Quantification of globotriaosylsphingosine in plasma and urine of fabry patients by stable isotope ultraperformance liquid chromatography-tandem mass spectrometry.

BACKGROUND: Biochemical markers that accurately reflect the severity and progression of disease in patients with Fabry disease and their response to treatment are urgently needed. Globotriaosylsphingosine, also called lysoglobotriaosylceramide (lysoGb3), is a promising candidate biomarker. METHODS: We synthesized lysoGb3 and isotope-labeled [5,6,7,8,9] (13)C5-lysoGb3 (internal standard). After addition of the internal standard to 25 µL plasma or 400 µL urine from patients with Fabry disease and healthy controls, samples were extracted with organic solvents and the lysoGb3 concentration was quantified by UPLC-ESI-MS/MS (ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry). Calibration curves were constructed with control plasma and urine supplemented with lysoGb3. In addition to lysoGb3, lyso-ene-Gb3 was quantified. Quantification was achieved by multiple reaction monitoring of the transitions m/z 786.4 > 282.3 [M+H](+) for lysoGb3, m/z 791.4 > 287.3 [M+H](+) for [5,6,7,8,9] (13)C5-lysoGb3, and 784.4 > 280.3 [M+H](+) for lyso-ene-Gb3. RESULTS: The mean (SD) plasma lysoGb3 concentration from 10 classically affected Fabry hemizygotes was 94.4 (25.8) pmol/mL (range 52.7-136.8 pmol/mL), from 10 classically affected Fabry heterozygotes 9.6 (5.8) pmol/mL (range 4.1-23.5 pmol/mL), and from 20 healthy controls 0.4 (0.1) pmol/mL (range 0.3-0.5 pmol/mL). Lyso-ene-Gb3 concentrations were 10%-25% of total lysoGb3. The urine concentration of lysoGb3 was 40-480 times lower than in corresponding plasma samples. Lyso-ene-Gb3 concentrations in urine were comparable or even higher than the corresponding lysoGb3 concentrations. CONCLUSIONS: This assay for the quantification of lysoGb3 and lyso-ene-Gb3 in human plasma and urine samples will be an important tool in the diagnosis of Fabry disease and for monitoring the effect of enzyme replacement therapy in patients with Fabry disease.

Elevated plasma glucosylsphingosine in Gaucher disease: relation to phenotype, storage cell markers, and therapeutic response.

Gaucher disease, caused by a deficiency of the lysosomal enzyme glucocerebrosidase, leads to prominent glucosylceramide accumulation in lysosomes of tissue macrophages (Gaucher cells). Here we show glucosylsphingosine, the deacylated form of glucosylceramide, to be markedly increased in plasma of symptomatic nonneuronopathic (type 1) Gaucher patients (n = 64, median = 230.7 nM, range 15.6-1035.2 nM; normal (n = 28): median 1.3 nM, range 0.8-2.7 nM). The method developed for mass spectrometric quantification of plasma glucosylsphingosine is sensitive and robust. Plasma glucosylsphingosine levels correlate with established plasma markers of Gaucher cells, chitotriosidase (ρ = 0.66) and CCL18 (ρ = 0.40). Treatment of Gaucher disease patients by supplementing macrophages with mannose-receptor targeted recombinant glucocerebrosidase results in glucosylsphingosine reduction, similar to protein markers of Gaucher cells. Since macrophages prominently accumulate the lysoglycosphingolipid on glucocerebrosidase inactivation, Gaucher cells seem a major source of the elevated plasma glucosylsphingosine. Our findings show that plasma glucosylsphingosine can qualify as a biomarker for type 1 Gaucher disease, but that further investigations are warranted regarding its relationship with clinical manifestations of Gaucher disease.

Effects of switching from a reduced dose imiglucerase to velaglucerase in type 1 Gaucher disease: clinical and biochemical outcomes.

