
This ESI-MS/MS-based assay for IDUA should be practical for high-throughput analysis appropriate for newborn-screening laboratories. The assay is compatible with microtiter plate and multichannel pipetting techniques (or robotics). Each IDUA assay requires only 13.3 µg of substrate, which can be readily prepared from commercially available heparin (to be published) and 0.1 µg of internal standard.
As with many assays that use synthetic substrates, endogenous IDUA substrate present in DBS can affect the enzymatic velocity measured with the IDUA-S assay, particularly because the concentration of IDUA-S is below its Km. Presumably this effect would enhance the difference seen between unaffected and affected patients because the amount of accumulated natural substrate would be higher in DBS from affected patients. Thus, the IDUA velocities measured are not deemed to be accurate measurements of the residual enzymatic activity and are used only as a screening guide.
In our previous study (8), we used a combination of parent-ion scanning and neutral-loss scanning to collect ESI-MS/MS data on five lysosomal enzymes. We can now analyze these five enzymes plus IDUA by use of the multiple-reaction monitoring mode, which simplifies the data acquisition protocol. For the IDUA assay, however, we had to resort to a reversed-phase C18-silica filter plate to remove buffer salts because the reaction product contains a polar anhydromannosyl-6-sulfate residue, which would bind tightly to the silica gel used in our previous method (8). We envision the combined use of silica and reversed-phase C18-silica plates for the assay of a large number of enzymes. After solid-phase extraction using these two media, the eluates can be combined for a single infusion into the mass spectrometer. Pilot studies with tens of thousands of samples are being initiated to develop these ESI-MS/MS assays for newborn screening of several lysosomal storage diseases.
Acknowledgments
This work was supported by grants from the NIH (DK67859) and from Genzyme Inc. We are grateful to Dr. Joan Keutzer and Helmut Kallwaas (Genzyme, Inc.) for helpful discussions. We are also grateful to Ana Maria Martins, Universidade Federal de Sao Paulo, Brazil, for supplying us with some of the DBS used in this study. The sponsors had no role in the design, conduct, data interpretation, or reporting of the study.
References
-L-iduronidase deficiency in dried blood spots on filter paper: the possibility of newborn diagnosis. Clin Chem 2001;47:780-781.-glucosidase activity in blood spots as a diagnostic test for Pompe disease. Clin Chem 2001;47:1378-1383.-L-iduronide for the estimation of -L-iduronidase activity and the detection of Hurler and Scheie syndromes. Clin Chim Acta 1979;92:257-265.[CrossRef][Web of Science][Medline]
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indicates the mean. IDUA activity values for each sample are given in Table 1 of the online Data Supplement. Data are for 14 MPS-I patients, 4 carriers, and 48 random unaffected newborns.