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One-step synthesis of carbon-13-labeled globotriaosylsphingosine (lyso-Gb3), an internal standard for biomarker analysis of Fabry disease.

ABSTRACT: Globotriaosylsphingosine (lyso-Gb3) is a well-established biomarker for diagnosis and prognosis of Fabry disease. This biomarker is measured in biological samples by liquid chromatography-tandem mass spectrometry using an internal standard. The ideal internal standard is a variant of lyso-Gb3 substituted with heavy isotopes, but the total synthesis of such a compound is very labor intensive. In this report, we describe a simple, one-step synthesis of lyso-Gb3 labeled with carbon-13 in all of the galactosyl carbons.

SUBMITTER: Hong X

PROVIDER: S-EPMC6239936 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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One-step synthesis of carbon-13-labeled globotriaosylsphingosine (lyso-Gb3), an internal standard for biomarker analysis of Fabry disease.

Hong Xinying X Gelb Michael H MH

Molecular genetics and metabolism 20180810 3

Globotriaosylsphingosine (lyso-Gb3) is a well-established biomarker for diagnosis and prognosis of Fabry disease. This biomarker is measured in biological samples by liquid chromatography-tandem mass spectrometry using an internal standard. The ideal internal standard is a variant of lyso-Gb3 substituted with heavy isotopes, but the total synthesis of such a compound is very labor intensive. In this report, we describe a simple, one-step synthesis of lyso-Gb3 labeled with carbon-13 in all of the ...[more]

PMID: 30126819

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Project description: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.

Project description:Fabry disease is an X-linked genetic disorder characterized by deficient activity of ?-galactosidase A (GLA) and accumulation of glycolipids, and various GLA gene mutations lead to a wide range of clinical phenotypes from the classic form to the later-onset one. To investigate the biochemical heterogeneity and elucidate the basis of the disease using available clinical samples, we measured GLA activity, GLA protein and accumulated globotriaosylsphingosine (Lyso-Gb3), a biomarker of this disease, in plasma samples from Fabry patients. The analysis revealed that both the enzyme activity and the protein level were apparently decreased, and the enzyme activity was well correlated with the protein level in many Fabry patients. In these cases, a defect of biosynthesis or excessive degradation of mutant GLAs should be involved in the pathogenesis, and the residual protein level would determine the accumulation of Lyso-Gb3 and the severity of the disease. However, there are some exceptional cases, i.e., ones harboring p.C142Y, p.R112H and p.M296I, who exhibit a considerable amount of GLA protein. Especially, a subset of Fabry patients with p.R112H or p.M296I has been attracted interest because the patients exhibit almost normal plasma Lyso-Gb3 concentration. Structural analysis revealed that C142Y causes a structural change at the entrance of the active site. It will lead to a complete enzyme activity deficiency, resulting in a high level of plasma Lyso-Gb3 and the classic Fabry disease. On the other hand, it is thought that R112H causes a relatively large structural change on the molecular surface, and M296I a small one in a restricted region from the core to the surface, both the structural changes being far from the active site. These changes will cause not only partial degradation but also degeneration of the mutant GLA proteins, and the degenerated enzymes exhibiting small and residual activity remain and probably facilitate degradation of Lyso-Gb3 in plasma, leading to the later-onset phenotype. The results of this comprehensive analysis will be useful for elucidation of the basis of Fabry disease.

Dataset Information

One-step synthesis of carbon-13-labeled globotriaosylsphingosine (lyso-Gb3), an internal standard for biomarker analysis of Fabry disease.

Publications

One-step synthesis of carbon-13-labeled globotriaosylsphingosine (lyso-Gb3), an internal standard for biomarker analysis of Fabry disease.

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