This paper describes the effects of a switch to velaglucerase alfa in a group of adult patients with type 1 Gaucher disease, all of whom had previously had their dose reduced as a consequence of the worldwide imiglucerase shortage. Thirty-two patients from two large European Gaucher centers switched to treatment with velaglucerase alfa after 1-8.5 months of dose reduction. The course of important Gaucher disease parameters was studied at four time points: one year before the shortage, just before the shortage, before a switch to velaglucerase and after up to one year of treatment with velaglucerase. These parameters included hemoglobin concentration, platelet count, plasma chitotriosidase activity in all patients, and spleen and liver volumes (as well as bone marrow fat fraction images) in 10 patients. Decreases in platelet counts as a result of reduced treatment with imiglucerase were quickly restored on treatment with velaglucerase alfa. Chitotriosidase activity declined overall after switching. Five out of 10 patients had an increase in liver volume of at least 10% after six months of velaglucerase treatment, which was reversible in 3. Most patients received infusions at home and no important side effects were observed. Velaglucerase alfa appears to be a safe and effective alternative for imiglucerase.

Gaucher disease due to saposin C deficiency, previously described as non-neuronopathic form--no positive effects after 2-years of miglustat therapy.

Gaucher disease occurs mainly as a result of a deficiency of the lysosomal enzyme beta-glucocerebrosidase activity. A rare variant form of Gaucher disease is known in which saposin C required for glucosylceramide degradation is deficient. In an earlier paper we described the first cases of two siblings with the non-neuronopathic form of Gaucher disease caused by saposin C deficiency [Tylki-Szymanska et al., 2007]. In this article, we present a follow up of clinical and biochemical findings in one patient who has been treated with miglustat for two years. We observed that administration of miglustat failed to exert any favorable effect on the clinical condition, haematological parameters and glucosylceramide level in the serum. In two individuals (described in this article) very slow deterioration of the peripheral and central nervous systems was observed.

Kupffer cells promote hepatic steatosis via interleukin-1beta-dependent suppression of peroxisome proliferator-activated receptor alpha activity.

UNLABELLED: Kupffer cells have been implicated in the pathogenesis of various liver diseases. However, their involvement in metabolic disorders of the liver, including fatty liver disease, remains unclear. The present study sought to determine the impact of Kupffer cells on hepatic triglyceride storage and to explore the possible mechanisms involved. To that end, C57Bl/6 mice rendered obese and steatotic by chronic high-fat feeding were treated for 1 week with clodronate liposomes, which cause depletion of Kupffer cells. Loss of expression of marker genes Cd68, F4/80, and Clec4f, and loss of Cd68 immunostaining verified almost complete removal of Kupffer cells from the liver. Also, expression of complement components C1, the chemokine (C-C motif) ligand 6 (Ccl6), and cytokines interleukin-15 (IL-15) and IL-1beta were markedly reduced. Importantly, Kupffer cell depletion significantly decreased liver triglyceride and glucosylceramide levels concurrent with increased expression of genes involved in fatty acid oxidation including peroxisome proliferator-activated receptor alpha (PPARalpha), carnitine palmitoyltransferase 1A (Cpt1alpha), and fatty acid transport protein 2 (Fatp2). Treatment of mice with IL-1beta decreased expression of PPARalpha and its target genes, which was confirmed in primary hepatocytes. Consistent with these data, IL-1beta suppressed human and mouse PPARalpha promoter activity. Suppression of PPARalpha promoter activity was recapitulated by overexpression of nuclear factor kappaB (NF-kappaB) subunit p50 and p65, and was abolished upon deletion of putative NF-kappaB binding sites. Finally, IL-1beta and NF-kappaB interfered with the ability of PPARalpha to activate gene transcription. CONCLUSION: Our data point toward important cross-talk between Kupffer cells and hepatocytes in the regulation of hepatic triglyceride storage. The effect of Kupffer cells on liver triglycerides are at least partially mediated by IL-1beta, which suppresses PPARalpha expression and activity.

Biomarkers in the diagnosis of lysosomal storage disorders: proteins, lipids, and inhibodies.

A biomarker is an analyte indicating the presence of a biological process linked to the clinical manifestations and outcome of a particular disease. In the case of lysosomal storage disorders (LSDs), primary and secondary accumulating metabolites or proteins specifically secreted by storage cells are good candidates for biomarkers. Clinical applications of biomarkers are found in improved diagnosis, monitoring disease progression, and assessing therapeutic correction. These are illustrated by reviewing the discovery and use of biomarkers for Gaucher disease and Fabry disease. In addition, recently developed chemical tools allowing specific visualization of enzymatically active lysosomal glucocerebrosidase are described. Such probes, coined inhibodies, offer entirely new possibilities for more sophisticated molecular diagnosis, enzyme replacement therapy monitoring, and fundamental research.

