Ann Lab Med.  2013 Jul;33(4):274-278. 10.3343/alm.2013.33.4.274.

Analysis of Lyso-Globotriaosylsphingosine in Dried Blood Spots

• Johnson B
• Mascher H
• Mascher D
• Legnini E
• Hung CY
• Dajnoki A
• Chien YH
• Marodi L
• Hwu WL
• Bodamer OA

• KMID: 1707235
• DOI: http://doi.org/10.3343/alm.2013.33.4.274

Abstract

Recently, lyso-globotriaosylsphingosine (lyso-Gb3) was found to be elevated in plasma of treatment naive male patients and some female patients with Fabry Disease (FD). This study tested whether lyso-Gb3 could be analyzed in dried blood spots (DBS) from filter cards and whether concentrations are elevated in newborn infants with FD. Lyso-Gb3 concentrations were analyzed in DBS following extraction using a novel HPLC-mass spectrometry (MS)/MS method. Lyso-Gb3 levels in DBS were above the lower limit of quantitation (0.28 ng/mL) in 5/17 newborn FD infants (16 males; range: 1.02-8.81 ng/mL), but in none of the newborn controls, in all 13 patients (4 males) with classic FD (range: 2.06-54.1 ng/mL), in 125/159 Taiwanese individuals with symptomatic or asymptomatic FD who carry the late onset alpha-galactosidase A (GLA) mutation c.936+919G>A (IVS4+919G>A) (3.75+/-0.69 ng/mL; range: 0.418-3.97 ng/mL) and in 20/29 healthy controls (0.77+/-0.24 ng/mL; range: 0.507-1.4 ng/mL). The HPLC-MS/MS method for analysis of lyso-Gb3 is robust and yields reproducible results in DBS in patients with FD. However, concentrations of lyso-Gb3 were below the limit of quantitation in most newborn infants with FD rendering this approach not suitable for newborn screening. In addition, most females with the late onset mutation have undetectable lyso-Gb3 concentrations.

Keyword

Fabry disease; Dried blood spot; Filter card; Tandem mass spectrometry

MeSH Terms

Adolescent
Adult
Blood Chemical Analysis/*methods
Child
Chromatography, High Pressure Liquid
*Dried Blood Spot Testing
Fabry Disease/blood/diagnosis
Female
Glycolipids/*blood
Humans
Infant, Newborn
Male
Sphingolipids/*blood
Tandem Mass Spectrometry
Young Adult
Glycolipids
Sphingolipids

Figure

• 

  Fig. 1 Representative chromatograms from a normal control individual (A) and a male with Fabry Disease (FD) (B). Increased levels of lyso-globotriaosylsphingosine (lyso-Gb3) are detected in males with FD.

• 

  Fig. 2 (A) Lyso-Gb3 levels in classical FD patients and newborns with FD. Classic FD patients have higher levels of lyso-Gb3 than newborns with FD (Kruskal-Wallis, P=1.154×10-4). (B) Lyso-Gb3 levels in healthy controls and late onset FD patients. Lyso-Gb3 levels are not statistically different between healthy controls and the Taiwanese patients with symptomatic or asymptomatic FD, who carry the late onset GLA mutation c.936+919G>A (IVS4+919G>A; Kruskal-Wallis, P=0.159). (C) Lyso-Gb3 levels in classic FD patients and late onset FD patients. Patients with classic FD have higher lyso-Gb3 levels than the Taiwanese patients with symptomatic or asymptomatic FD, who carry the late onset GLA mutation c.936+919G>A (IVS4+919G>A; Wilcoxon, P=7.756×10-8).Abbreviations: Lyso-Gb3, lyso-globotriaosylsphingosine; FD, Fabry Disease; LLOQ, Lower limit of quantitation; GLA, α-galactosidase A.

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