{"doi":"10.1373/clinchem.2003.028381","title":"Tandem Mass Spectrometry for the Direct Assay of Enzymes in Dried Blood Spots: Application to Newborn Screening for Krabbe Disease","abstract":null,"journal":"Clinical Chemistry","year":2004,"id":684886,"datarank":0.6554171778700533,"base_score":4.3694478524670215,"endowment":4.3694478524670215,"self_citation_contribution":0.6554171778700533,"citation_network_contribution":0.0,"self_endowment_contribution":0.6554171778700533,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":78,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":965912,"name":"Knut Brockmann","orcid":"0000-0001-6823-9091","position":1,"is_corresponding":false},{"id":1559468,"name":"Frantisek Turecek","orcid":null,"position":2,"is_corresponding":false},{"id":1789363,"name":"C Ronald Scott","orcid":null,"position":3,"is_corresponding":false},{"id":1743654,"name":"Michael H Gelb","orcid":null,"position":4,"is_corresponding":false},{"id":809018,"name":"Yijun Li","orcid":"0000-0003-0513-9565","position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Tandem Mass Spectrometry for the Direct Assay of Enzymes in Dried Blood Spots: Application to Newborn Screening for Krabbe Disease","abstract":"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 β-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 β-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. Reaction scheme for the assay of GALC by ESI-MS/MS. Shown is the GALC reaction, the monoprotonated ions derived from the GALC reaction product (C8-Cer) and internal standard (C10-Cer), and the dominant collision-induced dissociation (CID) of these ions to give the common ion of m/z 264.3, which is detected by ESI-MS/MS with a triple-quadrupole instrument. 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-β1-1′-ceramide (C8:0; β-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 × 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. GALC activities measured in 2-mm dried blood spots. The mean GALC activity [0.30 μmol · h−1 · (L blood)−1] measured in 2-mm blood-free paper disks [values from four determinations were 0.27, 0.31, 0.31, and 0.31 μmol · h−1 · (L blood)−1] has been subtracted from the observed GALC activities to give the values listed in the table. All GALC activities were calculated assuming that a 2-mm paper disk contains 1.27 μL of blood (13). GALC activities measured in 2-mm dried blood spots. The mean GALC activity [0.30 μmol · h−1 · (L blood)−1] measured in 2-mm blood-free paper disks [values from four determinations were 0.27, 0.31, 0.31, and 0.31 μmol · h−1 · (L blood)−1] has been subtracted from the observed GALC activities to give the values listed in the table. All GALC activities were calculated assuming that a 2-mm paper disk contains 1.27 μL of blood (13). 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 β-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. 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.","is_dataset_classified":null,"base_score":4.3694478524670215,"endowment":4.3694478524670215,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"14981030","pmcid":null,"openalex_id":"https://openalex.org/W2054921761","authors":[],"funders":[{"funder_name":"NIDDK NIH HHS","grant_id":"DK67859","title":null}],"total_grants":1,"fwci":2.1394,"citation_percentile":0.86364514,"influential_citations":0,"citation_trend":[{"year":2012,"count":4},{"year":2013,"count":3},{"year":2014,"count":3},{"year":2015,"count":2},{"year":2016,"count":4},{"year":2017,"count":5},{"year":2018,"count":8},{"year":2019,"count":2},{"year":2020,"count":3},{"year":2021,"count":1},{"year":2022,"count":3},{"year":2023,"count":3},{"year":2024,"count":1}],"oa_status":"closed","license":"https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model","oa_locations":[{"url":"http://academic.oup.com/clinchem/article-pdf/50/3/638/32735663/clinchem0638.pdf","host_type":"publisher"},{"url":"https://doi.org/10.1373/clinchem.2003.028381","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/14981030","host_type":"repository"}],"fields_of_study":["Lysosomal Storage Disorders Research","Metabolism and Genetic Disorders","Glycosylation and Glycoproteins Research"],"mesh_terms":["Blood Specimen Collection","Clinical Enzyme Tests","Humans","Infant, Newborn","Leukodystrophy, Globoid Cell","Neonatal Screening","Spectrometry, Mass, Electrospray Ionization"],"keywords":["Dried blood","Spots","Newborn screening","Krabbe disease","Dried blood spot","Chromatography","Mass spectrometry","Tandem mass spectrometry","Chemistry","Medicine","Disease","Biochemistry","Pathology"],"sdg_mappings":[{"sdg_number":0,"sdg_label":"Good health and well-being"}],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-18T15:15:05.982241Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}