Spontaneous regression of disease manifestations can occur in type 1 Gaucher disease; results of a retrospective cohort study.

Gaucher disease (GD) is a lysosomal storage disorder, caused by deficient activity of the enzyme glucocerebrosidase. GD is classically divided into three major phenotypes. The most prevailing form is type 1, which presents with variable hepatosplenomegaly, cytopenia, and/or bone disease. In adult patients with mild manifestations, progress of disease might be slow or even absent. As a consequence, treatment with intravenous enzyme replacement or substrate reduction is not always necessary. In the Netherlands, the follow-up of GD patients is centralized, which allows detailed investigation of untreated patients. A retrospective study was conducted in 18 type 1 GD patients, (2 teenagers: 15 and 16 years of age at first visit) who were not treated for at least one year. The chitotriosidase activity, platelet count, hemoglobin level, lumbar bone marrow fat content measured with quantitative chemical shift imaging (QCSI), liver ratio (ml/kg body weight), and spleen volume were recorded. Criteria were developed to score regression, stability or progression of disease. During a mean follow up of 4.5 years (range 1.1-12.2) seven patients (39%) showed spontaneous regression of GD. Eight patients (44%) were stable. Two patients had progressive disease, solely based upon a sustained increase in chitotriosidase activity. A pediatric patient had an increase in splenomegaly but an improvement in bone marrow fat fraction, probably due to aging. Nine patients fulfilled the local criteria to start treatment at first visit, of whom six started treatment within 1.1 to 6.8 years. The other three refused therapy, but nevertheless showed stability or even regression of the disease during a follow up of 4.6, 9.5 and 11.4 years respectively. None of the parameters was predictive of progression or regression of disease. In conclusion, GD in adults can, in some cases, regress spontaneously. No parameters for accurately predicting future disease course exist.

Deficiency in N-acetylgalactosamine-6-sulfate sulfatase results in collagen perturbations in cartilage of Morquio syndrome A patients.

AIM: To investigate extracellular matrix (ECM) characteristics of cortical bone and articular cartilage of patients with Morquio syndrome A, a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase. PATIENTS AND METHODS: Cartilage, bone, and fibroblasts from 2 unrelated patients with Morquio syndrome were used. Histological analysis on bone and cartilage was carried out by means of light and electron microscopy. Lysyl hydroxylation and cross-linking of collagen present in bone, cartilage, and fibroblast cultures was determined by reverse-phase high performance liquid chromatography. RESULTS: No histological or biochemical differences were seen in cortical bone; furthermore, no differences were seen in the amount and modification of collagen deposited by fibroblasts. Articular cartilage showed major differences: collagen fibrils show a wider range of fibril diameter, the fibrils are in mean thicker, the lysyl hydroxylation level of the triple helix is strongly decreased, the total amount of pyridinolines is in the lower ranges, and the ratio hydroxylysylpyridinoline to lysylpyridinoline is decreased. Changes were also observed with respect to the arrangement of proteoglycans in the extracellular matrix surrounding the chondrocytes. CONCLUSION: The collagen of bone and the collagen deposited by fibroblasts is normal, whereas the ECM of cartilage in Morquio syndrome A patients is affected. Thus, deficiency in N-acetylgalactosamine-6-sulfate sulfatase has an impact on the phenotypic properties of chondrocytes, resulting in the formation of cartilage that is more prone to degeneration, being an explanation for the occurrence of osteoarthritis in Morquio syndrome A patients at early age.

Detection of chitinase activity by 2-aminobenzoic acid labeling of chito-oligosaccharides.

