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Technical Briefs |
Departments of1 Chemistry, 3 Pediatrics, and 4 Biochemistry, University of Washington, Seattle, WA 2 Department of Pediatrics and Neuropediatrics, Children’s Hospital, University of Goettingen, Goettingen, Germany
aaddress correspondence to this author at: Departments of Chemistry and Biochemistry, University of Washington, Campus Box 351700, Seattle, WA 98195; fax 206-685-8665, e-mail gelb{at}chem.washington.edu
Tandem mass spectrometry (MS/MS) for newborn screening allows detection of abnormal or excess metabolites in dried blood spots (1)(2)(3)(4)(5). Dried blood spots provide a source of active enzymes for the detection of Fabry (6), Hunter(7), Hurler (7), Pompe(8), Gaucher(9), Niemann–Pick (9), and Tay–Sachs(10) diseases. We investigated whether MS/MS can be used to directly assay enzymes in dried blood spots, in this case, galactocerebroside ß-galactosidase (GALC; EC 3.2.1.46) for the detection of Krabbe disease. Development of a GALC assay is challenging because of the low GALC activity in cell lysates. It has been argued that a neonatal screening method for Krabbe disease is needed (11). This is the first report of an assay for Krabbe disease that uses dried blood spots and the first use of MS/MS for direct enzyme assay from newborn-screening cards.
The GALC assay uses commercially available reagents and is based on the reaction shown in Fig. 1
and the use of electrospray ionization (ESI) MS/MS. GALC converts ß-Gal-C8-Cer to C8-Cer. A known amount of substrate analog with two extra methylene groups, C10-Cer, is added as internal standard. After collision-induced dissociation, C8-Cer and C10-Cer give rise to the same fragment ion (m/z 264.3); thus C8-Cer and C10-Cer are distinguished by the Q1 quadrupole. The indicated fragmentation pathway is the major process, allowing for high-sensitivity detection of C8-Cer and C10-Cer.
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A simple, one-step, solid-phase extraction procedure was developed (see below) to remove the large amount of detergent before ESI-MS/MS. The product C8-Cer and internal standard C10-Cer have nearly identical ESI-MS/MS ionization efficiencies (see Fig. 1
in the online Data Supplement that accompanies this Technical Brief at http://www.clinchem.org/content/vol50/issue3/). Studies with a second internal standard, C12-Cer, showed essentially no loss of C8-Cer product and C10-Cer internal standard during sample workup and the assay incubation period.
Dried blood spots were obtained in compliance with Institutional Review Board review, were stored at -20 °C (no longer than 10 days at room temperature during transport) in zip-lock plastic bags (three layers), and were analyzed within 70 days. The GALC assay mixture was prepared as follows. D-Galactosyl-ß1-1'-ceramide (C8:0; ß-Gal-C8-Cer; Avanti) substrate stock solution (200 µL of 3 mmol/L solution in methanol) and ceramide (C10:0; C10-Cer; Avanti) internal standard stock solution (240 µL of 0.05 mmol/L solution in methanol) were added to a 5-mL glass vial, and the solvent was removed in a vacuum desiccator. We then added 240 µL of an aqueous solution of sodium taurocholate (120 g/L) and oleic acid (12 g/L) to the vial, vortex-mixed the sample until the residue dissolved, and then added 2.76 mL of citrate–phosphate buffer, pH 4.5 (0.2 mol/L Na2HPO4, 0.1 mol/L sodium citrate; pH adjusted with 6 mol/L HCl). The vial was vortex-mixed, and any emulsion was broken up by centrifugation. The stock assay solution (3.0 mL) contained 0.2 mmol/L substrate, 4.0 µmol/L internal standard, 9.6 g/L sodium taurocholate, 0.96 g/L oleic acid, and citrate–phosphate, pH 4.5 and was stored at -20 °C (multiple freeze–thaw cycles tolerated).
To each 1.7-mL Eppendorf tube we added a 2-mm diameter dried blood spot followed by 25 µL of GALC assay mixture. Tubes were briefly centrifuged at 5000g to pellet the liquid. Tubes were incubated for 20–24 h at 37 °C with orbital shaking (150 rpm) in an air shaker, after which 400 µL of methanol–CHCl3 (2:1 by volume) was added. The liquid was aspirated up and down five times with a Pipetman P1000, and 350 µL of CHCl3 and 350 µL of H2O were added. The tube was centrifuged at 5000g for 2 min, after which the bottom layer (300 µL) was removed with a Pipetman P1000 and transferred to a glass-wool-plugged 1-mL Pipetman tip that was filled with silica gel (90–110 mg; 230–400 mesh; Merck). The pipet tip was placed in a glass tube, and solvent was pushed through the silica gel for 
2 s with compressed air. Methanol–CHCl3 (600 µL; 1:9 by volume) was applied to the silica gel and pushed through with compressed air. The solvent was removed in a vacuum desiccator, and the residue was suspended in 200 µL of 5 mmol/L ammonium formate in methanol–CHCl3 (3:1 by volume) for ESI-MS/MS.
ESI-MS/MS was performed on a Sciex API-III Plus tandem quadrupole instrument operating in positive-ion, parent-ion scan mode with the following settings: needle voltage (ISV), 4300 V; counter electrode (IN), 650 V; nebulizer gas, 35 psi; curtain gas, 1.2 L/min; skimmer voltage (OR), 45 V; quadrupole d.c. offset (R0), 30 V; quadrupole 1 resolution (RE1), 110; quadrupole 3 resolution (RE3), 105; collision gas number density (CGT), 150 x 1012 molecules/cm3; Q2 collision gas, argon; parent ion, m/z 264.0; dwell time, 3.0 ms; pause time, 0.052 s; step size, 0.2 atomic mass units (amu); Q1 scan range (C8-Cer), m/z 425.4–428.4; scan range (C10-Cer), m/z 453.4–456.4. A sum of 200 scans over 0.3 min was used for quantification. Samples were introduced by infusion at 3.3 µL/min through a fused-silica capillary (100 µm i.d.) with a syringe drive. The capillary was flushed with acetonitrile (4 times 50 µL) after each sample. The amount of product was calculated from the ion abundance ratio of product (C8-Cer) to internal standard (C10-Cer) for a sample minus that of a blank, multiplied by the amount of added internal standard and divided by the response ratio of product to internal standard.
