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Clinical Chemistry 50: 638-640, 2004; 10.1373/clinchem.2003.028381
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(Clinical Chemistry. 2004;50:638-640.)
© 2004 American Association for Clinical Chemistry, Inc.

Technical Briefs

Tandem Mass Spectrometry for the Direct Assay of Enzymes in Dried Blood Spots: Application to Newborn Screening for Krabbe Disease

Yijun Li1, Knut Brockmann2, Frantisek Turecek1, C. Ronald Scott3 and Michael H. Gelb1,4,a

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@chem.washington.edu

The first 300 words of the full text of this article appear below.

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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  		Figure 1. 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 . . . [Full Text of this Article]




The following articles in journals at HighWire Press have cited this article:


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 Clin. Chem.Home page
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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 NeurologyHome page
H. W. Moser
Peripheral nerve involvement in Krabbe disease: a guide to therapy selection and evaluation.
Neurology, July 25, 2006; 67(2): 201 - 202.
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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]

Home page
 Clin. Chem.Home page
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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