Chitinases are hydrolases capable of hydrolyzing the abundant natural polysaccharide chitin. Next to artificial fluorescent substrates, more physiological chito-oligomers are commonly used in chitinase assays. Analysis of chito-oligosaccharides products is generally accomplished by UV detection. However, the relatively poor sensitivity poses a serious limitation. Here we report on a novel, much more sensitive assay for the detection of chito-oligosaccharide reaction products released by chitinases, based on fluorescent detection, following chemical labeling by 2-aminobenzoic acid. Comparison with existing UV-based assays, shows that the novel assay offers the same advantages yet allows detection of chito-oligosaccharides in the low picomolar range.

Type I Gaucher disease, a glycosphingolipid storage disorder, is associated with insulin resistance.

CONTEXT: Complex glycosphingolipids, in majority the ganglioside GM3, surround the insulin receptor in a special membrane compartment (raft) and modulate signaling through this receptor. Increased levels of GM3 in rafts impair insulin signaling, resulting in insulin resistance. Gaucher disease is a lysosomal storage disorder in which impaired breakdown of glucosylceramide leads to its accumulation in macrophages. Secondary to this defect, GM3 concentrations, for which glucosylceramide is the precursor, in plasma and several cell types are elevated. OBJECTIVE: We studied the influence of glycosphingolipid storage on whole body glucose and fat metabolism by measuring insulin-mediated (IMGU) and noninsulin-mediated glucose uptake (NIMGU) and suppression of free fatty acids by insulin. DESIGN AND MAIN OUTCOME MEASURES: We studied six Gaucher patients, either naive to treatment or with considerable remaining burden of disease, and six matched healthy control subjects in the basal state, during an euglycemic and a hyperglycemic clamp with somatostatin measuring NIMGU and during an euglycemic hyperinsulinemic clamp measuring IMGU, using stable isotopes. RESULTS: NIMGU (both during euglycemia and hyperglycemia) did not differ between patients and control subjects. IMGU was lower in Gaucher patients, compared with controls. Suppression of lipolysis by insulin tended to be less effective in Gaucher patients. CONCLUSION: Gaucher disease, a lysosomal glycosphingolipid storage disorder, is associated with (peripheral) insulin resistance, possibly through the influence of glycosphingolipids on insulin receptor functioning.

Treatment of Fabry disease with different dosing regimens of agalsidase: effects on antibody formation and GL-3.

Two different enzyme preparations are used for the treatment of Fabry disease patients, agalsidase alpha (Replagal, Shire) and agalsidase beta (Fabrazyme, Genzyme). Therapeutic efficacy of both products has been variable probably due to differences in gender, severity, age and other patient characteristics. We studied the occurrence of alpha-Gal A antibodies and their effect on urinary and plasma globotriaosylceramide (GL-3), plasma chitotriosidase and clinical outcome in 52 patients after 12 months of treatment with either 0.2mg/kg agalsidase alppha (10 males, 8 females) or beta (8 males, 5 females) or 1.0mg/kg agalsidase beta (10 males, 11 females). Antibodies were detected in 18/28 male patients after 6 months. None of the females developed antibodies. Following 12 months of 0.2mg/kg treatment, urinary GL-3 decreased in antibody negative (AB-) but increased in antibody positive (AB+) patients. Treatment with 1.0mg/kg gave a reduction in urinary GL-3 in both AB- and AB+ patients. Levels of plasma GL-3 and chitotriosidase decreased in all patient groups. Twelve months of 0.2mg/kg treatment did not change renal function or left ventricular mass. Further, no change in renal function was seen following 1.0mg/kg treatment and left ventricular mass decreased in both AB- and AB+ patients. In summary, alpha-Gal A antibodies frequently develop in male Fabry disease patients and interfere with urinary GL-3 excretion. Infusion of a dose of 1.0mg/kg results in a more robust decline in GL-3, less impact, if any of antibodies, stable renal function and reduction of LVMass.

Prominent increase in plasma ganglioside GM3 is associated with clinical manifestations of type I Gaucher disease.

