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Liquid Chromatography–Tandem Mass Spectrometry Quantification of Globotriaosylceramide in Plasma for Long-Term Monitoring of Fabry Patients Treated with Enzyme Replacement Therapy

¹ Clinical Laboratory Science, and ² Protein Characterization & Modification, Genzyme Corporation, One Mountain Road, Framingham, MA 01701-9322

^aauthor for correspondence: fax 508-820-7664, e-mail Crystal.Sung@genzyme.com

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

Fabry disease is a rare X-linked lysosomal storage disorder resulting from a deficiency in the -galactosidase A enzyme. Deficiency in the activity of this enzyme causes an accumulation of neutral glycosphingolipids, predominantly globotriaosylceramide (GL-3), in most nonneural tissues and in body fluids (1). Recent clinical studies indicate that tissue and plasma GL-3 concentrations in Fabry patients can be significantly reduced by enzyme replacement therapy (2)(3)(4)(5).

GL-3 (see Fig. 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue1/) exists as a mixture of structural isoforms containing acyl chains ranging from 16 to 24 carbons in length with various degrees of saturation and hydroxylation. These variations make the extraction and quantification of GL-3 challenging (6)(7). Moreover, no well-characterized reference standards of known purity are available.

GL-3 in tissues and plasma has been measured by thin-layer chromatography (8)(9), liquid chromatography (LC) (10)(11)(12)(13)(14)(15), and gas chromatography(16), but the methods are labor-intensive and slow. An enzyme-linked immunosorbent assay (17) requires recombinant verotoxin B and polyclonal rabbit anti-verotoxin B. To date, the most rapid quantitative assays for total GL-3 have used flow-injection tandem mass spectrometry (MS/MS) (18)(19). We now report the development and use of a rapid LC/MS/MS method for the quantitative determination of total plasma GL-3.

Porcine GL-3 and porcine globotriaosylsphingosine A (lyso-GL-3), from Matreya, were 98% pure by thin-layer chromatographic analysis. C16:0-GL-3 and C17:0-GL-3 were enzymatically synthesized from lyso-GL-3 at Genzyme Pharmaceuticals (18). C16:0-enriched GL-3 was prepared by gravimetrically combining C16:0-GL-3 and porcine GL-3 in a 9:25 g/g mass ratio. Methanol, water, and . . . [Full Text of this Article]