ESI-MS/MS parent ion scans for the GALC assay performed with a single 2-mm dried blood spot as well as a control assay containing a 2-mm blood-free paper disk are shown in Fig. 2 in the online Data Supplement. The product-to-internal standard mole ratio was typically approximately eightfold greater in the blood-containing sample than in the control. Assay optimization showed maximum GALC activity at pH 4.5 and 9 g/L sodium taurocholate (Fig. 3 in the online Data Supplement). The amount of C8-Cer product increased linearly with the incubation time from 0 to 24 h, and the reaction velocity showed saturation kinetics with variation of the substrate concentration [Km = 0.24 mmol/L; Vmax = 4.0 µmol · h-1 · (L blood)-1; Fig. 4 in the online Data Supplement].
We carried out 14 assays using 2-mm blood spots from the same individual. The CV for the amount of product after 24 h was 9.6%. We submitted the extract from a single assay to 17 ESI-MS/MS measurements over a 2-month period (extract was stored at room temperature); the CV was 5.6%. Thus the GALC assay is highly precise, and the MS signals for C8-Cer and C10-Cer are highly reproducible. When dried blood spots from four individuals were stored at -20 °C or room temperature for 70 days, the GALC activity decreased by a mean (SD) of 9.6 (3.8)% and 40.5 (5.1)%, respectively. Storage of dried blood spots at room temperature for 10 days led to a 9.5 (3.9)% decrease in GALC activity (data obtained from dried blood spots from two healthy individuals and two patients with Krabbe disease). Thus, GALC activity in dried blood spots is sufficiently stable to permit transport of samples at ambient temperature.
We measured the GALC enzyme activity in dried blood spots from 16 healthy individuals and from 5 individuals who were previously diagnosed with Krabbe disease. We also carried out GALC assays on samples from four patients with Fabry disease and three with Gaucher disease. The results are summarized in Table 1
. Activity among healthy individuals ranged from 1.4 to 3.7 µmol · h-1 · (L blood)-1, whereas activity for Krabbe patients was much lower [0.05–0.23 µmol · h-1 · (L blood)-1]. For the patients with Fabry and Gaucher disease, the GALC assay gave values that are typical of those obtained for healthy individuals, although a larger range of specific activity values was observed (Table 1
). These results suggest that an assay for GALC in dried blood spots from newborns can be implemented and evaluated for the early detection of Krabbe disease.
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The GALC assay most often reported in the literature is based on a radiometric assay (12). The ESI-MS/MS assay avoids the use of radioactive material, which may not be practical for high-throughput newborn screening. Measurement of GALC activity with a series of substrates that differed in the lengths of their fatty acyl groups revealed that C8:0 ceramide gives fivefold higher activity than substrates containing ceramides with the longer chain fatty acids found in natural ceramides (12), a factor that was important in our ability to detect GALC in dried blood spots. The use of ß-Gal-C8-Cer also has an advantage in that it generates the unnatural product C8-Cer, thus avoiding interference from the natural ceramides present in biological specimens.
Because the enzymatic products are resolved on the basis of their m/z ratios, we are exploring the use of MS/MS for the quantification of multiple enzymes in a single dried blood spot by use of a single-platform analytical method in much the same way that MS/MS is currently used to determine the concentrations of multiple metabolites in a single dried blood spot.
Acknowledgments
This work was supported by grants from Genzyme Inc. and from the NIH (Grant DK67859). We are grateful to Dr. Martin Sadilek for technical assistance with ESI-MS/MS.
References
-glucosidase activity in blood spots as a diagnostic test for Pompe disease. Clin Chem 2001;47:1378-1383.The following articles in journals at HighWire Press have cited this article:
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  S. Blanchard, M. Sadilek, C. R. Scott, F. Turecek, and M. H. Gelb Tandem Mass Spectrometry for the Direct Assay of Lysosomal Enzymes in Dried Blood Spots: Application to Screening Newborns for Mucopolysaccharidosis I Clin. Chem., December 1, 2008; 54(12): 2067 - 2070. [Abstract] [Full Text] [PDF]
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D. Wang, T. Wood, M. Sadilek, C. R. Scott, F. Turecek, and M. H. Gelb Tandem Mass Spectrometry for the Direct Assay of Enzymes in Dried Blood Spots: Application to Newborn Screening for Mucopolysaccharidosis II (Hunter Disease) Clin. Chem., January 1, 2007; 53(1): 137 - 140. [Abstract] [Full Text] [PDF]
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 H. W. Moser Peripheral nerve involvement in Krabbe disease: a guide to therapy selection and evaluation. Neurology, July 25, 2006; 67(2): 201 - 202. [Full Text] [PDF]
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D. S. Millington Newborn Screening for Lysosomal Storage Disorders Clin. Chem., May 1, 2005; 51(5): 808 - 809. [Full Text] [PDF]
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Y. Li, C. R. Scott, N. A. Chamoles, A. Ghavami, B. M. Pinto, F. Turecek, and M. H. Gelb Direct Multiplex Assay of Lysosomal Enzymes in Dried Blood Spots for Newborn Screening Clin. Chem., October 1, 2004; 50(10): 1785 - 1796. [Abstract] [Full Text] [PDF]
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