BACKGROUND: Patients with Gaucher disease show signs of insulin resistance. The ganglioside GM3 has recently shown to be a negative regulator of insulin sensitivity. In fibroblasts of Gaucher patients, deficient in degradation of glucosylceramide, an increased anabolism of this lipid to gangliosides occurs. The goal of the current study was to establish whether GM3 is elevated in plasma of type I Gaucher disease patients, and is related to disease manifestations. METHODS: Plasma GM3, glucosylceramide, and ceramide were determined and compared to overall severity of disease, hepatomegaly, and plasma chitotriosidase activity. RESULTS: The ceramide concentration in plasma of untreated Gaucher patients was slightly but not significantly lower than in controls (median: 9.8 micromol/L, range: 5.7-14.7 micromol/L, (n=40) vs. median: 11.0 micromol/L, range: 5.1-18.0 micromol/L, (n=30)). Glucosylceramide was significantly (p<0.0001) elevated. GM3 was also significantly (p<0.0001) increased (median: 10.2 micromol/L, range: 4.3-19.1 micromol/L, (n=40) vs. median: 3.6 micromol/L, range: 2.7-5.4 micromol/L, (n=30)). Plasma GM3 concentrations correlated with those of plasma chitotriosidase activity (rho=0.45, p=0.0036), overall severity of disease (rho=0.39, p=0.012), and hepatomegaly (rho=0.49, p=0.0015). CONCLUSIONS: GM3 is strikingly elevated in plasma of most Gaucher patients. The increase is comparable to that of glucosylceramide, the primary storage lipid. The marked elevations in GM3 may play a role in the insulin resistance of Gaucher patients.

Low frequency maintenance therapy with imiglucerase in adult type I Gaucher disease: a prospective randomized controlled trial.

BACKGROUND AND OBJECTIVES: Gaucher disease type I can be successfully treated with enzyme replacement therapy (ERT). In order to reduce the burden of the intravenously administered enzyme, a low frequency of administration was prospectively studied in patients with stable and minor disease following ERT. DESIGN AND METHODS: Eleven patients were randomly assigned either to continue their original regimen of a dose of ERT once every week or fortnight (five patients) or to lower the frequency of administration to once every 4 weeks, at the same cumulative dose (six patients). The primary end-point was change in liver ratio (mL/kg body weight). Secondary end-points were spleen volume, hemoglobin level, platelet count, lumbar bone marrow fat content measured with quantitative chemical shift imaging (QCSI), white cell count, and plasma levels of ferritin, chitotriosidase, liver enzymes and angiotensin-converting enzyme (ACE). RESULTS: There were no significant mean differences between the two treatment arms in liver ratio or any of the other end-points. However, there were two treatment failures in the low frequency of administration group. These patients showed progression of disease as evidenced by a reduction of QCSI in one patient and an increase in liver ratio as well as a slow decrease in QCSI in the other. Both patients already had relatively low baseline QCSI values. One patient switched back to the original regimen at 6 months because of subjective complaints. INTERPRETATION AND CONCLUSIONS: Low frequency ERT in adult Gaucher type I patients maintains stable disease in most, but not all patients with stable and minimal disease. Close monitoring of all disease parameters remains mandatory.

Substrate reduction therapy of glycosphingolipid storage disorders.

In the last 15 years enormous progress has been made regarding therapy of type I Gaucher disease, a severely disabling disorder characterized by intralysosomal storage of glucosylceramide in tissue macrophages. Effective enzyme replacement therapy of type I Gaucher disease, based on chronic intravenous administration of mannose-terminated recombinant human glucocerebrosidase, has been available since 1990 and has been applied in several thousand patients without serious adverse effects. An alternative therapeutic approach, so-called substrate reduction therapy, is based on partial reduction of the synthesis of glucosylceramide and hence of subsequent metabolites. Oral administration of an inhibitor of glucosylceramide synthesis (N-butyldeoxynojirimycin, registered in Europe since 2002 as miglustat (Zavesca)), is effective in reversing clinical symptoms in type I Gaucher patients with mild to moderate disease manifestations. The growing long-term experience with substrate reduction therapy indicates that this treatment is also without major adverse effects. Substrate reduction therapy, in conjunction with enzyme replacement therapy, may play an important role in the future clinical management of patients suffering from type I Gaucher disease. Clinical trials are under way that should reveal the value of substrate reduction for maintenance therapy of type I Gaucher disease and for treatment of neuronopathic variants of Gaucher disease, Niemann-Pick disease type C, late-onset Tay-Sachs disease and Sandhoff disease.

