scholarly journals Tandem Mass Spectrometry for the Direct Assay of Lysosomal Enzymes in Dried Blood Spots: Application to Screening Newborns for Mucopolysaccharidosis I

2008 ◽  
Vol 54 (12) ◽  
pp. 2067-2070 ◽  
Author(s):  
Sophie Blanchard ◽  
Martin Sadilek ◽  
C Ronald Scott ◽  
Frantisek Turecek ◽  
Michael H Gelb

Abstract Background: Treatments now available for mucopolysaccharidosis I require early detection for optimum therapy. Therefore, we have developed an assay appropriate for newborn screening of the activity of the relevant enzyme, α-L-iduronidase. Methods: We synthesized a new α-L-iduronidase substrate that can be used to assay the enzyme by use of tandem mass spectrometry together with an internal standard or by fluorometry. The assay uses a dried blood spot on a newborn screening card as the enzyme source. The assay protocol uses a simple liquid-liquid extraction step before mass spectrometry. We optimized enzyme reaction conditions and procedures for the assay, including the concentration of substrate, the reaction pH, the incubation time, and mass spectrometer operation. We also assessed inter- and intraassay imprecision. Results: When the assay was tested on dried blood spots, the α-L-iduronidase activity measured for 5 patients with mucopolysaccharidosis I was well below the interval found for 10 randomly chosen newborns. Inter- and intraassay imprecision were <10%. The synthesis of the α-L-iduronidase substrate is practical for use on a scale needed to support newborn screening demands. Conclusions: This newly developed tandem mass spectrometry assay has the potential to be adopted for newborn screening of mucopolysaccharidosis I. This assay has advantages over a previously reported assay also developed in this laboratory and has the potential to be performed in a multiplex fashion to measure several lysosomal enzymes relevant to treatable lysosomal storage diseases.

2010 ◽  
Vol 56 (12) ◽  
pp. 1854-1861 ◽  
Author(s):  
Trisha A Duffey ◽  
Garland Bellamy ◽  
Susan Elliott ◽  
Angela C Fox ◽  
Michael Glass ◽  
...  

BACKGROUND We sought to develop a tandem mass spectrometry assay in which the enzymatic activities of 3 lysosomal enzymes (α-glucosidase, α-galactosidase A, and α-l-iduronidase) could be quantified in dried blood spots by using a single assay buffer. METHODS A 3-mm dried blood spot punch was incubated in a single assay buffer with 3 different substrates and internal standards. The sample was processed by a simple liquid-liquid extraction by using ethyl acetate. The extract was dried down and resuspended in solvent for injection into the tandem mass spectrometer. Products and internal standards were monitored by multiple reaction monitoring. RESULTS Assay for the 3 lysosomal enzymes was successfully achieved with acceptable statistics. The assay can be performed by using a minimal quantity of disposable supplies and equipment. The entire procedure fits into a 48-h cycle including data analysis. Data from 5990 anonymous newborn dried blood spots showed an approximate bell-shaped distribution of enzymatic activities (mean values of 19.0, 11.5, and 3.5 μmol · h−1 · (L blood)−1 for α-glucosidase, α-galactosidase A, and α-l-iduronidase, respectively. Blank values obtained in the absence of blood were 0.13, 0.24, and 0.45 μmol · h−1 · (L blood)−1, respectively). By assaying 3 enzymes at once, problematic samples are spotted for reanalysis if enzyme activity values are low for all enzymes (for example, if insufficient blood is present in the assay). CONCLUSIONS This method demonstrates that a triplex assay in a single buffer and with minimal supplies and labor can be adapted to a high-throughput newborn screening laboratory for the analysis of Pompe, Fabry, and mucopolysaccharidosis-I (Hurler) diseases.


2007 ◽  
Vol 53 (1) ◽  
pp. 137-140 ◽  
Author(s):  
Ding Wang ◽  
Tim Wood ◽  
Martin Sadilek ◽  
C Ronald Scott ◽  
Frantisek Turecek ◽  
...  

Abstract Background: A treatment for mucopolysaccharidosis II (Hunter syndrome) has recently become available. Therefore, we developed a high-throughput assay method appropriate for newborn screening for the relevant enzyme, iduronate 2-sulfatase. Methods: We synthesized a new iduronate 2-sulfatase substrate that can be used to assay the enzyme by use of tandem mass spectrometry together with an internal standard. The assay uses a dried blood spot on a newborn screening card as the enzyme source. Results: When the assay was tested on dried blood spots, the iduronate 2-sulfatase activity measured for 13 patients with Hunter syndrome was well below the interval found for 57 randomly chosen newborns. The assay was more sensitive than previously reported iduronate 2-sulfatase assays. Conclusions: This newly developed tandem mass spectrometry assay has the potential to be adopted for newborn screening of Hunter syndrome. This method also has the potential to be carried out in multiplex fashion to assay several different enzymes relevant to lysosomal storage diseases that are assayed in a single infusion into the mass spectrometer.


2014 ◽  
Vol 47 (15) ◽  
pp. 144
Author(s):  
Lawrence J. Fisher ◽  
Osama Y. Al-Dirbashi ◽  
Svetlana Ogrel ◽  
Nathan McIntosh ◽  
Michael T. Geraghty ◽  
...  

1999 ◽  
Vol 45 (8) ◽  
pp. 1269-1277 ◽  
Author(s):  
Donald H Chace ◽  
Barbara W Adam ◽  
S Jay Smith ◽  
J Richard Alexander ◽  
Steven L Hillman ◽  
...  

Abstract Background: Advances in technology and the earlier release of newborns from hospitals have pressed the demand for accurate calibration and improved interlaboratory performance for newborn screening tests. As a first step toward standardization of newborn screening aminoacidopathy tests, we have produced six-pool sets of multianalyte dried-blood-spot amino acid reference materials (AARMs) containing predetermined quantities of five amino acids. We describe here the production of the AARMs, validation of their amino acid contents, and characterization of their homogeneity and their stability in storage. Methods: To each of six portions of a pool of washed erythrocytes suspended in serum we added Phe (0–200 mg/L), Leu (0–200 mg/L), Met (0–125 mg/L), Tyr (0–125 mg/L), and Val (0–125 mg/L). Six-pool sets (1300) were prepared, dried, and packaged. We used isotope-dilution mass spectrometry to estimate the endogenous amino acid concentrations of the AARMs and validate their final amino acid concentrations. We used additional tandem mass spectrometry analyses to examine the homogeneity of amino acid distribution in each AARM, and HPLC analyses to evaluate the stability of the amino acid contents of the AARMs. Results: The absolute mean biases across the analytic range for five amino acids were 2.8–9.4%. One-way ANOVAs of the homogeneity results predicted no statistically significant differences in amino acid concentrations within the blood spots or within the pools (P >0.05). Regression slopes (0 ± 0.01) for amino acid concentrations vs storage times and their P values (>0.05) showed no evidence of amino acid degradation at ambient temperatures, 4 °C, or −20 °C during the intervals tested. Conclusion: The validation, homogeneity, and stability of these blood spots support their use as a candidate national reference material for calibration of assays that measure amino acids in dried-blood spots.


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