Nano-LC-MS/MS for Quantification of Lyso-Gb3 and Its Analogues Reveals a Useful Biomarker for Fabry Disease.

Hideaki Sueoka,Junji Ichihara,Takahiro Tsukimura,Tadayasu Togawa,Hitoshi Sakuraba,Claudia Sommer

doi:10.1371/journal.pone.0127048

Hideaki Sueoka, Junji Ichihara + Show 4 more

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https://doi.org/10.1371/journal.pone.0127048

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Abstract

Biomarkers useful for diagnosis and evaluation of treatment for patients with Fabry disease are urgently needed. Recently, plasma globotriaosylsphingosine (lyso-Gb3) and lyso-Gb3-related analogues have attracted attention as promising biomarkers of Fabry disease. However, the plasma concentrations of lyso-Gb3 and its analogues are extremely low or below the detection limits in some Fabry patients as well as in healthy subjects. In this paper, we introduce the novel application of a nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) system to the measurement of lyso-Gb3 and its analogues in plasma. Nano-LC-MS/MS requires smaller amounts of samples and is more sensitive than conventional techniques. Using this method, we measured the plasma concentrations of lyso-Gb3 and its analogues in 40 healthy subjects, 5 functional variants (males with E66Q), and various Fabry patients (9 classic Fabry males/9 mutations; 7 later-onset Fabry males/5 mutations; and 10 Fabry females/9 mutations). The results revealed that the mean lyso-Gb3 and lyso-Gb3(-2) concentrations in all the Fabry patient subgroups were statistically higher, especially in the classic Fabry males, than those in the functional variants and healthy subjects. The plasma concentrations of lyso-Gb3 and its analogues in healthy subjects, functional variants, and some Fabry patients with specific mutations (R112H and M296I) that cannot be established by conventional techniques were successfully determined by means of nano-LC-MS/MS. The lyso-Gb3 and lyso-Gb3(-2) concentrations in male patients with these mutations were lower than those in most Fabry patients having other mutations, but higher than those in the functional variants and healthy subjects. This new method is expected to be useful for sensitive determination of the plasma concentrations of lyso-Gb3 and its analogues. This study also revealed that not only lyso-Gb3 but also lyso-Gb3(-2) in plasma is a useful biomarker for the diagnosis of Fabry disease.

Highlights

Fabry disease (OMIM No 301500) is an X-linked lysosomal storage disorder caused by a deficiency of an enzyme, α-galactosidase A (GLA, EC 3.2.1.22)
These results showed that nano-liquid chromatographymass spectrometry (LC-MS)/MS for quantification of plasma lyso-Gb3 was accurate, reproducible, and highly sensitive
We have developed a new sensitive method for measuring plasma lyso-Gb3 involving nanoLC-MS/MS

Summary

Introduction

Fabry disease (OMIM No 301500) is an X-linked lysosomal storage disorder caused by a deficiency of an enzyme, α-galactosidase A (GLA, EC 3.2.1.22). The GLA activity deficiency results in progressive accumulation of glycosphingolipids, predominantly globotriaosylceramide (Gb3), in organ tissues and body fluids [1]. Fabry disease exhibits clinical phenotypes ranging from the early-onset “classic form” to the “later-onset form”. Affected males with the classic form exhibit little or no GLA activity, and pain in the peripheral extremities, hypohidrosis, angiokeratomas, and corneal opacities in childhood or adolescence, and develop kidney, cardiac and cerebrovascular disorders in adulthood. Patients with the later-onset form usually have low residual GLA activity, and show milder clinical manifestations limited to the heart and/or kidneys [2]. The disease expression in Fabry females depends on random X-chromosomal inactivation and the clinical phenotypes are more heterogeneous than in males [3]

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