Clinical variability in mucolipidosis III (pseudo-Hurler polydystrophy).

Mucolipidosis III (MLIII) is caused by a deficiency of UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine 1-phosphotransferase (phosphotransferase) activity, an enzyme responsible for the formation of the recognition marker on most lysosomal enzymes. The consequences of this defect are impairment of many lysosomal catabolic processes. A deficiency of phosphotransferase activity causes two phenotypically different diseases: mucolipidosis II and a rare form, mucolipidosis III (pseudo-Hurler polydystrophy). The purpose of this article is to report three patients with ML III, presenting quite different clinical courses: Patient 1 is a 13-year-old girl in whom the only symptoms of ML III were joint stiffness of the hands. Patients 2 and 3 are sibs: a 5-year-old boy with a severe form of ML III and his 2-year-old sister, who is less affected than her brother at the same age. A comparison of biochemical results and the clinical picture of our patients with cases in the literature is presented.

Long-term effect of antibodies against infused alpha-galactosidase A in Fabry disease on plasma and urinary (lyso)Gb3 reduction and treatment outcome.

INTRODUCTION: Enzyme replacement therapy (ERT) with alpha-Galactosidase A (aGal A) may cause antibody (AB) formation against aGal A in males with Fabry disease (FD). Anti agalsidase ABs negatively influence globotriaosylceramide (Gb3) reduction. We investigated the impact of agalsidase AB on Gb3 and lysoGb3 and clinical outcome in Fabry patients on ERT. METHODS: Adult male and female patients on ERT for at least one year were included. Urinary Gb3 was measured by HPLC, plasma lysoGb3 by LC-ESI-MS/MS and AB with a neutralization assay. RESULTS: Of the 59 patients evaluable patients, 0/30 females and 17/29 males developed anti-agalsidase antibodies (AB+). Only 3/17 males had transient (low) titers (tolerized). All AB+ patients developed antibodies during the first year of treatment. Change of agalsidase preparation (or dose) did not induce antibody formation. AB+ males had significant less decline in plasma lysoGb3 compared to AB- males (p = 0.04). Urinary Gb3 levels decreased markedly in AB- but remained comparable to baseline in AB+ males (p<0.01). (Lyso)Gb3 reduction in plasma and urine on ERT was correlated with LVmass reduction in females and development white matter lesions and stroke. CONCLUSION: In male patients antibodies against aGal A remained present up to 10 years of ERT. The presence of these antibodies is associated with a less robust decrease in plasma lysoGb3 and a profound negative effect on urinary Gb3 reduction, which may reflect worse treatment outcome.

Vascular aspects of Fabry disease in relation to clinical manifestations and elevations in plasma globotriaosylsphingosine.

Fabry disease is an X-linked hereditary lysosomal storage disorder attributed to a deficiency of α-galactosidase A leading to increased plasma levels of globotriaosylsphingosine (lysoGb3). The disease presents as a vascular disease, with cerebral, cardiac, and renal complications. Carotid intima-media thickness (IMT), brachial flow-mediated dilation (FMD), pulse wave velocity, and advanced glycation end products were measured in 57 classically affected patients (22 men and 35 women), 55 healthy matched controls (20 men and 35 women), and 10 atypical Fabry disease patients (5 men and 5 women). Most patients received enzyme replacement therapy. In classically affected male patients, brachial FMD was decreased (2.9% [95% CI, 0.8% to 7.9%] versus 5.9% [2.1% to 8.5%] in controls; P=0.01), and carotid IMT was increased (0.67 mm [95% CI, 0.50-0.96 mm] versus 0.59 mm [95% CI, 0.40-0.76 mm] in controls; P=0.01). In women and atypical patients these vascular parameters were comparable with controls. Pulse wave velocity was not different; advanced glycation end products were only slightly increased in atypical patients. In classically affected women, a small increase in lysoGb3 was associated with an increase in IMT independent of age. In the classically affected men, all with increased IMT and high levels of plasma lysoGb3, lysoGb3 levels did not add to a higher IMT, suggestive of a ceiling effect. For FMD, elevated lysoGb3 levels (>7 nmol/L) contributed to a 2.9% lower FMD independent of age and sex (P=0.02). Increased carotid IMT and decreased brachial FMD occur in classic Fabry disease, which is associated with plasma lysoGb3 level independent of age and sex. These observations still exist despite enzyme replacement therapy.

