Mass spectrometric quantification of glucosylsphingosine in plasma and urine of type 1 Gaucher patients using an isotope standard
Introduction
Gaucher disease (GD) is caused by deficiency of the enzyme glucocerebrosidase (GBA) resulting in lysosomal accumulation of its lipid substrate glucosylceramide (GlcCer) in tissue macrophages. These lipid-laden phagocytes, named Gaucher cells, prominently accumulate in liver, spleen, and bone marrow of GD patients. Their presence is thought to cause the hepatosplenomegaly, pancytopenia and bone complications expressed by GD patients [1]. The majority of GD patients develop a non-neuropathic (type 1) course of disease. More rarely, in severely affected patients the clinical manifestation involves the central nervous system at infantile or juvenile age (type 2 and type 3 GD, respectively). Two therapeutic interventions are available for treatment of visceral manifestations of GD. Firstly, enzyme replacement therapy (ERT), based on chronic intravenous administration of recombinant GBA, results in prominent improvement of visceral symptoms [2], [3], [4]. With substrate reduction therapy (SRT), oral administration of the iminosugar N-butyl-deoxynojirimycin aims to reduce GlcCer synthesis and thus reduce its accumulation [5]. SRT with Miglustat is used for mildly to moderately affected patients in whom ERT is not a therapeutic option [6]. Very recently the improved inhibitor Eliglustat has also been registered by the FDA as drug for treatment of type 1 GD [7], [8]. The availability of costly therapies for type 1 GD has stimulated the search for biomarkers that can assist in diagnosis and individualised patient management. Several protein biomarkers for type 1 GD have been identified in the blood of patients. At least two of these, chitotriosidase and CCL18, are known to be produced by Gaucher cells and directly secreted into the blood [9], [10]. Plasma chitotriosidase and CCL18 levels reflect disease progression and are presently used to monitor disease progression and response to therapeutic intervention [11]. Of note, the primary storage lipid GlcCer is only modestly increased in blood of symptomatic type 1 GD patients [12]. In contrast, glucosylsphingosine (GlcSph), the deacylated form of GlcCer, has been found to be markedly increased in plasma of type 1 GD patients [13]. This finding was confirmed in a more recent study by Rolfs et al. [14]. Our initial investigation showed an average 200 fold elevation in plasma GlcSph levels in symptomatic type 1 GD patients examined prior to ERT. In response to therapy, plasma GlcSph was found to decrease, mimicking corrections of chitotriosidase and CCL18 [13]. Inhibition of GBA activity in cultured macrophages with a highly specific irreversible inhibitor led within one day to a sharp increase in GlcSph, again supporting the idea that visceral Gaucher cells are a major source of plasma GlcSph [13]. Recent investigations with an induced Gaucher mouse model also detected elevations in plasma GlcSph in symptomatic animals [15], [16], [17].
Given the interest in GlcSph as potential biomarker for GD, we improved its mass spectrometric detection. For this purpose we synthesised [5–9] 13C5-GlcSph for use as internal standard with quantitative LC-ESI-MS/MS. We here report on the improved quantification of GlcSph manifestation and therapy of type 1 GD. In addition, we here describe the prominent occurrence in GD urine, but not blood, of additional elevated m/z transitions reflecting isoforms of GlcSph differing in the sphingosine moiety from regular GlcSph. This finding resembles observations earlier made for heterogeneity in lysoGb3 in urine of Fabry disease patients [18].
Section snippets
Materials and standards
LC-MS grade methanol, water, formic acid, and HPLC grade chloroform were purchased from Biosolve (Valkenswaard, The Netherlands).and LC-MS grade butanol from Merck Millipore, Billerica, USA. Ammonium formate and GlcSph (D-glucosyl-β1-1′-D-erythro-sphingosine) were obtained from Sigma-Aldrich and Avanti Polar Lipids (Alabaster, USA), respectively.
Patients, plasma and urine samples
EDTA plasma (28 males and 27 females) and 24 hour urine samples were collected prior to therapy from Dutch patients (17 males, 13 females) suffering
[5–9] 13C5-labelled GlcSph detection by LC-MS/MS
To set up an improved LC-tandem mass spectrometric assay to quantify GlcSph in plasma and urine, we evaluated the use of [5–9] 13C5-labelled GlcSph as internal standard. First the detection conditions were optimised by direct infusion of unlabelled and isotope labelled glucosylsphingosine. Two abundant peaks with m/z of 462.3 and 467.3 were detected, corresponding to the single protonated form (M + H)+ of unlabelled and isotope labelled GlcSph, respectively (Fig. 1A). Fragmentation of either
Discussion
Kanfer et al. were the first to identify GlcSph in spleen of GD patients [27]. Next, accumulation of this sphingoid base in brain of GD patients was found to correlate with central nervous system (CNS) involvement [28], [29]. Following the availability of more sensitive detection methods, we again looked into the presence of GlcSph in plasma of type 1 GD patients. We and Rolfs et al. demonstrated a several hundred fold increase in GlcSph levels in plasma of GD patients [13], [14]. It was
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