Consequences of a global enzyme shortage of agalsidase beta in adult Dutch Fabry patients.

BACKGROUND: Enzyme replacement therapy is currently the only approved therapy for Fabry disease. From June 2009 on, viral contamination of Genzyme's production facility resulted in a worldwide shortage of agalsidase beta leading to involuntary dose reductions (approved dose 1 mg/kg/eow, reduced dose 0.5 mg/kg/m), or switch to agalsidase alpha (administered dose 0.2 mg/kg/eow). An assessment report from the European Medicines Agency (EMA) raised serious concerns about an increase in adverse events at lower dosages of agalsidase beta. We determined the influence of the shortage on clinical event incidence and the most sensitive biochemical marker (lysoGb3) in Dutch Fabry patients. METHODS: The incidence of clinical events per person per year was calculated from start of agalsidase beta treatment until the shortage, and was compared to the incidence of clinical events during the shortage period. In addition, plasma lysoGb3, eGFR, quality of life (SF-36) and brief pain inventory (BPI) questionnaires were analysed. RESULTS: All thirty-five Dutch Fabry patients using agalsidase beta (17 males) were included. Mean clinical event incidence was unchanged: 0.15 events per person per year before versus 0.15 during the shortage (p = 0.68). In total 28 clinical events occurred in 14 patients during 4.6 treatment years, compared to 7 events in 6 patients during the 1.3 year shortage period. eGFR and BPI scores were not significantly altered. Two SF-36 subscales were significantly but minimally reduced in females. In males, lysoGb3 increased with a median of 8.1 nM (range 2.5-29.2) after 1 year of shortage (p = 0.001). Increases in lysoGb3 were found in both patients switching to agalsidase alpha and on a reduced agalsidase beta dose. Antibody status, treatment duration or clinical event incidence showed no clear correlation to lysoGb3 increases. CONCLUSIONS: No increase in clinical event incidence was found in the adult Dutch Fabry cohort during the agalsidase beta shortage. Increases in lysoGb3, however, suggest recurrence of disease activity.

HPLC for simultaneous quantification of total ceramide, glucosylceramide, and ceramide trihexoside concentrations in plasma.

BACKGROUND: Simple, reproducible assays are needed for the quantification of sphingolipids, ceramide (Cer), and sphingoid bases. We developed an HPLC method for simultaneous quantification of total plasma concentrations of Cer, glucosylceramide (GlcCer), and ceramide trihexoside (CTH). METHODS: After addition of sphinganine as internal calibrator, we extracted lipids from 50 microL plasma. We deacylated Cer and glycosphingolipids by use of microwave-assisted hydrolysis in methanolic NaOH, followed by derivatization of the liberated amino-group with o-phthaldialdehyde. We separated the derivatized sphingoid bases and lysoglycosphingolipids by HPLC on a C18 reversed-phase column with a methanol/water mobile phase (88:12, vol/vol) and quantified them by use of a fluorescence detector at lambda(ex) 340 nm and lambda(em) 435 nm. RESULTS: Optimal conditions in the Solids/Moisture System SAM-155 microwave oven (CEM Corp.) for the complete deacylation of Cer and neutral glycosphingolipids without decomposition were 60 min at 85% power, fan setting 7. Intra- and interassay CVs were <4% and <14%, respectively, and recovery rates were 87%-113%. The limit of quantification was 2 pmol (0.1 pmol on column), and the method was linear over the interval of 2-200 microL plasma. In samples from 40 healthy individuals, mean (SD) concentrations were 9.0 (2.3) micromol/L for Cer, 6.3 (1.9) micromol/L for GlcCer, and 1.7 (0.5) micromol/L for CTH. Plasma concentrations of GlcCer were higher in Gaucher disease patient samples and of CTH in Fabry disease patient samples. CONCLUSIONS: HPLC enables quantification of total Cer, GlcCer, and CTH in plasma and is useful for the follow-up of patients on therapy for Gaucher or Fabry disease.